Research ArticlePPAR-120574 Ligand Inhibits Nasopharyngeal Carcinoma CellProliferation and Metastasis by Regulating E2F2

Ping-Li Yang 1 Jia-ShunWang1 Xiao-Mei Cheng2 Jing-Cai Chen1 Hui Zhu1

Xiao-Lan Li1 Li Cao1 andWei Tang 1

1Department of Otorhinolaryngology e First Affiliated Hospital Shihezi University School of MedicineShihezi Xinjiang China2Outpatient Department of Xinjiang Production and Construction Corp Urumchi Xinjiang China

Correspondence should be addressed to Ping-Li Yang entyplshzueducn andWei Tang twentshzsinacom

Received 24 March 2019 Revised 23 June 2019 Accepted 3 July 2019 Published 1 August 2019

Academic Editor Antonio Brunetti

Copyright copy 2019 Ping-Li Yang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Purpose Peroxisome proliferator-activated receptor-120574 (PPAR-120574) is a nuclear hormone receptor with a key role in lipid metabolismPrevious studies have identified various roles of PPAR-120574 in cell cycle progression cellular proliferation and tumor progressionHowever no report has described a role for PPAR-120574 in human nasopharyngeal carcinoma (NPC) Notably some studies havereported a relationship between PPAR-120574 and E2F transcription factor 2 (E2F2) which has been identified as a regulator of cell cycleapoptosis and the DNA damage response Notably E2F2 has also been reported to correlate with a poor prognosis in patients withvarious malignancies Methods We used immunohistochemical (IHC) and western blot methods to evaluate PPAR-120574 and E2F2expression and function in nonkeratinizing NPC and nasopharyngitis (NPG) tissue samples as well as western blotting and CCK8analyses in the NPC cell lines CNE1 and CNE2 Results We observed lower levels of PPAR-120574 expression in nonkeratinizing NPCtissues compared with NPG tissues and determined an association between a low level of PPAR-120574 expression with a more advancedtumor stage Furthermore strong E2F2 expression was detected in nonkeratinizing NPC tissues We further demonstrated thatrosiglitazone a PPAR-120574agonist reducedE2F2 expression andproliferation inNPC cell linesConclusions Our study results revealeda novel role for the PPAR-120574ndashE2F2 pathway in controlling NPC cell proliferation and metastasis

1 Introduction

Nasopharyngeal carcinoma (NPC) arises from the epithe-lial and columnar cells of the nasopharyngeal mucosaHistopathologically the World Health Organization (WHO)has classified NPCs into three subtypes type I keratinizingsquamous carcinoma type II nonkeratinizing carcinomaand type III basaloid squamous carcinoma Although NPC isa rare disease worldwide it has a high incidence in SoutheastAsia and type II is the most common form on both scales[1 2] Although NPC is a radiosensitive tumor it is associatedwith relatively high rates of recurrence and distant metastasisat 2 years after radiotherapy These factors underscore theneed to identify factors related to the proliferation andmetastasis of NPC as well as potential therapeutic targets

E2F transcription factor 2 (E2F2) has been identified as aregulator of cell proliferation differentiation and apoptosis

[3] Although many recent studies have associated E2F2activity with inappropriate cell proliferation andor apoptosisin various tumor types [4ndash6] the role of E2F2 inNPC remainsunknown Several research groups have identified E2F2 as amediator in the ability of peroxisome proliferator-activatedreceptors (PPARs) 120572 120573 and 120574 to regulate cell proliferation[7ndash9] PPAR-120574 is expressed strongly in adipose tissue andhas been shown to play a key role in the onset of obesity[10] T2DM[11]metabolic syndrome [12] and cardiovasculardisease [13] and the activated form plays an inhibitory role incell growth and proliferation [14] Increasing evidence sug-gests that PPAR-120574 protects against tumors by inhibiting cellproliferation However the underlying mechanism remainsunknown Based on the abovementioned reports that PPAR-120574affects tumor proliferation and metastasis by targeting E2F2in this study we investigated the expression patterns andactivities of these proteins in NPC tissue samples and cell

HindawiPPAR ResearchVolume 2019 Article ID 8679271 9 pageshttpsdoiorg10115520198679271

2 PPAR Research

lines to determine the effects on tumor cell proliferation anddifferentiation

2 Materials and Methods

21 Patient Tissue Samples Fifty-two diagnosed nonkera-tinizing NPC tissues and 34 diagnosed NPG tissues ana-lyzed by the Department of Pathology of the First Affil-iated Hospital of Shihezi University School of Medicinewere collected from the Department of Otolaryngology ofthe same institution between April 2015 and January 2019For each tissue sample a portion was stored at minus80∘Cprior to Western blotting and another portion was storedin formalin prior to immunohistochemistry (IHC) analy-sis

22 Immunohistochemical Staining Nonkeratinizing NPCand NPG tissues were fixed in 4 formalin for 12 h dehy-drated in a graded ethanol series (75 85 95 and 100)soaked in xylene and embedded in paraffin Subsequentlythe samples were cut into 4120583m sections and placed on glassslides deparaffinized in xylene and hydrated in a gradedethanol series (100 95 85 and 75) All slides wereincubated in a citrate solution for heat-induced antigenretrieval and exposed to 3 H2O2 for 10min to blockendogenous peroxidases After three washes with phosphate-buffered saline (PBS) the sections were incubated in a block-ing solution containing bovine serum albumin (BSA) for20min followed by exposure to primary antibodies againstE2F2 (YT1443 Immunoway rabbit polyclonal 1200 dilution)and PPAR-120574 (Novusbio rabbit polyclonal 1100 dilution) at4∘C overnight The antigenic sites were visualized using adiaminobenzidine (DAB) kitThe nuclei were counterstainedwith hematoxylin Finally the slides were dehydrated ina graded ethanol series and xylene and observed under amicroscope The expression levels of E2F2 and PPAR-120574 inthe tissues were classified as follows IOD SUMArea SUMlt002 low expression or IOD SUM Area SUM ge002 highexpression

23 Real-Time RT-PCR Total RNA was isolated from theNPC andNPG tissues which was prepared using an EZNAtotal RNA kit I (Omega Bio-Tek) The harvested tissue washomogenized in TRK lysis buffer followed by RNA isolationand purification A TaKaRa LA Taq reverse transcriptionkit was applied to reverse transcribe mRNA from NPCand NPG tissues into cDNA After that quantitative real-time-PCR was performed The primer sequences for humanGAPDH E2F2 and PPAR-120574 used in this study were GAPDHforward 51015840-TCAAGAAGGTGGTGAAGCAGG-31015840 GAPDHreverse 51015840-TCAAGGTGGAGGAGTGGGT E2F2 forward51015840-CTGAAGGAGCTGATGAACACG-31015840 E2F2 reverse51015840-CCCTTGGGTGCTCTTGAGATA-31015840 15-PGDH forward51015840-GCATAGTTGGATTCACACGCT-31015840 15-PGDH reverse51015840-TTGGCAATCAATGGTGGGTC-31015840 Relative expressionlevel was calculated for each gene by the 2minusΔΔCT method withGAPDH for normalization

24 Cell Lines and Cell Culture The human NPC cell linesCNE1 (well differentiated) and CNE2 (poorly differenti-ated) which can represent the characteristics of NPC celllines with different degree of differentiation were culturedin Dulbeccorsquos modified Eaglersquos medium (DMEM) medium(Hyclone USA) supplemented with 10 fetal bovine serum(Gibco USA) and 1 penicillinstreptomycin (HycloneUSA) All cells were cultured in an atmosphere of 5CO2 at 37∘C NPC cell lines CNE1 and CNE2 wereobtained from the cancer center of Union Hospital (WuhanChina)

25 Western Blotting Nonkeratinizing NPC and NPG sam-ples and CNE1 and CNE2 cells incubated with or withoutPPAR-120574 ligand were collected and lysed in radioimmuno-precipitation assay (RIPA) buffer (Beyotime China) con-taining a phosphatase inhibitor and phenylmethanesulfonylfluoride The protein amounts in the lysates were quantifiedusing a BCA protein assay kit (Beyotime Haimen JiangsuChina) and 20ndash50 120583g of total proteins per sample wereloaded onto 10 sodium dodecyl sulfate-polyacrylamide gelsand transferred to polyvinylidene difluoride membranesSubsequently the membranes were blocked in a 5 solu-tion of nonfat milk powder at room temperature for 1 hand subsequently incubated overnight at 4∘C in dilutionsof the following primary antibodies E2F2 (YT1443 rabbitpolyclonal 11000 dilution) PPAR-120574 (NR1C3 rabbit poly-clonal 1500 dilution) and GAPDH (KM9002 mouse mon-oclonal 14000 dilution) Next the membranes were washedand incubated with species-specific horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperatureThe proteins were finally visualized using the EnhancedChemiluminescent Plus reagent (Beyotime China) The tar-get protein levels in each sample were quantified densitomet-rically and normalized against GAPDH levels in the samesample

26 Cell Counting Kit-8 Proliferation Assay CNE1 and CNE2cells were seeded in 96-well plates at a density of 103 cellswelland incubated overnight Next 5 10 and 20120583mol rosigli-tazone (Rog) a PPAR-120574 agonist andor 100 nmol GW9662a PPAR-120574 antagonist were added to the wells At 24 4872 and 96 h indicated wells were washed with PBS andthe medium was replaced with 100120583L of fresh medium plus10120583L of CCK-8 kit reagent The cells were further culturedfor 4 h and the optical density (OD) of each well was mea-sured at 450 nm using a SynergyMx MultiMode MicroplateReader (Biotek USA) at 0 05 1 and 2 h OD values ofdifferent density Rog groups against control group were ana-lyzed

27 Flow Cytometric Analysis The quantification of apop-totic cells was performed with the rh Annexin V-FITCDetection Kit (Antgene) according to the manufacturerrsquosinstructions CNE1 and CNE2 cell lines were harvested bytrypsinization and washed twice in cold PBS Staining wasperformed with rh Annexin V-FITC and propidium iodideApoptosis was determined by calculating the percentage of

PPAR Research 3

apoptotic cells relative to the total number of cells Theresults were confirmed in at least three independent experi-ments

28 Statistical Analyses All statistical analyses were per-formed using SPSS (version 170 IBM Corp USA) The chi-square test was used to compare the levels of expression ofE2F2 and PPAR-120574 in nonkeratinizing NPC and NPG samplesand the respective correlations with the clinicopathologicalfeatures of NPC patients The unpaired Studentrsquos t-test wasused to compare E2F2 and PPAR-120574 expression betweengroups of CNE1 and CNE2 cells For all analyses a P valueof lt005 was considered to indicate a statistically significantdifference

3 Results

31 Expression of E2F2 and PPAR-120574 in Undifferentiated NPCandNPGTissues andCorrelations with the ClinicopathologicalFeatures of NPC Patients IHC was used to evaluate theexpression of E2F2 and PPAR-120574 proteins in 52 nonkeratiniz-ing NPC and 34 NPG tissue samples Strong E2F2 expres-sion (IODArea ge02) was observed in 6346 (3352) ofnonkeratinizing NPC tissues compared with 1765 (634) ofNPG tissues (P lt001) Strong PPAR-120574 expression (IODAreage02) was also observed in 2885 (1552) of nonkeratiniz-ing NPC tissues compared with 7647 (2634) of NPGtissues (P lt001) (Table 1) Both differences were signifi-cant

We further analyzed the associations of E2F2 andPPAR-120574 expression with the clinical parameters in patientswith nonkeratinizing NPC Although no correlations wereobserved with sex age or T (tumor) classification weobserved a positive correlation between E2F2 and nonkera-tinizing NPC staging namely the N (lymph node metastasis)(N0ndashN1 2240(5500) vs N2ndashN3 1112(9167) P lt005)andM (distant metastasis) classifications (M0 2352(5476)vs M1 1010(100) P lt005) PPAR-120574 expression was onlyfound to correlate positively with the M classification (M01542 (3571)vsM1 010(0) Plt005)These results stronglyindicate that E2F2 contributes to the proliferation andmetas-tasis of nonkeratinizing NPC and is associated with thedisease stage

32 Immunohistochemical Staining of E2F2 and PPAR-120574 inNonkeratinizing NPC and NPG Tissues IHC was used toevaluate the expression and localization of E2F2 and PPAR-120574in tissue samples Notably we observed strong E2F2 expres-sion in nonkeratinizing NPC tissues (Figure 1(a)) comparedwith NPG tissues (Figure 1(c)) Although E2F2 localized inboth the nuclei and cytoplasm of the former tissues it wasmost strongly expressed in the nuclei We further observeda trend toward decreased PPAR-120574 expression in nonker-atinizing NPC tissues (Figure 1(b)) compared with NPGtissues (Figure 1(d)) A semiquantitative evaluation revealedsignificantly stronger E2F2 expression in nonkeratinizingNPC tissues compared toNPG tissues (Figure 1(e)) (Plt001)as well as a significant difference in PPAR-120574 expressionbetween these tissue types (Figure 1(f)) (P lt001)

33 Expression of E2F2 and PPAR-120574 in Nonkeratinizing NPCand NPG Tissues The mRNA expression of E2F2 in NPC issignificantly higher than NPG while the mRNA expressionof PPAR-120574 in NPC is lower than NPG (Figure 2(a)) West-ern blotting revealed stronger E2F2 expression in nonker-atinizing NPC tissues and weaker PPAR-120574 expression innonkeratinizing NPC tissues when compared with NPGtissues (Figure 2(b)) consistent with the IHC findings Aquantitative analysis of the E2F2 and PPAR-120574 protein expres-sion levels revealed significant differences between the tissuetypes (P lt001 and lt005 respectively) (Figures 2(c) and2(d))

34 PPAR-120574 Ligand Inhibits the Proliferation of CNE1 andCNE2 Cell Lines To analyze the functional role of PPAR-120574 in NPC cell lines we performed CCK-8 assays to detectthe proliferation of NPC cell lines The results showed thatPPAR-120574 ligand Rog inhibited the proliferation of both celllines in a dose-dependent manner (Figures 3(c) and 2(d))To determine if proliferation was blocked we performedapoptosis analysis by flow cytometry The results showed thatthe number of apoptotic cells did not increase after treatmentwith Rog (Figures 3(a) and 3(b))

35 Expression of E2F2 and PPAR-120574 in CNE1 and CNE2Cell Lines a13er Treatment with a PPAR-120574 Ligand Finallywe incubated cell lines with a PPAR-120574 ligand to analyze thefunctional role of PPAR-120574 in NPC cell lines The expressionof E2F2 decreased while PPAR-120574 increased after treatmentwith Rog and the effect was dose-dependent (Figures 4(a)and 4(b)) We further verified the protein expression levelsusing western blotting and found that after treatment withthe PPAR-120574 agonist Rog the expression of E2F2 decreasedwhereas that of PPAR-120574 increased To confirm the inhibitoryeffect of PPAR-120574 on E2F2 we also treated cells with thePPAR-120574 antagonist GW9662 and observed an increase inthe expression of E2F2 (Figures 4(c) and 4(d)) These resultssuggest that PPAR-120574 contributes to the inhibition of NPC cellproliferation and metastasis

4 Discussion

NPC is a polygenic hereditary malignant tumor that mostcommonly arises from the lateral wall of the nasophar-ynx particularly the fossa of Rosenmuller and around theEustachian cushion (and occult locations) As the primarytumor location is the base of the skull the local invasionof NPC may have serious consequences and most patientspresent with lymph nodes or distant tissue metastasis atthe time of diagnosis [15] Currently the proliferation andmetastasis of tumor tissues are closely related to the differen-tiation and proliferation rates of cells understanding that thefactors that regulate the cell cycle help us to understand themechanisms that cause cell proliferation and differentiationas well as look for factors related to treatment and progno-sis

Previously we identified that E2F2 could induce thetranscription of target genes and drive cell cycle progressionand proliferation [16] Cell cycle deregulation is a common

4 PPAR Research

Table1Ex

pressio

nsof

E2F2

andPP

AR-120574in

clinicalsamples

ofun

differentiatedNPC

andNPG

andassociations

with

clinicop

atho

logicalcharacteristicso

fund

ifferentia

tedNPC

samples

Parameter

NE2

F2a

NPP

AR-120574b

Lowexpressio

nHighexpressio

nPvaluec

Lowexpressio

nHighexpressio

nPvaluec

Histologictype

lt001

lt001

NPC

5219

(3654)

33(6346

)52

37(7115)

15(2885)

NPG

3428

(8235)

6(176

5)34

8(2353)

26(7647)

Gender

0868

0377

Male

3914

(3590)

25(6410)

3929

(7436)

10(2564

)Female

135(3846

)8(615

4)13

8(615

4)5(3846

)Age

(y)

0253

01

lt50

2210

(4545)

12(5455)

2213

(590

9)9(4091)

≧50

309(3000

)21

(7000

)30

24(8000

)6(2000

)Tclassifi

catio

n0457

033

I-II

3514

(4000

)21

(6000

)35

23(6571)

12(3429)

III-IV

175(294

1)12

(7059)

1714

(8235)

3(176

5Nclassifi

catio

n0037

0071

N0-N1

4018

(4500

)22

(5500

)40

26(6500

)14

(3500

)N2-N3

121(833)

11(916

7)12

11(916

7)1(833)

Mclassifi

catio

n000

4004

6M0

4219

(4524)

23(5476)

4227

(6429)

15(3571)

M1

100(0)

10(100

)10

10(100

)0(0)

a E2F2lowexpressio

nIO

DA

realt

002highexpressio

nIO

DA

rea⩾

002

b PPA

R-120574lowexpressio

nIO

DA

realt

002highexpressio

nIO

DA

rea⩾

002

c Chi-squ

aretest

PPAR Research 5

50 Ｇ

E2F2

NPC

(a)

(a)

50 Ｇ

PPAR-

NPC

(b)

(b)E2F2

NPG

(c)

50 Ｇ

(c)

50 Ｇ

PPAR-

NPG

(d)

(d)

000

001

002

003

004

005

E2F2

(IOD

Are

a)

NPC NPG

lowast

(e)

000

002

004

006

008

PPA

R-

(IOD

Are

a)

NPC NPG

lowastlowast

(f)

Figure 1 E2F2 and PPAR-120574 protein expression in nonkeratinizing nasopharyngeal carcinoma (NPC) and nasopharyngitis (NPG) tissuesImmunohistochemistrywas performed to detect E2F2 and PPAR-120574 protein expression in nonkeratinizing NPC tissues (a b) andNPG tissues(c d) Scale bar 50120583m(e f)The immunohistochemistry datawere analyzed semiquantitatively to determine the E2F2 andPPAR-120574expressionlevels in nonkeratinizing NPC and NPG tissues Data are shown as means plusmn standard deviations lowastlowastP lt001

feature of human cancer The function of proproliferationwhich lies in the ability enables cell cycle entry into the Sphase via E2F2 [17] and strong E2F2 expression was detectedin various types of tumors [18ndash21] There is no report aboutE2F2 in NPC In this study we use IHC method to analysis52 nonkeratinizing NPC tissues and 34 NPG tissues revealedstronger E2F2 expression in the former which was associatedwith the clinical tumor stage Specifically we observed sig-nificantly higher levels of E2F2 expression in N2ndashN3 and M1

stage nonkeratinizing NPC tissues This finding suggests anassociation of E2F2 expressionwith the degree ofmalignancywhich was confirmed using western blotting and RT-PCRThis finding indicates that E2F2 is a meaningful marker ofthe degree of tumor malignancy Furthermore we found thatE2F2 is localizedmainly in the nuclei of nonkeratinizingNPCtissues suggesting that this transcription factor translocatesto the nucleus to promote DNA synthesis and subsequent cellproliferation and division This important role of E2F2 in cell

6 PPAR Research

00

05

10

15

20

Relat

ive m

RNA

leve

l (E

2F2)

NPC NPG0

2

4

6

Relat

ive m

RNA

leve

l(P

PAR-

)

NPGNPC

lowastlowastlowastlowast

(a)

NPG

E2F2

PPAR-

GAPDH

NPC(b)

NPC

E2F2

GA

PDH

NPG06

08

10

12lowastlowast

(c)

PPA

R-

GA

PDH

NPC NPG06

07

08

09

10 lowast

(d)

Figure 2 E2F2 and PPAR-120574 protein expression in nonkeratinizing nasopharyngeal carcinoma (NPC) and nasopharyngitis (NPG) tissue lysates(a b) RT-PCR and Western blotting were performed to detect the expression of E2F2 PPAR-120574 and GAPDH in lysates of nonkeratinizingNPC and NPG tissues (c d) Quantitative analyses of the E2F2 and PPAR-120574 expression levels in nonkeratinizing NPC and NPG tissues Dataare expressed as means plusmn standard deviations lowastP lt005 lowastlowastP lt001

proliferation suggests that treatment with an E2F2 inhibitormay inhibit proliferation

PPAR-120574 a member of the ligand-activated superfamilyof nuclear transcription factors is an important regulator ofinflammation glucosemetabolism and cell proliferation [22]and was previously shown to inhibit tumor cell proliferation[23ndash25] In this study we used IHC western blotting and RT-PCR to demonstrate reduced PPAR-120574 expression in nonker-atinizing NPC tissues compared to NPG tissues especiallyin cases of the former that involved distant metastasis Itsuggests that expression of PPAR-120574 related to NPC andrelated to the malignancy degree of tumor The antitumoraffections of PPAR-120574 were thought to be associated with

inhibition of angiogenesis stemness process and cell cyclearrest [26 27] In our study we observed a decrease inE2F2 expression and reductions in the proliferation anddifferentiation rates in NPC cells treated with the PPAR-120574agonist Rog and it was dose-dependent Notably this effectwas offset by incubation with the PPAR-120574 agonist GW9662Supporting our result Komatsu et al reported that thedownregulation of E2F2 led to PPAR-120574 agonist-mediated cellcycle withdrawal leading to a cell cycle blockade at the G1Stransition Numerous studies had demonstrated that ligandactivation of PPAR-120574 activation inhibits the phosphorylationof Rb protein which has been reported to lead to abrogationof E2F2 [28] or directly decrease the DNA-binding activity

PPAR Research 7

CNE1

Com

p-PE

-A

Comp-FITC-A

Rog(0) Rog(5M) Rog(10M) Rog(20M)

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

(a)

CNE2

Com

p-PE

-A

Comp-FITC-A

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

Rog(0) Rog(5M) Rog(10M) Rog(20M)

(b)

24 48 72 96

Rog5ctlRog10ctlRog20ctl

Rela

tive p

rolif

erat

ion

(Rog

ctl)

CNE1

00

02

04

06

08

10

12

lowastlowast

lowastlowastlowastlowast

lowast

(c)

24 48 72 96

Rela

tive p

rolif

erat

ion

(Rog

ctl)

CNE2

Rog5ctlRog10ctlRog20ctl

00

02

04

06

08

10

12

lowastlowast

lowastlowastlowastlowastlowast

lowast

(d)

Figure 3 PPAR-120574 ligand inhibits the proliferation of CNE1 and CNE2 cell lines (a b) rh Annexin V-FITC assay was performed to detect thepercentage of apoptotic cells relative to the total number of cells in CNE1 and CNE2 cell lines after treatment with dose-dependent Rog (cd) Analysis of a CCK-8 proliferation assay of CNE1 and CNE2 cell lines

of E2FDP transcription factors [29] Wu et al found thatcombined inactivation of E2F2 is sufficient to block cellularproliferation completely [30]

Potentially PPAR-120574 partly downregulates the expressionof E2F2 and thus slows the proliferation of tumor cells ThusPPAR-120574may be a new therapeutic target for NPC

Abbreviations

PPAR-120574 Peroxisome proliferator-activated receptor-120574E2F2 E2F transcription factor 2NPC Nasopharyngeal carcinomaNPG NasopharyngitisRog Rosiglitazone

8 PPAR Research

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE1(a)

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE2(b)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE1

minus

minus

minus

minus

(c)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE2

minus minus

minus minus

(d)Figure 4 E2F2 and PPAR-120574 expression in CNE1 and CNE2 nasopharyngeal carcinoma cells a13er treatment with PPAR-120574 ligand (a b) Westernblotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cells after treatment with dose-dependentPPAR-120574 agonist Rog (c d) Western blotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cellsafter treatment with PPAR-120574 agonist (rosiglitazone Rog) and PPAR-120574 antagonist (GW9662)

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

None of the authors has conflicts of interest with thissubmission

Acknowledgments

This paper was supported by grants from the First AffiliatedHospital of Shihezi University School of Medicine (noZZZC201828A)

References

[1] M Altun A Fandi O Dupuis E Cvitkovic Z Krajina and FEschwege ldquoUndifferentiated nasopharyngeal cancer (UCNT)current diagnostic and therapeutic aspectsrdquo International Jour-nal of Radiation Oncology ∙ Biology ∙ Physics vol 32 no 3 pp859ndash877 1995

[2] M L K Chua J T S Wee E P Hui and A T C ChanldquoNasopharyngeal carcinomardquoeLancet vol 387 no 10022 pp1012ndash1024 2016

[3] D K Dimova and N J Dyson ldquoThe E2F transcriptional net-work old acquaintances with new facesrdquo Oncogene vol 24 no17 pp 2810ndash2826 2005

[4] J DeGregori and D G Johnson ldquoDistinct and overlappingroles for E2F family members in transcription proliferation andapoptosisrdquo Current Molecular Medicine vol 6 no 7 pp 739ndash748 2006

[5] Q Dong P Meng T Wang et al ldquoMicroRNA let-7a inhibitsproliferation of human prostate cancer cells in vitro and in vivoby targeting E2F2 and CCND2rdquo PLoS ONE vol 5 no 4 ArticleID e10147 2010

[6] S Hayami M Yoshimatsu A Veerakumarasivam et al ldquoOver-expression of the JmjC histone demethylase KDM5B in humancarcinogenesis involvement in the proliferation of cancer cellsthrough the E2FRB pathwayrdquoMolecular Cancer vol 9 no 1 p59 2010

[7] Y Gao D Han L Sun et al ldquoPPAR120572 regulates the proliferationof human glioma cells through miR-214 and E2F2rdquo BioMedResearch International vol 2018 Article ID 3842753 10 pages2018

[8] H-X Liu Y Fang YHu F J Gonzalez J Fang andY-J YWanldquoPPAR120573 regulates liver regeneration bymodulating Akt and E2fsignalingrdquo PLoS ONE vol 8 no 6 Article ID e65644 2013

[9] Y Komatsu I Ito M Wayama et al ldquoPPAR120574 ligands suppressthe feedback loop between E2F2 and cyclin-E1rdquo Biochemicaland Biophysical Research Communications vol 370 no 1 pp145ndash148 2008

[10] Y Zhang O S Dallner T Nakadai et al ldquoA noncanonicalPPAR120574RXR120572-binding sequence regulates leptin expression inresponse to changes in adipose tissue massrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 115 no 26 pp E6039ndashE6047 2018

PPAR Research 9

[11] D Aleman-Gonzalez-Duhart F Tamay-Cach S Alvarez-Almazan and J E Mendieta-Wejebe ldquoCurrent advances in thebiochemical and physiological aspects of the treatment of type 2diabetes mellitus with thiazolidinedionesrdquo PPAR Research vol2016 Article ID 7614270 10 pages 2016

[12] S H Kim H S Park M J Hong H J Hur D Y Kwon andMS Kim ldquoCaffeic acid phenethyl ester improves metabolic syn-drome by activating ppar-gamma and inducing adipose tissueremodeling in diet-induced obese micerdquoMolecular Nutrition ampFood Research vol 62 Article ID e1700701 2018

[13] M Kukida M Mogi H Kan-no et al ldquoAT2 receptor stimula-tion inhibits phosphate-induced vascular calcificationrdquo KidneyInternational vol 95 no 1 pp 138ndash148 2019

[14] L Michalik B Desvergne and W Wahli ldquoPeroxisome-prolif-erator-activated receptors and cancers complex storiesrdquoNatureReviews Cancer vol 4 no 1 pp 61ndash70 2004

[15] M Senba X Zhong M Senba and H Itakura ldquoEBV andnasopharyngeal carcinomardquo e Lancet vol 343 no 8905 p1104 1994

[16] Y Li J Huang D Yang et al ldquoExpression patterns of E2Ftranscription factors and their potential prognostic roles inbreast cancerrdquoOncology Letters vol 15 pp 9216ndash9230 2018

[17] M N Gnanapragasam and J J Bieker ldquoOrchestration of lateevents in erythropoiesis by KLF1EKLFrdquo Current Opinion inHematology vol 24 no 3 pp 183ndash190 2017

[18] C Sun Q Zhou W Hu et al ldquoTranscriptional E2F1258 aspotential targets and transcriptional E2F367 as new biomark-ers for the prognosis of human lung carcinomardquo AGING vol10 no 5 pp 973ndash987 2018

[19] L Chen X Huang and X Chen ldquoMiR-365 suppresses cholan-giocarcinoma cell proliferation and induces apoptosis by target-ing E2F2rdquoOncology Research Featuring Preclinical and ClinicalCancer erapeutics vol 26 no 9 pp 1375ndash1382 2018

[20] A Feliciano Y Garcia-Mayea L Jubierre et al ldquomiR-99areveals two novel oncogenic proteins E2F2 and EMR2 andrepresses stemness in lung cancerrdquo Cell Death amp Disease vol8 no 10 p e3141 2017

[21] T Tao Q Shen J Luo Y Xu and W Liang ldquoMicroRNA-125a regulates cell proliferation via directly targeting E2F2 inosteosarcomardquoCellular Physiology andBiochemistry vol 43 no2 pp 768ndash774 2017

[22] H K Kim J-Y Hwang J K Hyun et al ldquoExpression ofa peroxisome proliferator-activated receptor 1205741 splice variantthat was identified in human lung cancers suppresses celldeath induced by cisplatin and oxidative stressrdquo Clinical CancerResearch vol 13 no 9 pp 2577ndash2583 2007

[23] M Monami I Dicembrini and E Mannucci ldquoThiazolidine-diones and cancer results of a meta-analysis of randomizedclinical trialsrdquo Acta Diabetologica vol 51 no 1 pp 91ndash101 2014

[24] S Chang and H Hu ldquoAssociation of thiazolidinediones withgastric cancer in type 2 diabetes mellitus a population-basedcasendashcontrol studyrdquo BMC Cancer vol 13 no 1 article no 4202013

[25] M T Bazelier F de Vries P Vestergaard H Leufkens and MDe Bruin ldquoUse of thiazolidinediones and risk of bladder cancerdisease or drugsrdquo Current Drug Safety vol 8 no 5 pp 364ndash370 2013

[26] VVellaM LNicolosi S GiulianoM BellomoA Belfiore andR Malaguarnera ldquoPPAR-120574 agonists as antineoplastic agents incancers with dysregulated IGF axisrdquo Frontiers in Endocrinologyvol 8 article no 31 2017

[27] V Costa D Foti F Paonessa et al ldquoThe insulin receptorA new anticancer target for peroxisome proliferator-activatedreceptor-120574 (PPAR120574) and thiazolidinedione- PPAR120574 agonistsrdquoEndocrine-Related Cancer vol 15 no 1 pp 325ndash335 2008

[28] S Wakino U Kintscher S Kim F Yin W A Hsueh and RE Law ldquoPeroxisome proliferator-activated receptor 120574 ligandsinhibit retinoblastoma phosphorylation andG1 997888rarr S transitionin vascular smooth muscle cellsrdquo e Journal of BiologicalChemistry vol 275 no 29 pp 22435ndash22441 2000

[29] S Altiok M Xu and B M Spiegelman ldquoPPAR120574 induces cellcycle withdrawal Inhibition of E2fDP DNA-binding activityvia down-regulation of PP2ArdquoGenesampDevelopment vol 11 no15 pp 1987ndash1998 1997

[30] L Wu C Timmers B Malti et al ldquoThe E2F1-3 transcriptionfactors are essential for cellular proliferationrdquo Nature vol 414no 6862 pp 457ndash462 2001

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

PPAR Research 3

apoptotic cells relative to the total number of cells Theresults were confirmed in at least three independent experi-ments

28 Statistical Analyses All statistical analyses were per-formed using SPSS (version 170 IBM Corp USA) The chi-square test was used to compare the levels of expression ofE2F2 and PPAR-120574 in nonkeratinizing NPC and NPG samplesand the respective correlations with the clinicopathologicalfeatures of NPC patients The unpaired Studentrsquos t-test wasused to compare E2F2 and PPAR-120574 expression betweengroups of CNE1 and CNE2 cells For all analyses a P valueof lt005 was considered to indicate a statistically significantdifference

3 Results

31 Expression of E2F2 and PPAR-120574 in Undifferentiated NPCandNPGTissues andCorrelations with the ClinicopathologicalFeatures of NPC Patients IHC was used to evaluate theexpression of E2F2 and PPAR-120574 proteins in 52 nonkeratiniz-ing NPC and 34 NPG tissue samples Strong E2F2 expres-sion (IODArea ge02) was observed in 6346 (3352) ofnonkeratinizing NPC tissues compared with 1765 (634) ofNPG tissues (P lt001) Strong PPAR-120574 expression (IODAreage02) was also observed in 2885 (1552) of nonkeratiniz-ing NPC tissues compared with 7647 (2634) of NPGtissues (P lt001) (Table 1) Both differences were signifi-cant

We further analyzed the associations of E2F2 andPPAR-120574 expression with the clinical parameters in patientswith nonkeratinizing NPC Although no correlations wereobserved with sex age or T (tumor) classification weobserved a positive correlation between E2F2 and nonkera-tinizing NPC staging namely the N (lymph node metastasis)(N0ndashN1 2240(5500) vs N2ndashN3 1112(9167) P lt005)andM (distant metastasis) classifications (M0 2352(5476)vs M1 1010(100) P lt005) PPAR-120574 expression was onlyfound to correlate positively with the M classification (M01542 (3571)vsM1 010(0) Plt005)These results stronglyindicate that E2F2 contributes to the proliferation andmetas-tasis of nonkeratinizing NPC and is associated with thedisease stage

32 Immunohistochemical Staining of E2F2 and PPAR-120574 inNonkeratinizing NPC and NPG Tissues IHC was used toevaluate the expression and localization of E2F2 and PPAR-120574in tissue samples Notably we observed strong E2F2 expres-sion in nonkeratinizing NPC tissues (Figure 1(a)) comparedwith NPG tissues (Figure 1(c)) Although E2F2 localized inboth the nuclei and cytoplasm of the former tissues it wasmost strongly expressed in the nuclei We further observeda trend toward decreased PPAR-120574 expression in nonker-atinizing NPC tissues (Figure 1(b)) compared with NPGtissues (Figure 1(d)) A semiquantitative evaluation revealedsignificantly stronger E2F2 expression in nonkeratinizingNPC tissues compared toNPG tissues (Figure 1(e)) (Plt001)as well as a significant difference in PPAR-120574 expressionbetween these tissue types (Figure 1(f)) (P lt001)

33 Expression of E2F2 and PPAR-120574 in Nonkeratinizing NPCand NPG Tissues The mRNA expression of E2F2 in NPC issignificantly higher than NPG while the mRNA expressionof PPAR-120574 in NPC is lower than NPG (Figure 2(a)) West-ern blotting revealed stronger E2F2 expression in nonker-atinizing NPC tissues and weaker PPAR-120574 expression innonkeratinizing NPC tissues when compared with NPGtissues (Figure 2(b)) consistent with the IHC findings Aquantitative analysis of the E2F2 and PPAR-120574 protein expres-sion levels revealed significant differences between the tissuetypes (P lt001 and lt005 respectively) (Figures 2(c) and2(d))

34 PPAR-120574 Ligand Inhibits the Proliferation of CNE1 andCNE2 Cell Lines To analyze the functional role of PPAR-120574 in NPC cell lines we performed CCK-8 assays to detectthe proliferation of NPC cell lines The results showed thatPPAR-120574 ligand Rog inhibited the proliferation of both celllines in a dose-dependent manner (Figures 3(c) and 2(d))To determine if proliferation was blocked we performedapoptosis analysis by flow cytometry The results showed thatthe number of apoptotic cells did not increase after treatmentwith Rog (Figures 3(a) and 3(b))

35 Expression of E2F2 and PPAR-120574 in CNE1 and CNE2Cell Lines a13er Treatment with a PPAR-120574 Ligand Finallywe incubated cell lines with a PPAR-120574 ligand to analyze thefunctional role of PPAR-120574 in NPC cell lines The expressionof E2F2 decreased while PPAR-120574 increased after treatmentwith Rog and the effect was dose-dependent (Figures 4(a)and 4(b)) We further verified the protein expression levelsusing western blotting and found that after treatment withthe PPAR-120574 agonist Rog the expression of E2F2 decreasedwhereas that of PPAR-120574 increased To confirm the inhibitoryeffect of PPAR-120574 on E2F2 we also treated cells with thePPAR-120574 antagonist GW9662 and observed an increase inthe expression of E2F2 (Figures 4(c) and 4(d)) These resultssuggest that PPAR-120574 contributes to the inhibition of NPC cellproliferation and metastasis

4 Discussion

NPC is a polygenic hereditary malignant tumor that mostcommonly arises from the lateral wall of the nasophar-ynx particularly the fossa of Rosenmuller and around theEustachian cushion (and occult locations) As the primarytumor location is the base of the skull the local invasionof NPC may have serious consequences and most patientspresent with lymph nodes or distant tissue metastasis atthe time of diagnosis [15] Currently the proliferation andmetastasis of tumor tissues are closely related to the differen-tiation and proliferation rates of cells understanding that thefactors that regulate the cell cycle help us to understand themechanisms that cause cell proliferation and differentiationas well as look for factors related to treatment and progno-sis

Previously we identified that E2F2 could induce thetranscription of target genes and drive cell cycle progressionand proliferation [16] Cell cycle deregulation is a common

4 PPAR Research

Table1Ex

pressio

nsof

E2F2

andPP

AR-120574in

clinicalsamples

ofun

differentiatedNPC

andNPG

andassociations

with

clinicop

atho

logicalcharacteristicso

fund

ifferentia

tedNPC

samples

Parameter

NE2

F2a

NPP

AR-120574b

Lowexpressio

nHighexpressio

nPvaluec

Lowexpressio

nHighexpressio

nPvaluec

Histologictype

lt001

lt001

NPC

5219

(3654)

33(6346

)52

37(7115)

15(2885)

NPG

3428

(8235)

6(176

5)34

8(2353)

26(7647)

Gender

0868

0377

Male

3914

(3590)

25(6410)

3929

(7436)

10(2564

)Female

135(3846

)8(615

4)13

8(615

4)5(3846

)Age

(y)

0253

01

lt50

2210

(4545)

12(5455)

2213

(590

9)9(4091)

≧50

309(3000

)21

(7000

)30

24(8000

)6(2000

)Tclassifi

catio

n0457

033

I-II

3514

(4000

)21

(6000

)35

23(6571)

12(3429)

III-IV

175(294

1)12

(7059)

1714

(8235)

3(176

5Nclassifi

catio

n0037

0071

N0-N1

4018

(4500

)22

(5500

)40

26(6500

)14

(3500

)N2-N3

121(833)

11(916

7)12

11(916

7)1(833)

Mclassifi

catio

n000

4004

6M0

4219

(4524)

23(5476)

4227

(6429)

15(3571)

M1

100(0)

10(100

)10

10(100

)0(0)

a E2F2lowexpressio

nIO

DA

realt

002highexpressio

nIO

DA

rea⩾

002

b PPA

R-120574lowexpressio

nIO

DA

realt

002highexpressio

nIO

DA

rea⩾

002

c Chi-squ

aretest

PPAR Research 5

50 Ｇ

E2F2

NPC

(a)

(a)

50 Ｇ

PPAR-

NPC

(b)

(b)E2F2

NPG

(c)

50 Ｇ

(c)

50 Ｇ

PPAR-

NPG

(d)

(d)

000

001

002

003

004

005

E2F2

(IOD

Are

a)

NPC NPG

lowast

(e)

000

002

004

006

008

PPA

R-

(IOD

Are

a)

NPC NPG

lowastlowast

(f)

Figure 1 E2F2 and PPAR-120574 protein expression in nonkeratinizing nasopharyngeal carcinoma (NPC) and nasopharyngitis (NPG) tissuesImmunohistochemistrywas performed to detect E2F2 and PPAR-120574 protein expression in nonkeratinizing NPC tissues (a b) andNPG tissues(c d) Scale bar 50120583m(e f)The immunohistochemistry datawere analyzed semiquantitatively to determine the E2F2 andPPAR-120574expressionlevels in nonkeratinizing NPC and NPG tissues Data are shown as means plusmn standard deviations lowastlowastP lt001

feature of human cancer The function of proproliferationwhich lies in the ability enables cell cycle entry into the Sphase via E2F2 [17] and strong E2F2 expression was detectedin various types of tumors [18ndash21] There is no report aboutE2F2 in NPC In this study we use IHC method to analysis52 nonkeratinizing NPC tissues and 34 NPG tissues revealedstronger E2F2 expression in the former which was associatedwith the clinical tumor stage Specifically we observed sig-nificantly higher levels of E2F2 expression in N2ndashN3 and M1

stage nonkeratinizing NPC tissues This finding suggests anassociation of E2F2 expressionwith the degree ofmalignancywhich was confirmed using western blotting and RT-PCRThis finding indicates that E2F2 is a meaningful marker ofthe degree of tumor malignancy Furthermore we found thatE2F2 is localizedmainly in the nuclei of nonkeratinizingNPCtissues suggesting that this transcription factor translocatesto the nucleus to promote DNA synthesis and subsequent cellproliferation and division This important role of E2F2 in cell

6 PPAR Research

00

05

10

15

20

Relat

ive m

RNA

leve

l (E

2F2)

NPC NPG0

2

4

6

Relat

ive m

RNA

leve

l(P

PAR-

)

NPGNPC

lowastlowastlowastlowast

(a)

NPG

E2F2

PPAR-

GAPDH

NPC(b)

NPC

E2F2

GA

PDH

NPG06

08

10

12lowastlowast

(c)

PPA

R-

GA

PDH

NPC NPG06

07

08

09

10 lowast

(d)

Figure 2 E2F2 and PPAR-120574 protein expression in nonkeratinizing nasopharyngeal carcinoma (NPC) and nasopharyngitis (NPG) tissue lysates(a b) RT-PCR and Western blotting were performed to detect the expression of E2F2 PPAR-120574 and GAPDH in lysates of nonkeratinizingNPC and NPG tissues (c d) Quantitative analyses of the E2F2 and PPAR-120574 expression levels in nonkeratinizing NPC and NPG tissues Dataare expressed as means plusmn standard deviations lowastP lt005 lowastlowastP lt001

proliferation suggests that treatment with an E2F2 inhibitormay inhibit proliferation

PPAR-120574 a member of the ligand-activated superfamilyof nuclear transcription factors is an important regulator ofinflammation glucosemetabolism and cell proliferation [22]and was previously shown to inhibit tumor cell proliferation[23ndash25] In this study we used IHC western blotting and RT-PCR to demonstrate reduced PPAR-120574 expression in nonker-atinizing NPC tissues compared to NPG tissues especiallyin cases of the former that involved distant metastasis Itsuggests that expression of PPAR-120574 related to NPC andrelated to the malignancy degree of tumor The antitumoraffections of PPAR-120574 were thought to be associated with

inhibition of angiogenesis stemness process and cell cyclearrest [26 27] In our study we observed a decrease inE2F2 expression and reductions in the proliferation anddifferentiation rates in NPC cells treated with the PPAR-120574agonist Rog and it was dose-dependent Notably this effectwas offset by incubation with the PPAR-120574 agonist GW9662Supporting our result Komatsu et al reported that thedownregulation of E2F2 led to PPAR-120574 agonist-mediated cellcycle withdrawal leading to a cell cycle blockade at the G1Stransition Numerous studies had demonstrated that ligandactivation of PPAR-120574 activation inhibits the phosphorylationof Rb protein which has been reported to lead to abrogationof E2F2 [28] or directly decrease the DNA-binding activity

PPAR Research 7

CNE1

Com

p-PE

-A

Comp-FITC-A

Rog(0) Rog(5M) Rog(10M) Rog(20M)

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

(a)

CNE2

Com

p-PE

-A

Comp-FITC-A

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

Rog(0) Rog(5M) Rog(10M) Rog(20M)

(b)

24 48 72 96

Rog5ctlRog10ctlRog20ctl

Rela

tive p

rolif

erat

ion

(Rog

ctl)

CNE1

00

02

04

06

08

10

12

lowastlowast

lowastlowastlowastlowast

lowast

(c)

24 48 72 96

Rela

tive p

rolif

erat

ion

(Rog

ctl)

CNE2

Rog5ctlRog10ctlRog20ctl

00

02

04

06

08

10

12

lowastlowast

lowastlowastlowastlowastlowast

lowast

(d)

Figure 3 PPAR-120574 ligand inhibits the proliferation of CNE1 and CNE2 cell lines (a b) rh Annexin V-FITC assay was performed to detect thepercentage of apoptotic cells relative to the total number of cells in CNE1 and CNE2 cell lines after treatment with dose-dependent Rog (cd) Analysis of a CCK-8 proliferation assay of CNE1 and CNE2 cell lines

of E2FDP transcription factors [29] Wu et al found thatcombined inactivation of E2F2 is sufficient to block cellularproliferation completely [30]

Potentially PPAR-120574 partly downregulates the expressionof E2F2 and thus slows the proliferation of tumor cells ThusPPAR-120574may be a new therapeutic target for NPC

Abbreviations

PPAR-120574 Peroxisome proliferator-activated receptor-120574E2F2 E2F transcription factor 2NPC Nasopharyngeal carcinomaNPG NasopharyngitisRog Rosiglitazone

8 PPAR Research

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE1(a)

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE2(b)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE1

minus

minus

minus

minus

(c)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE2

minus minus

minus minus

(d)Figure 4 E2F2 and PPAR-120574 expression in CNE1 and CNE2 nasopharyngeal carcinoma cells a13er treatment with PPAR-120574 ligand (a b) Westernblotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cells after treatment with dose-dependentPPAR-120574 agonist Rog (c d) Western blotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cellsafter treatment with PPAR-120574 agonist (rosiglitazone Rog) and PPAR-120574 antagonist (GW9662)

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

None of the authors has conflicts of interest with thissubmission

Acknowledgments

This paper was supported by grants from the First AffiliatedHospital of Shihezi University School of Medicine (noZZZC201828A)

References

[1] M Altun A Fandi O Dupuis E Cvitkovic Z Krajina and FEschwege ldquoUndifferentiated nasopharyngeal cancer (UCNT)current diagnostic and therapeutic aspectsrdquo International Jour-nal of Radiation Oncology ∙ Biology ∙ Physics vol 32 no 3 pp859ndash877 1995

[2] M L K Chua J T S Wee E P Hui and A T C ChanldquoNasopharyngeal carcinomardquoeLancet vol 387 no 10022 pp1012ndash1024 2016

[3] D K Dimova and N J Dyson ldquoThe E2F transcriptional net-work old acquaintances with new facesrdquo Oncogene vol 24 no17 pp 2810ndash2826 2005

[4] J DeGregori and D G Johnson ldquoDistinct and overlappingroles for E2F family members in transcription proliferation andapoptosisrdquo Current Molecular Medicine vol 6 no 7 pp 739ndash748 2006

[5] Q Dong P Meng T Wang et al ldquoMicroRNA let-7a inhibitsproliferation of human prostate cancer cells in vitro and in vivoby targeting E2F2 and CCND2rdquo PLoS ONE vol 5 no 4 ArticleID e10147 2010

[6] S Hayami M Yoshimatsu A Veerakumarasivam et al ldquoOver-expression of the JmjC histone demethylase KDM5B in humancarcinogenesis involvement in the proliferation of cancer cellsthrough the E2FRB pathwayrdquoMolecular Cancer vol 9 no 1 p59 2010

[7] Y Gao D Han L Sun et al ldquoPPAR120572 regulates the proliferationof human glioma cells through miR-214 and E2F2rdquo BioMedResearch International vol 2018 Article ID 3842753 10 pages2018

[8] H-X Liu Y Fang YHu F J Gonzalez J Fang andY-J YWanldquoPPAR120573 regulates liver regeneration bymodulating Akt and E2fsignalingrdquo PLoS ONE vol 8 no 6 Article ID e65644 2013

[9] Y Komatsu I Ito M Wayama et al ldquoPPAR120574 ligands suppressthe feedback loop between E2F2 and cyclin-E1rdquo Biochemicaland Biophysical Research Communications vol 370 no 1 pp145ndash148 2008

[10] Y Zhang O S Dallner T Nakadai et al ldquoA noncanonicalPPAR120574RXR120572-binding sequence regulates leptin expression inresponse to changes in adipose tissue massrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 115 no 26 pp E6039ndashE6047 2018

PPAR Research 9

[11] D Aleman-Gonzalez-Duhart F Tamay-Cach S Alvarez-Almazan and J E Mendieta-Wejebe ldquoCurrent advances in thebiochemical and physiological aspects of the treatment of type 2diabetes mellitus with thiazolidinedionesrdquo PPAR Research vol2016 Article ID 7614270 10 pages 2016

[12] S H Kim H S Park M J Hong H J Hur D Y Kwon andMS Kim ldquoCaffeic acid phenethyl ester improves metabolic syn-drome by activating ppar-gamma and inducing adipose tissueremodeling in diet-induced obese micerdquoMolecular Nutrition ampFood Research vol 62 Article ID e1700701 2018

[13] M Kukida M Mogi H Kan-no et al ldquoAT2 receptor stimula-tion inhibits phosphate-induced vascular calcificationrdquo KidneyInternational vol 95 no 1 pp 138ndash148 2019

[14] L Michalik B Desvergne and W Wahli ldquoPeroxisome-prolif-erator-activated receptors and cancers complex storiesrdquoNatureReviews Cancer vol 4 no 1 pp 61ndash70 2004

[15] M Senba X Zhong M Senba and H Itakura ldquoEBV andnasopharyngeal carcinomardquo e Lancet vol 343 no 8905 p1104 1994

[16] Y Li J Huang D Yang et al ldquoExpression patterns of E2Ftranscription factors and their potential prognostic roles inbreast cancerrdquoOncology Letters vol 15 pp 9216ndash9230 2018

[17] M N Gnanapragasam and J J Bieker ldquoOrchestration of lateevents in erythropoiesis by KLF1EKLFrdquo Current Opinion inHematology vol 24 no 3 pp 183ndash190 2017

[18] C Sun Q Zhou W Hu et al ldquoTranscriptional E2F1258 aspotential targets and transcriptional E2F367 as new biomark-ers for the prognosis of human lung carcinomardquo AGING vol10 no 5 pp 973ndash987 2018

[19] L Chen X Huang and X Chen ldquoMiR-365 suppresses cholan-giocarcinoma cell proliferation and induces apoptosis by target-ing E2F2rdquoOncology Research Featuring Preclinical and ClinicalCancer erapeutics vol 26 no 9 pp 1375ndash1382 2018

[20] A Feliciano Y Garcia-Mayea L Jubierre et al ldquomiR-99areveals two novel oncogenic proteins E2F2 and EMR2 andrepresses stemness in lung cancerrdquo Cell Death amp Disease vol8 no 10 p e3141 2017

[21] T Tao Q Shen J Luo Y Xu and W Liang ldquoMicroRNA-125a regulates cell proliferation via directly targeting E2F2 inosteosarcomardquoCellular Physiology andBiochemistry vol 43 no2 pp 768ndash774 2017

[22] H K Kim J-Y Hwang J K Hyun et al ldquoExpression ofa peroxisome proliferator-activated receptor 1205741 splice variantthat was identified in human lung cancers suppresses celldeath induced by cisplatin and oxidative stressrdquo Clinical CancerResearch vol 13 no 9 pp 2577ndash2583 2007

[23] M Monami I Dicembrini and E Mannucci ldquoThiazolidine-diones and cancer results of a meta-analysis of randomizedclinical trialsrdquo Acta Diabetologica vol 51 no 1 pp 91ndash101 2014

[24] S Chang and H Hu ldquoAssociation of thiazolidinediones withgastric cancer in type 2 diabetes mellitus a population-basedcasendashcontrol studyrdquo BMC Cancer vol 13 no 1 article no 4202013

[25] M T Bazelier F de Vries P Vestergaard H Leufkens and MDe Bruin ldquoUse of thiazolidinediones and risk of bladder cancerdisease or drugsrdquo Current Drug Safety vol 8 no 5 pp 364ndash370 2013

[26] VVellaM LNicolosi S GiulianoM BellomoA Belfiore andR Malaguarnera ldquoPPAR-120574 agonists as antineoplastic agents incancers with dysregulated IGF axisrdquo Frontiers in Endocrinologyvol 8 article no 31 2017

[27] V Costa D Foti F Paonessa et al ldquoThe insulin receptorA new anticancer target for peroxisome proliferator-activatedreceptor-120574 (PPAR120574) and thiazolidinedione- PPAR120574 agonistsrdquoEndocrine-Related Cancer vol 15 no 1 pp 325ndash335 2008

[28] S Wakino U Kintscher S Kim F Yin W A Hsueh and RE Law ldquoPeroxisome proliferator-activated receptor 120574 ligandsinhibit retinoblastoma phosphorylation andG1 997888rarr S transitionin vascular smooth muscle cellsrdquo e Journal of BiologicalChemistry vol 275 no 29 pp 22435ndash22441 2000

[29] S Altiok M Xu and B M Spiegelman ldquoPPAR120574 induces cellcycle withdrawal Inhibition of E2fDP DNA-binding activityvia down-regulation of PP2ArdquoGenesampDevelopment vol 11 no15 pp 1987ndash1998 1997

[30] L Wu C Timmers B Malti et al ldquoThe E2F1-3 transcriptionfactors are essential for cellular proliferationrdquo Nature vol 414no 6862 pp 457ndash462 2001

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

4 PPAR Research

Table1Ex

pressio

nsof

E2F2

andPP

AR-120574in

clinicalsamples

ofun

differentiatedNPC

andNPG

andassociations

with

clinicop

atho

logicalcharacteristicso

fund

ifferentia

tedNPC

samples

Parameter

NE2

F2a

NPP

AR-120574b

Lowexpressio

nHighexpressio

nPvaluec

Lowexpressio

nHighexpressio

nPvaluec

Histologictype

lt001

lt001

NPC

5219

(3654)

33(6346

)52

37(7115)

15(2885)

NPG

3428

(8235)

6(176

5)34

8(2353)

26(7647)

Gender

0868

0377

Male

3914

(3590)

25(6410)

3929

(7436)

10(2564

)Female

135(3846

)8(615

4)13

8(615

4)5(3846

)Age

(y)

0253

01

lt50

2210

(4545)

12(5455)

2213

(590

9)9(4091)

≧50

309(3000

)21

(7000

)30

24(8000

)6(2000

)Tclassifi

catio

n0457

033

I-II

3514

(4000

)21

(6000

)35

23(6571)

12(3429)

III-IV

175(294

1)12

(7059)

1714

(8235)

3(176

5Nclassifi

catio

n0037

0071

N0-N1

4018

(4500

)22

(5500

)40

26(6500

)14

(3500

)N2-N3

121(833)

11(916

7)12

11(916

7)1(833)

Mclassifi

catio

n000

4004

6M0

4219

(4524)

23(5476)

4227

(6429)

15(3571)

M1

100(0)

10(100

)10

10(100

)0(0)

a E2F2lowexpressio

nIO

DA

realt

002highexpressio

nIO

DA

rea⩾

002

b PPA

R-120574lowexpressio

nIO

DA

realt

002highexpressio

nIO

DA

rea⩾

002

c Chi-squ

aretest

PPAR Research 5

50 Ｇ

E2F2

NPC

(a)

(a)

50 Ｇ

PPAR-

NPC

(b)

(b)E2F2

NPG

(c)

50 Ｇ

(c)

50 Ｇ

PPAR-

NPG

(d)

(d)

000

001

002

003

004

005

E2F2

(IOD

Are

a)

NPC NPG

lowast

(e)

000

002

004

006

008

PPA

R-

(IOD

Are

a)

NPC NPG

lowastlowast

(f)

Figure 1 E2F2 and PPAR-120574 protein expression in nonkeratinizing nasopharyngeal carcinoma (NPC) and nasopharyngitis (NPG) tissuesImmunohistochemistrywas performed to detect E2F2 and PPAR-120574 protein expression in nonkeratinizing NPC tissues (a b) andNPG tissues(c d) Scale bar 50120583m(e f)The immunohistochemistry datawere analyzed semiquantitatively to determine the E2F2 andPPAR-120574expressionlevels in nonkeratinizing NPC and NPG tissues Data are shown as means plusmn standard deviations lowastlowastP lt001

feature of human cancer The function of proproliferationwhich lies in the ability enables cell cycle entry into the Sphase via E2F2 [17] and strong E2F2 expression was detectedin various types of tumors [18ndash21] There is no report aboutE2F2 in NPC In this study we use IHC method to analysis52 nonkeratinizing NPC tissues and 34 NPG tissues revealedstronger E2F2 expression in the former which was associatedwith the clinical tumor stage Specifically we observed sig-nificantly higher levels of E2F2 expression in N2ndashN3 and M1

stage nonkeratinizing NPC tissues This finding suggests anassociation of E2F2 expressionwith the degree ofmalignancywhich was confirmed using western blotting and RT-PCRThis finding indicates that E2F2 is a meaningful marker ofthe degree of tumor malignancy Furthermore we found thatE2F2 is localizedmainly in the nuclei of nonkeratinizingNPCtissues suggesting that this transcription factor translocatesto the nucleus to promote DNA synthesis and subsequent cellproliferation and division This important role of E2F2 in cell

6 PPAR Research

00

05

10

15

20

Relat

ive m

RNA

leve

l (E

2F2)

NPC NPG0

2

4

6

Relat

ive m

RNA

leve

l(P

PAR-

)

NPGNPC

lowastlowastlowastlowast

(a)

NPG

E2F2

PPAR-

GAPDH

NPC(b)

NPC

E2F2

GA

PDH

NPG06

08

10

12lowastlowast

(c)

PPA

R-

GA

PDH

NPC NPG06

07

08

09

10 lowast

(d)

Figure 2 E2F2 and PPAR-120574 protein expression in nonkeratinizing nasopharyngeal carcinoma (NPC) and nasopharyngitis (NPG) tissue lysates(a b) RT-PCR and Western blotting were performed to detect the expression of E2F2 PPAR-120574 and GAPDH in lysates of nonkeratinizingNPC and NPG tissues (c d) Quantitative analyses of the E2F2 and PPAR-120574 expression levels in nonkeratinizing NPC and NPG tissues Dataare expressed as means plusmn standard deviations lowastP lt005 lowastlowastP lt001

proliferation suggests that treatment with an E2F2 inhibitormay inhibit proliferation

PPAR-120574 a member of the ligand-activated superfamilyof nuclear transcription factors is an important regulator ofinflammation glucosemetabolism and cell proliferation [22]and was previously shown to inhibit tumor cell proliferation[23ndash25] In this study we used IHC western blotting and RT-PCR to demonstrate reduced PPAR-120574 expression in nonker-atinizing NPC tissues compared to NPG tissues especiallyin cases of the former that involved distant metastasis Itsuggests that expression of PPAR-120574 related to NPC andrelated to the malignancy degree of tumor The antitumoraffections of PPAR-120574 were thought to be associated with

inhibition of angiogenesis stemness process and cell cyclearrest [26 27] In our study we observed a decrease inE2F2 expression and reductions in the proliferation anddifferentiation rates in NPC cells treated with the PPAR-120574agonist Rog and it was dose-dependent Notably this effectwas offset by incubation with the PPAR-120574 agonist GW9662Supporting our result Komatsu et al reported that thedownregulation of E2F2 led to PPAR-120574 agonist-mediated cellcycle withdrawal leading to a cell cycle blockade at the G1Stransition Numerous studies had demonstrated that ligandactivation of PPAR-120574 activation inhibits the phosphorylationof Rb protein which has been reported to lead to abrogationof E2F2 [28] or directly decrease the DNA-binding activity

PPAR Research 7

CNE1

Com

p-PE

-A

Comp-FITC-A

Rog(0) Rog(5M) Rog(10M) Rog(20M)

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

(a)

CNE2

Com

p-PE

-A

Comp-FITC-A

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

Rog(0) Rog(5M) Rog(10M) Rog(20M)

(b)

24 48 72 96

Rog5ctlRog10ctlRog20ctl

Rela

tive p

rolif

erat

ion

(Rog

ctl)

CNE1

00

02

04

06

08

10

12

lowastlowast

lowastlowastlowastlowast

lowast

(c)

24 48 72 96

Rela

tive p

rolif

erat

ion

(Rog

ctl)

CNE2

Rog5ctlRog10ctlRog20ctl

00

02

04

06

08

10

12

lowastlowast

lowastlowastlowastlowastlowast

lowast

(d)

Figure 3 PPAR-120574 ligand inhibits the proliferation of CNE1 and CNE2 cell lines (a b) rh Annexin V-FITC assay was performed to detect thepercentage of apoptotic cells relative to the total number of cells in CNE1 and CNE2 cell lines after treatment with dose-dependent Rog (cd) Analysis of a CCK-8 proliferation assay of CNE1 and CNE2 cell lines

of E2FDP transcription factors [29] Wu et al found thatcombined inactivation of E2F2 is sufficient to block cellularproliferation completely [30]

Potentially PPAR-120574 partly downregulates the expressionof E2F2 and thus slows the proliferation of tumor cells ThusPPAR-120574may be a new therapeutic target for NPC

Abbreviations

PPAR-120574 Peroxisome proliferator-activated receptor-120574E2F2 E2F transcription factor 2NPC Nasopharyngeal carcinomaNPG NasopharyngitisRog Rosiglitazone

8 PPAR Research

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE1(a)

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE2(b)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE1

minus

minus

minus

minus

(c)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE2

minus minus

minus minus

(d)Figure 4 E2F2 and PPAR-120574 expression in CNE1 and CNE2 nasopharyngeal carcinoma cells a13er treatment with PPAR-120574 ligand (a b) Westernblotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cells after treatment with dose-dependentPPAR-120574 agonist Rog (c d) Western blotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cellsafter treatment with PPAR-120574 agonist (rosiglitazone Rog) and PPAR-120574 antagonist (GW9662)

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

None of the authors has conflicts of interest with thissubmission

Acknowledgments

This paper was supported by grants from the First AffiliatedHospital of Shihezi University School of Medicine (noZZZC201828A)

References

[1] M Altun A Fandi O Dupuis E Cvitkovic Z Krajina and FEschwege ldquoUndifferentiated nasopharyngeal cancer (UCNT)current diagnostic and therapeutic aspectsrdquo International Jour-nal of Radiation Oncology ∙ Biology ∙ Physics vol 32 no 3 pp859ndash877 1995

[2] M L K Chua J T S Wee E P Hui and A T C ChanldquoNasopharyngeal carcinomardquoeLancet vol 387 no 10022 pp1012ndash1024 2016

[3] D K Dimova and N J Dyson ldquoThe E2F transcriptional net-work old acquaintances with new facesrdquo Oncogene vol 24 no17 pp 2810ndash2826 2005

[4] J DeGregori and D G Johnson ldquoDistinct and overlappingroles for E2F family members in transcription proliferation andapoptosisrdquo Current Molecular Medicine vol 6 no 7 pp 739ndash748 2006

[5] Q Dong P Meng T Wang et al ldquoMicroRNA let-7a inhibitsproliferation of human prostate cancer cells in vitro and in vivoby targeting E2F2 and CCND2rdquo PLoS ONE vol 5 no 4 ArticleID e10147 2010

[6] S Hayami M Yoshimatsu A Veerakumarasivam et al ldquoOver-expression of the JmjC histone demethylase KDM5B in humancarcinogenesis involvement in the proliferation of cancer cellsthrough the E2FRB pathwayrdquoMolecular Cancer vol 9 no 1 p59 2010

[7] Y Gao D Han L Sun et al ldquoPPAR120572 regulates the proliferationof human glioma cells through miR-214 and E2F2rdquo BioMedResearch International vol 2018 Article ID 3842753 10 pages2018

[8] H-X Liu Y Fang YHu F J Gonzalez J Fang andY-J YWanldquoPPAR120573 regulates liver regeneration bymodulating Akt and E2fsignalingrdquo PLoS ONE vol 8 no 6 Article ID e65644 2013

[9] Y Komatsu I Ito M Wayama et al ldquoPPAR120574 ligands suppressthe feedback loop between E2F2 and cyclin-E1rdquo Biochemicaland Biophysical Research Communications vol 370 no 1 pp145ndash148 2008

[10] Y Zhang O S Dallner T Nakadai et al ldquoA noncanonicalPPAR120574RXR120572-binding sequence regulates leptin expression inresponse to changes in adipose tissue massrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 115 no 26 pp E6039ndashE6047 2018

PPAR Research 9

[11] D Aleman-Gonzalez-Duhart F Tamay-Cach S Alvarez-Almazan and J E Mendieta-Wejebe ldquoCurrent advances in thebiochemical and physiological aspects of the treatment of type 2diabetes mellitus with thiazolidinedionesrdquo PPAR Research vol2016 Article ID 7614270 10 pages 2016

[12] S H Kim H S Park M J Hong H J Hur D Y Kwon andMS Kim ldquoCaffeic acid phenethyl ester improves metabolic syn-drome by activating ppar-gamma and inducing adipose tissueremodeling in diet-induced obese micerdquoMolecular Nutrition ampFood Research vol 62 Article ID e1700701 2018

[13] M Kukida M Mogi H Kan-no et al ldquoAT2 receptor stimula-tion inhibits phosphate-induced vascular calcificationrdquo KidneyInternational vol 95 no 1 pp 138ndash148 2019

[14] L Michalik B Desvergne and W Wahli ldquoPeroxisome-prolif-erator-activated receptors and cancers complex storiesrdquoNatureReviews Cancer vol 4 no 1 pp 61ndash70 2004

[15] M Senba X Zhong M Senba and H Itakura ldquoEBV andnasopharyngeal carcinomardquo e Lancet vol 343 no 8905 p1104 1994

[16] Y Li J Huang D Yang et al ldquoExpression patterns of E2Ftranscription factors and their potential prognostic roles inbreast cancerrdquoOncology Letters vol 15 pp 9216ndash9230 2018

[17] M N Gnanapragasam and J J Bieker ldquoOrchestration of lateevents in erythropoiesis by KLF1EKLFrdquo Current Opinion inHematology vol 24 no 3 pp 183ndash190 2017

[18] C Sun Q Zhou W Hu et al ldquoTranscriptional E2F1258 aspotential targets and transcriptional E2F367 as new biomark-ers for the prognosis of human lung carcinomardquo AGING vol10 no 5 pp 973ndash987 2018

[19] L Chen X Huang and X Chen ldquoMiR-365 suppresses cholan-giocarcinoma cell proliferation and induces apoptosis by target-ing E2F2rdquoOncology Research Featuring Preclinical and ClinicalCancer erapeutics vol 26 no 9 pp 1375ndash1382 2018

[20] A Feliciano Y Garcia-Mayea L Jubierre et al ldquomiR-99areveals two novel oncogenic proteins E2F2 and EMR2 andrepresses stemness in lung cancerrdquo Cell Death amp Disease vol8 no 10 p e3141 2017

[21] T Tao Q Shen J Luo Y Xu and W Liang ldquoMicroRNA-125a regulates cell proliferation via directly targeting E2F2 inosteosarcomardquoCellular Physiology andBiochemistry vol 43 no2 pp 768ndash774 2017

[22] H K Kim J-Y Hwang J K Hyun et al ldquoExpression ofa peroxisome proliferator-activated receptor 1205741 splice variantthat was identified in human lung cancers suppresses celldeath induced by cisplatin and oxidative stressrdquo Clinical CancerResearch vol 13 no 9 pp 2577ndash2583 2007

[23] M Monami I Dicembrini and E Mannucci ldquoThiazolidine-diones and cancer results of a meta-analysis of randomizedclinical trialsrdquo Acta Diabetologica vol 51 no 1 pp 91ndash101 2014

[24] S Chang and H Hu ldquoAssociation of thiazolidinediones withgastric cancer in type 2 diabetes mellitus a population-basedcasendashcontrol studyrdquo BMC Cancer vol 13 no 1 article no 4202013

[25] M T Bazelier F de Vries P Vestergaard H Leufkens and MDe Bruin ldquoUse of thiazolidinediones and risk of bladder cancerdisease or drugsrdquo Current Drug Safety vol 8 no 5 pp 364ndash370 2013

[26] VVellaM LNicolosi S GiulianoM BellomoA Belfiore andR Malaguarnera ldquoPPAR-120574 agonists as antineoplastic agents incancers with dysregulated IGF axisrdquo Frontiers in Endocrinologyvol 8 article no 31 2017

[27] V Costa D Foti F Paonessa et al ldquoThe insulin receptorA new anticancer target for peroxisome proliferator-activatedreceptor-120574 (PPAR120574) and thiazolidinedione- PPAR120574 agonistsrdquoEndocrine-Related Cancer vol 15 no 1 pp 325ndash335 2008

[28] S Wakino U Kintscher S Kim F Yin W A Hsueh and RE Law ldquoPeroxisome proliferator-activated receptor 120574 ligandsinhibit retinoblastoma phosphorylation andG1 997888rarr S transitionin vascular smooth muscle cellsrdquo e Journal of BiologicalChemistry vol 275 no 29 pp 22435ndash22441 2000

[29] S Altiok M Xu and B M Spiegelman ldquoPPAR120574 induces cellcycle withdrawal Inhibition of E2fDP DNA-binding activityvia down-regulation of PP2ArdquoGenesampDevelopment vol 11 no15 pp 1987ndash1998 1997

[30] L Wu C Timmers B Malti et al ldquoThe E2F1-3 transcriptionfactors are essential for cellular proliferationrdquo Nature vol 414no 6862 pp 457ndash462 2001

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

PPAR Research 5

50 Ｇ

E2F2

NPC

(a)

(a)

50 Ｇ

PPAR-

NPC

(b)

(b)E2F2

NPG

(c)

50 Ｇ

(c)

50 Ｇ

PPAR-

NPG

(d)

(d)

000

001

002

003

004

005

E2F2

(IOD

Are

a)

NPC NPG

lowast

(e)

000

002

004

006

008

PPA

R-

(IOD

Are

a)

NPC NPG

lowastlowast

(f)

Figure 1 E2F2 and PPAR-120574 protein expression in nonkeratinizing nasopharyngeal carcinoma (NPC) and nasopharyngitis (NPG) tissuesImmunohistochemistrywas performed to detect E2F2 and PPAR-120574 protein expression in nonkeratinizing NPC tissues (a b) andNPG tissues(c d) Scale bar 50120583m(e f)The immunohistochemistry datawere analyzed semiquantitatively to determine the E2F2 andPPAR-120574expressionlevels in nonkeratinizing NPC and NPG tissues Data are shown as means plusmn standard deviations lowastlowastP lt001

feature of human cancer The function of proproliferationwhich lies in the ability enables cell cycle entry into the Sphase via E2F2 [17] and strong E2F2 expression was detectedin various types of tumors [18ndash21] There is no report aboutE2F2 in NPC In this study we use IHC method to analysis52 nonkeratinizing NPC tissues and 34 NPG tissues revealedstronger E2F2 expression in the former which was associatedwith the clinical tumor stage Specifically we observed sig-nificantly higher levels of E2F2 expression in N2ndashN3 and M1

stage nonkeratinizing NPC tissues This finding suggests anassociation of E2F2 expressionwith the degree ofmalignancywhich was confirmed using western blotting and RT-PCRThis finding indicates that E2F2 is a meaningful marker ofthe degree of tumor malignancy Furthermore we found thatE2F2 is localizedmainly in the nuclei of nonkeratinizingNPCtissues suggesting that this transcription factor translocatesto the nucleus to promote DNA synthesis and subsequent cellproliferation and division This important role of E2F2 in cell

6 PPAR Research

00

05

10

15

20

Relat

ive m

RNA

leve

l (E

2F2)

NPC NPG0

2

4

6

Relat

ive m

RNA

leve

l(P

PAR-

)

NPGNPC

lowastlowastlowastlowast

(a)

NPG

E2F2

PPAR-

GAPDH

NPC(b)

NPC

E2F2

GA

PDH

NPG06

08

10

12lowastlowast

(c)

PPA

R-

GA

PDH

NPC NPG06

07

08

09

10 lowast

(d)

Figure 2 E2F2 and PPAR-120574 protein expression in nonkeratinizing nasopharyngeal carcinoma (NPC) and nasopharyngitis (NPG) tissue lysates(a b) RT-PCR and Western blotting were performed to detect the expression of E2F2 PPAR-120574 and GAPDH in lysates of nonkeratinizingNPC and NPG tissues (c d) Quantitative analyses of the E2F2 and PPAR-120574 expression levels in nonkeratinizing NPC and NPG tissues Dataare expressed as means plusmn standard deviations lowastP lt005 lowastlowastP lt001

proliferation suggests that treatment with an E2F2 inhibitormay inhibit proliferation

PPAR-120574 a member of the ligand-activated superfamilyof nuclear transcription factors is an important regulator ofinflammation glucosemetabolism and cell proliferation [22]and was previously shown to inhibit tumor cell proliferation[23ndash25] In this study we used IHC western blotting and RT-PCR to demonstrate reduced PPAR-120574 expression in nonker-atinizing NPC tissues compared to NPG tissues especiallyin cases of the former that involved distant metastasis Itsuggests that expression of PPAR-120574 related to NPC andrelated to the malignancy degree of tumor The antitumoraffections of PPAR-120574 were thought to be associated with

inhibition of angiogenesis stemness process and cell cyclearrest [26 27] In our study we observed a decrease inE2F2 expression and reductions in the proliferation anddifferentiation rates in NPC cells treated with the PPAR-120574agonist Rog and it was dose-dependent Notably this effectwas offset by incubation with the PPAR-120574 agonist GW9662Supporting our result Komatsu et al reported that thedownregulation of E2F2 led to PPAR-120574 agonist-mediated cellcycle withdrawal leading to a cell cycle blockade at the G1Stransition Numerous studies had demonstrated that ligandactivation of PPAR-120574 activation inhibits the phosphorylationof Rb protein which has been reported to lead to abrogationof E2F2 [28] or directly decrease the DNA-binding activity

PPAR Research 7

CNE1

Com

p-PE

-A

Comp-FITC-A

Rog(0) Rog(5M) Rog(10M) Rog(20M)

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

(a)

CNE2

Com

p-PE

-A

Comp-FITC-A

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

Rog(0) Rog(5M) Rog(10M) Rog(20M)

(b)

24 48 72 96

Rog5ctlRog10ctlRog20ctl

Rela

tive p

rolif

erat

ion

(Rog

ctl)

CNE1

00

02

04

06

08

10

12

lowastlowast

lowastlowastlowastlowast

lowast

(c)

24 48 72 96

Rela

tive p

rolif

erat

ion

(Rog

ctl)

CNE2

Rog5ctlRog10ctlRog20ctl

00

02

04

06

08

10

12

lowastlowast

lowastlowastlowastlowastlowast

lowast

(d)

Figure 3 PPAR-120574 ligand inhibits the proliferation of CNE1 and CNE2 cell lines (a b) rh Annexin V-FITC assay was performed to detect thepercentage of apoptotic cells relative to the total number of cells in CNE1 and CNE2 cell lines after treatment with dose-dependent Rog (cd) Analysis of a CCK-8 proliferation assay of CNE1 and CNE2 cell lines

of E2FDP transcription factors [29] Wu et al found thatcombined inactivation of E2F2 is sufficient to block cellularproliferation completely [30]

Potentially PPAR-120574 partly downregulates the expressionof E2F2 and thus slows the proliferation of tumor cells ThusPPAR-120574may be a new therapeutic target for NPC

Abbreviations

PPAR-120574 Peroxisome proliferator-activated receptor-120574E2F2 E2F transcription factor 2NPC Nasopharyngeal carcinomaNPG NasopharyngitisRog Rosiglitazone

8 PPAR Research

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE1(a)

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE2(b)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE1

minus

minus

minus

minus

(c)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE2

minus minus

minus minus

(d)Figure 4 E2F2 and PPAR-120574 expression in CNE1 and CNE2 nasopharyngeal carcinoma cells a13er treatment with PPAR-120574 ligand (a b) Westernblotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cells after treatment with dose-dependentPPAR-120574 agonist Rog (c d) Western blotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cellsafter treatment with PPAR-120574 agonist (rosiglitazone Rog) and PPAR-120574 antagonist (GW9662)

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

None of the authors has conflicts of interest with thissubmission

Acknowledgments

This paper was supported by grants from the First AffiliatedHospital of Shihezi University School of Medicine (noZZZC201828A)

References

[1] M Altun A Fandi O Dupuis E Cvitkovic Z Krajina and FEschwege ldquoUndifferentiated nasopharyngeal cancer (UCNT)current diagnostic and therapeutic aspectsrdquo International Jour-nal of Radiation Oncology ∙ Biology ∙ Physics vol 32 no 3 pp859ndash877 1995

[2] M L K Chua J T S Wee E P Hui and A T C ChanldquoNasopharyngeal carcinomardquoeLancet vol 387 no 10022 pp1012ndash1024 2016

[3] D K Dimova and N J Dyson ldquoThe E2F transcriptional net-work old acquaintances with new facesrdquo Oncogene vol 24 no17 pp 2810ndash2826 2005

[4] J DeGregori and D G Johnson ldquoDistinct and overlappingroles for E2F family members in transcription proliferation andapoptosisrdquo Current Molecular Medicine vol 6 no 7 pp 739ndash748 2006

[5] Q Dong P Meng T Wang et al ldquoMicroRNA let-7a inhibitsproliferation of human prostate cancer cells in vitro and in vivoby targeting E2F2 and CCND2rdquo PLoS ONE vol 5 no 4 ArticleID e10147 2010

[6] S Hayami M Yoshimatsu A Veerakumarasivam et al ldquoOver-expression of the JmjC histone demethylase KDM5B in humancarcinogenesis involvement in the proliferation of cancer cellsthrough the E2FRB pathwayrdquoMolecular Cancer vol 9 no 1 p59 2010

[7] Y Gao D Han L Sun et al ldquoPPAR120572 regulates the proliferationof human glioma cells through miR-214 and E2F2rdquo BioMedResearch International vol 2018 Article ID 3842753 10 pages2018

[8] H-X Liu Y Fang YHu F J Gonzalez J Fang andY-J YWanldquoPPAR120573 regulates liver regeneration bymodulating Akt and E2fsignalingrdquo PLoS ONE vol 8 no 6 Article ID e65644 2013

[9] Y Komatsu I Ito M Wayama et al ldquoPPAR120574 ligands suppressthe feedback loop between E2F2 and cyclin-E1rdquo Biochemicaland Biophysical Research Communications vol 370 no 1 pp145ndash148 2008

[10] Y Zhang O S Dallner T Nakadai et al ldquoA noncanonicalPPAR120574RXR120572-binding sequence regulates leptin expression inresponse to changes in adipose tissue massrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 115 no 26 pp E6039ndashE6047 2018

PPAR Research 9

[11] D Aleman-Gonzalez-Duhart F Tamay-Cach S Alvarez-Almazan and J E Mendieta-Wejebe ldquoCurrent advances in thebiochemical and physiological aspects of the treatment of type 2diabetes mellitus with thiazolidinedionesrdquo PPAR Research vol2016 Article ID 7614270 10 pages 2016

[12] S H Kim H S Park M J Hong H J Hur D Y Kwon andMS Kim ldquoCaffeic acid phenethyl ester improves metabolic syn-drome by activating ppar-gamma and inducing adipose tissueremodeling in diet-induced obese micerdquoMolecular Nutrition ampFood Research vol 62 Article ID e1700701 2018

[13] M Kukida M Mogi H Kan-no et al ldquoAT2 receptor stimula-tion inhibits phosphate-induced vascular calcificationrdquo KidneyInternational vol 95 no 1 pp 138ndash148 2019

[14] L Michalik B Desvergne and W Wahli ldquoPeroxisome-prolif-erator-activated receptors and cancers complex storiesrdquoNatureReviews Cancer vol 4 no 1 pp 61ndash70 2004

[15] M Senba X Zhong M Senba and H Itakura ldquoEBV andnasopharyngeal carcinomardquo e Lancet vol 343 no 8905 p1104 1994

[16] Y Li J Huang D Yang et al ldquoExpression patterns of E2Ftranscription factors and their potential prognostic roles inbreast cancerrdquoOncology Letters vol 15 pp 9216ndash9230 2018

[17] M N Gnanapragasam and J J Bieker ldquoOrchestration of lateevents in erythropoiesis by KLF1EKLFrdquo Current Opinion inHematology vol 24 no 3 pp 183ndash190 2017

[18] C Sun Q Zhou W Hu et al ldquoTranscriptional E2F1258 aspotential targets and transcriptional E2F367 as new biomark-ers for the prognosis of human lung carcinomardquo AGING vol10 no 5 pp 973ndash987 2018

[19] L Chen X Huang and X Chen ldquoMiR-365 suppresses cholan-giocarcinoma cell proliferation and induces apoptosis by target-ing E2F2rdquoOncology Research Featuring Preclinical and ClinicalCancer erapeutics vol 26 no 9 pp 1375ndash1382 2018

[20] A Feliciano Y Garcia-Mayea L Jubierre et al ldquomiR-99areveals two novel oncogenic proteins E2F2 and EMR2 andrepresses stemness in lung cancerrdquo Cell Death amp Disease vol8 no 10 p e3141 2017

[21] T Tao Q Shen J Luo Y Xu and W Liang ldquoMicroRNA-125a regulates cell proliferation via directly targeting E2F2 inosteosarcomardquoCellular Physiology andBiochemistry vol 43 no2 pp 768ndash774 2017

[22] H K Kim J-Y Hwang J K Hyun et al ldquoExpression ofa peroxisome proliferator-activated receptor 1205741 splice variantthat was identified in human lung cancers suppresses celldeath induced by cisplatin and oxidative stressrdquo Clinical CancerResearch vol 13 no 9 pp 2577ndash2583 2007

[23] M Monami I Dicembrini and E Mannucci ldquoThiazolidine-diones and cancer results of a meta-analysis of randomizedclinical trialsrdquo Acta Diabetologica vol 51 no 1 pp 91ndash101 2014

[24] S Chang and H Hu ldquoAssociation of thiazolidinediones withgastric cancer in type 2 diabetes mellitus a population-basedcasendashcontrol studyrdquo BMC Cancer vol 13 no 1 article no 4202013

[25] M T Bazelier F de Vries P Vestergaard H Leufkens and MDe Bruin ldquoUse of thiazolidinediones and risk of bladder cancerdisease or drugsrdquo Current Drug Safety vol 8 no 5 pp 364ndash370 2013

[26] VVellaM LNicolosi S GiulianoM BellomoA Belfiore andR Malaguarnera ldquoPPAR-120574 agonists as antineoplastic agents incancers with dysregulated IGF axisrdquo Frontiers in Endocrinologyvol 8 article no 31 2017

[27] V Costa D Foti F Paonessa et al ldquoThe insulin receptorA new anticancer target for peroxisome proliferator-activatedreceptor-120574 (PPAR120574) and thiazolidinedione- PPAR120574 agonistsrdquoEndocrine-Related Cancer vol 15 no 1 pp 325ndash335 2008

[28] S Wakino U Kintscher S Kim F Yin W A Hsueh and RE Law ldquoPeroxisome proliferator-activated receptor 120574 ligandsinhibit retinoblastoma phosphorylation andG1 997888rarr S transitionin vascular smooth muscle cellsrdquo e Journal of BiologicalChemistry vol 275 no 29 pp 22435ndash22441 2000

[29] S Altiok M Xu and B M Spiegelman ldquoPPAR120574 induces cellcycle withdrawal Inhibition of E2fDP DNA-binding activityvia down-regulation of PP2ArdquoGenesampDevelopment vol 11 no15 pp 1987ndash1998 1997

[30] L Wu C Timmers B Malti et al ldquoThe E2F1-3 transcriptionfactors are essential for cellular proliferationrdquo Nature vol 414no 6862 pp 457ndash462 2001

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

6 PPAR Research

00

05

10

15

20

Relat

ive m

RNA

leve

l (E

2F2)

NPC NPG0

2

4

6

Relat

ive m

RNA

leve

l(P

PAR-

)

NPGNPC

lowastlowastlowastlowast

(a)

NPG

E2F2

PPAR-

GAPDH

NPC(b)

NPC

E2F2

GA

PDH

NPG06

08

10

12lowastlowast

(c)

PPA

R-

GA

PDH

NPC NPG06

07

08

09

10 lowast

(d)

Figure 2 E2F2 and PPAR-120574 protein expression in nonkeratinizing nasopharyngeal carcinoma (NPC) and nasopharyngitis (NPG) tissue lysates(a b) RT-PCR and Western blotting were performed to detect the expression of E2F2 PPAR-120574 and GAPDH in lysates of nonkeratinizingNPC and NPG tissues (c d) Quantitative analyses of the E2F2 and PPAR-120574 expression levels in nonkeratinizing NPC and NPG tissues Dataare expressed as means plusmn standard deviations lowastP lt005 lowastlowastP lt001

proliferation suggests that treatment with an E2F2 inhibitormay inhibit proliferation

PPAR-120574 a member of the ligand-activated superfamilyof nuclear transcription factors is an important regulator ofinflammation glucosemetabolism and cell proliferation [22]and was previously shown to inhibit tumor cell proliferation[23ndash25] In this study we used IHC western blotting and RT-PCR to demonstrate reduced PPAR-120574 expression in nonker-atinizing NPC tissues compared to NPG tissues especiallyin cases of the former that involved distant metastasis Itsuggests that expression of PPAR-120574 related to NPC andrelated to the malignancy degree of tumor The antitumoraffections of PPAR-120574 were thought to be associated with

inhibition of angiogenesis stemness process and cell cyclearrest [26 27] In our study we observed a decrease inE2F2 expression and reductions in the proliferation anddifferentiation rates in NPC cells treated with the PPAR-120574agonist Rog and it was dose-dependent Notably this effectwas offset by incubation with the PPAR-120574 agonist GW9662Supporting our result Komatsu et al reported that thedownregulation of E2F2 led to PPAR-120574 agonist-mediated cellcycle withdrawal leading to a cell cycle blockade at the G1Stransition Numerous studies had demonstrated that ligandactivation of PPAR-120574 activation inhibits the phosphorylationof Rb protein which has been reported to lead to abrogationof E2F2 [28] or directly decrease the DNA-binding activity

PPAR Research 7

CNE1

Com

p-PE

-A

Comp-FITC-A

Rog(0) Rog(5M) Rog(10M) Rog(20M)

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

(a)

CNE2

Com

p-PE

-A

Comp-FITC-A

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

Rog(0) Rog(5M) Rog(10M) Rog(20M)

(b)

24 48 72 96

Rog5ctlRog10ctlRog20ctl

Rela

tive p

rolif

erat

ion

(Rog

ctl)

CNE1

00

02

04

06

08

10

12

lowastlowast

lowastlowastlowastlowast

lowast

(c)

24 48 72 96

Rela

tive p

rolif

erat

ion

(Rog

ctl)

CNE2

Rog5ctlRog10ctlRog20ctl

00

02

04

06

08

10

12

lowastlowast

lowastlowastlowastlowastlowast

lowast

(d)

Figure 3 PPAR-120574 ligand inhibits the proliferation of CNE1 and CNE2 cell lines (a b) rh Annexin V-FITC assay was performed to detect thepercentage of apoptotic cells relative to the total number of cells in CNE1 and CNE2 cell lines after treatment with dose-dependent Rog (cd) Analysis of a CCK-8 proliferation assay of CNE1 and CNE2 cell lines

of E2FDP transcription factors [29] Wu et al found thatcombined inactivation of E2F2 is sufficient to block cellularproliferation completely [30]

Potentially PPAR-120574 partly downregulates the expressionof E2F2 and thus slows the proliferation of tumor cells ThusPPAR-120574may be a new therapeutic target for NPC

Abbreviations

PPAR-120574 Peroxisome proliferator-activated receptor-120574E2F2 E2F transcription factor 2NPC Nasopharyngeal carcinomaNPG NasopharyngitisRog Rosiglitazone

8 PPAR Research

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE1(a)

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE2(b)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE1

minus

minus

minus

minus

(c)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE2

minus minus

minus minus

(d)Figure 4 E2F2 and PPAR-120574 expression in CNE1 and CNE2 nasopharyngeal carcinoma cells a13er treatment with PPAR-120574 ligand (a b) Westernblotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cells after treatment with dose-dependentPPAR-120574 agonist Rog (c d) Western blotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cellsafter treatment with PPAR-120574 agonist (rosiglitazone Rog) and PPAR-120574 antagonist (GW9662)

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

None of the authors has conflicts of interest with thissubmission

Acknowledgments

This paper was supported by grants from the First AffiliatedHospital of Shihezi University School of Medicine (noZZZC201828A)

References

[1] M Altun A Fandi O Dupuis E Cvitkovic Z Krajina and FEschwege ldquoUndifferentiated nasopharyngeal cancer (UCNT)current diagnostic and therapeutic aspectsrdquo International Jour-nal of Radiation Oncology ∙ Biology ∙ Physics vol 32 no 3 pp859ndash877 1995

[2] M L K Chua J T S Wee E P Hui and A T C ChanldquoNasopharyngeal carcinomardquoeLancet vol 387 no 10022 pp1012ndash1024 2016

[3] D K Dimova and N J Dyson ldquoThe E2F transcriptional net-work old acquaintances with new facesrdquo Oncogene vol 24 no17 pp 2810ndash2826 2005

[4] J DeGregori and D G Johnson ldquoDistinct and overlappingroles for E2F family members in transcription proliferation andapoptosisrdquo Current Molecular Medicine vol 6 no 7 pp 739ndash748 2006

[5] Q Dong P Meng T Wang et al ldquoMicroRNA let-7a inhibitsproliferation of human prostate cancer cells in vitro and in vivoby targeting E2F2 and CCND2rdquo PLoS ONE vol 5 no 4 ArticleID e10147 2010

[6] S Hayami M Yoshimatsu A Veerakumarasivam et al ldquoOver-expression of the JmjC histone demethylase KDM5B in humancarcinogenesis involvement in the proliferation of cancer cellsthrough the E2FRB pathwayrdquoMolecular Cancer vol 9 no 1 p59 2010

[7] Y Gao D Han L Sun et al ldquoPPAR120572 regulates the proliferationof human glioma cells through miR-214 and E2F2rdquo BioMedResearch International vol 2018 Article ID 3842753 10 pages2018

[8] H-X Liu Y Fang YHu F J Gonzalez J Fang andY-J YWanldquoPPAR120573 regulates liver regeneration bymodulating Akt and E2fsignalingrdquo PLoS ONE vol 8 no 6 Article ID e65644 2013

[9] Y Komatsu I Ito M Wayama et al ldquoPPAR120574 ligands suppressthe feedback loop between E2F2 and cyclin-E1rdquo Biochemicaland Biophysical Research Communications vol 370 no 1 pp145ndash148 2008

[10] Y Zhang O S Dallner T Nakadai et al ldquoA noncanonicalPPAR120574RXR120572-binding sequence regulates leptin expression inresponse to changes in adipose tissue massrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 115 no 26 pp E6039ndashE6047 2018

PPAR Research 9

[11] D Aleman-Gonzalez-Duhart F Tamay-Cach S Alvarez-Almazan and J E Mendieta-Wejebe ldquoCurrent advances in thebiochemical and physiological aspects of the treatment of type 2diabetes mellitus with thiazolidinedionesrdquo PPAR Research vol2016 Article ID 7614270 10 pages 2016

[12] S H Kim H S Park M J Hong H J Hur D Y Kwon andMS Kim ldquoCaffeic acid phenethyl ester improves metabolic syn-drome by activating ppar-gamma and inducing adipose tissueremodeling in diet-induced obese micerdquoMolecular Nutrition ampFood Research vol 62 Article ID e1700701 2018

[13] M Kukida M Mogi H Kan-no et al ldquoAT2 receptor stimula-tion inhibits phosphate-induced vascular calcificationrdquo KidneyInternational vol 95 no 1 pp 138ndash148 2019

[14] L Michalik B Desvergne and W Wahli ldquoPeroxisome-prolif-erator-activated receptors and cancers complex storiesrdquoNatureReviews Cancer vol 4 no 1 pp 61ndash70 2004

[15] M Senba X Zhong M Senba and H Itakura ldquoEBV andnasopharyngeal carcinomardquo e Lancet vol 343 no 8905 p1104 1994

[16] Y Li J Huang D Yang et al ldquoExpression patterns of E2Ftranscription factors and their potential prognostic roles inbreast cancerrdquoOncology Letters vol 15 pp 9216ndash9230 2018

[17] M N Gnanapragasam and J J Bieker ldquoOrchestration of lateevents in erythropoiesis by KLF1EKLFrdquo Current Opinion inHematology vol 24 no 3 pp 183ndash190 2017

[18] C Sun Q Zhou W Hu et al ldquoTranscriptional E2F1258 aspotential targets and transcriptional E2F367 as new biomark-ers for the prognosis of human lung carcinomardquo AGING vol10 no 5 pp 973ndash987 2018

[19] L Chen X Huang and X Chen ldquoMiR-365 suppresses cholan-giocarcinoma cell proliferation and induces apoptosis by target-ing E2F2rdquoOncology Research Featuring Preclinical and ClinicalCancer erapeutics vol 26 no 9 pp 1375ndash1382 2018

[20] A Feliciano Y Garcia-Mayea L Jubierre et al ldquomiR-99areveals two novel oncogenic proteins E2F2 and EMR2 andrepresses stemness in lung cancerrdquo Cell Death amp Disease vol8 no 10 p e3141 2017

[21] T Tao Q Shen J Luo Y Xu and W Liang ldquoMicroRNA-125a regulates cell proliferation via directly targeting E2F2 inosteosarcomardquoCellular Physiology andBiochemistry vol 43 no2 pp 768ndash774 2017

[22] H K Kim J-Y Hwang J K Hyun et al ldquoExpression ofa peroxisome proliferator-activated receptor 1205741 splice variantthat was identified in human lung cancers suppresses celldeath induced by cisplatin and oxidative stressrdquo Clinical CancerResearch vol 13 no 9 pp 2577ndash2583 2007

[23] M Monami I Dicembrini and E Mannucci ldquoThiazolidine-diones and cancer results of a meta-analysis of randomizedclinical trialsrdquo Acta Diabetologica vol 51 no 1 pp 91ndash101 2014

[24] S Chang and H Hu ldquoAssociation of thiazolidinediones withgastric cancer in type 2 diabetes mellitus a population-basedcasendashcontrol studyrdquo BMC Cancer vol 13 no 1 article no 4202013

[25] M T Bazelier F de Vries P Vestergaard H Leufkens and MDe Bruin ldquoUse of thiazolidinediones and risk of bladder cancerdisease or drugsrdquo Current Drug Safety vol 8 no 5 pp 364ndash370 2013

[26] VVellaM LNicolosi S GiulianoM BellomoA Belfiore andR Malaguarnera ldquoPPAR-120574 agonists as antineoplastic agents incancers with dysregulated IGF axisrdquo Frontiers in Endocrinologyvol 8 article no 31 2017

[27] V Costa D Foti F Paonessa et al ldquoThe insulin receptorA new anticancer target for peroxisome proliferator-activatedreceptor-120574 (PPAR120574) and thiazolidinedione- PPAR120574 agonistsrdquoEndocrine-Related Cancer vol 15 no 1 pp 325ndash335 2008

[28] S Wakino U Kintscher S Kim F Yin W A Hsueh and RE Law ldquoPeroxisome proliferator-activated receptor 120574 ligandsinhibit retinoblastoma phosphorylation andG1 997888rarr S transitionin vascular smooth muscle cellsrdquo e Journal of BiologicalChemistry vol 275 no 29 pp 22435ndash22441 2000

[29] S Altiok M Xu and B M Spiegelman ldquoPPAR120574 induces cellcycle withdrawal Inhibition of E2fDP DNA-binding activityvia down-regulation of PP2ArdquoGenesampDevelopment vol 11 no15 pp 1987ndash1998 1997

[30] L Wu C Timmers B Malti et al ldquoThe E2F1-3 transcriptionfactors are essential for cellular proliferationrdquo Nature vol 414no 6862 pp 457ndash462 2001

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

PPAR Research 7

CNE1

Com

p-PE

-A

Comp-FITC-A

Rog(0) Rog(5M) Rog(10M) Rog(20M)

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

(a)

CNE2

Com

p-PE

-A

Comp-FITC-A

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

105

105

104

104

103

103

102

102

0

0

Rog(0) Rog(5M) Rog(10M) Rog(20M)

(b)

24 48 72 96

Rog5ctlRog10ctlRog20ctl

Rela

tive p

rolif

erat

ion

(Rog

ctl)

CNE1

00

02

04

06

08

10

12

lowastlowast

lowastlowastlowastlowast

lowast

(c)

24 48 72 96

Rela

tive p

rolif

erat

ion

(Rog

ctl)

CNE2

Rog5ctlRog10ctlRog20ctl

00

02

04

06

08

10

12

lowastlowast

lowastlowastlowastlowastlowast

lowast

(d)

Figure 3 PPAR-120574 ligand inhibits the proliferation of CNE1 and CNE2 cell lines (a b) rh Annexin V-FITC assay was performed to detect thepercentage of apoptotic cells relative to the total number of cells in CNE1 and CNE2 cell lines after treatment with dose-dependent Rog (cd) Analysis of a CCK-8 proliferation assay of CNE1 and CNE2 cell lines

of E2FDP transcription factors [29] Wu et al found thatcombined inactivation of E2F2 is sufficient to block cellularproliferation completely [30]

Potentially PPAR-120574 partly downregulates the expressionof E2F2 and thus slows the proliferation of tumor cells ThusPPAR-120574may be a new therapeutic target for NPC

Abbreviations

PPAR-120574 Peroxisome proliferator-activated receptor-120574E2F2 E2F transcription factor 2NPC Nasopharyngeal carcinomaNPG NasopharyngitisRog Rosiglitazone

8 PPAR Research

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE1(a)

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE2(b)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE1

minus

minus

minus

minus

(c)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE2

minus minus

minus minus

(d)Figure 4 E2F2 and PPAR-120574 expression in CNE1 and CNE2 nasopharyngeal carcinoma cells a13er treatment with PPAR-120574 ligand (a b) Westernblotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cells after treatment with dose-dependentPPAR-120574 agonist Rog (c d) Western blotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cellsafter treatment with PPAR-120574 agonist (rosiglitazone Rog) and PPAR-120574 antagonist (GW9662)

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

None of the authors has conflicts of interest with thissubmission

Acknowledgments

This paper was supported by grants from the First AffiliatedHospital of Shihezi University School of Medicine (noZZZC201828A)

References

[1] M Altun A Fandi O Dupuis E Cvitkovic Z Krajina and FEschwege ldquoUndifferentiated nasopharyngeal cancer (UCNT)current diagnostic and therapeutic aspectsrdquo International Jour-nal of Radiation Oncology ∙ Biology ∙ Physics vol 32 no 3 pp859ndash877 1995

[2] M L K Chua J T S Wee E P Hui and A T C ChanldquoNasopharyngeal carcinomardquoeLancet vol 387 no 10022 pp1012ndash1024 2016

[3] D K Dimova and N J Dyson ldquoThe E2F transcriptional net-work old acquaintances with new facesrdquo Oncogene vol 24 no17 pp 2810ndash2826 2005

[4] J DeGregori and D G Johnson ldquoDistinct and overlappingroles for E2F family members in transcription proliferation andapoptosisrdquo Current Molecular Medicine vol 6 no 7 pp 739ndash748 2006

[5] Q Dong P Meng T Wang et al ldquoMicroRNA let-7a inhibitsproliferation of human prostate cancer cells in vitro and in vivoby targeting E2F2 and CCND2rdquo PLoS ONE vol 5 no 4 ArticleID e10147 2010

[6] S Hayami M Yoshimatsu A Veerakumarasivam et al ldquoOver-expression of the JmjC histone demethylase KDM5B in humancarcinogenesis involvement in the proliferation of cancer cellsthrough the E2FRB pathwayrdquoMolecular Cancer vol 9 no 1 p59 2010

[7] Y Gao D Han L Sun et al ldquoPPAR120572 regulates the proliferationof human glioma cells through miR-214 and E2F2rdquo BioMedResearch International vol 2018 Article ID 3842753 10 pages2018

[8] H-X Liu Y Fang YHu F J Gonzalez J Fang andY-J YWanldquoPPAR120573 regulates liver regeneration bymodulating Akt and E2fsignalingrdquo PLoS ONE vol 8 no 6 Article ID e65644 2013

[9] Y Komatsu I Ito M Wayama et al ldquoPPAR120574 ligands suppressthe feedback loop between E2F2 and cyclin-E1rdquo Biochemicaland Biophysical Research Communications vol 370 no 1 pp145ndash148 2008

[10] Y Zhang O S Dallner T Nakadai et al ldquoA noncanonicalPPAR120574RXR120572-binding sequence regulates leptin expression inresponse to changes in adipose tissue massrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 115 no 26 pp E6039ndashE6047 2018

PPAR Research 9

[11] D Aleman-Gonzalez-Duhart F Tamay-Cach S Alvarez-Almazan and J E Mendieta-Wejebe ldquoCurrent advances in thebiochemical and physiological aspects of the treatment of type 2diabetes mellitus with thiazolidinedionesrdquo PPAR Research vol2016 Article ID 7614270 10 pages 2016

[12] S H Kim H S Park M J Hong H J Hur D Y Kwon andMS Kim ldquoCaffeic acid phenethyl ester improves metabolic syn-drome by activating ppar-gamma and inducing adipose tissueremodeling in diet-induced obese micerdquoMolecular Nutrition ampFood Research vol 62 Article ID e1700701 2018

[13] M Kukida M Mogi H Kan-no et al ldquoAT2 receptor stimula-tion inhibits phosphate-induced vascular calcificationrdquo KidneyInternational vol 95 no 1 pp 138ndash148 2019

[14] L Michalik B Desvergne and W Wahli ldquoPeroxisome-prolif-erator-activated receptors and cancers complex storiesrdquoNatureReviews Cancer vol 4 no 1 pp 61ndash70 2004

[15] M Senba X Zhong M Senba and H Itakura ldquoEBV andnasopharyngeal carcinomardquo e Lancet vol 343 no 8905 p1104 1994

[16] Y Li J Huang D Yang et al ldquoExpression patterns of E2Ftranscription factors and their potential prognostic roles inbreast cancerrdquoOncology Letters vol 15 pp 9216ndash9230 2018

[17] M N Gnanapragasam and J J Bieker ldquoOrchestration of lateevents in erythropoiesis by KLF1EKLFrdquo Current Opinion inHematology vol 24 no 3 pp 183ndash190 2017

[18] C Sun Q Zhou W Hu et al ldquoTranscriptional E2F1258 aspotential targets and transcriptional E2F367 as new biomark-ers for the prognosis of human lung carcinomardquo AGING vol10 no 5 pp 973ndash987 2018

[19] L Chen X Huang and X Chen ldquoMiR-365 suppresses cholan-giocarcinoma cell proliferation and induces apoptosis by target-ing E2F2rdquoOncology Research Featuring Preclinical and ClinicalCancer erapeutics vol 26 no 9 pp 1375ndash1382 2018

[20] A Feliciano Y Garcia-Mayea L Jubierre et al ldquomiR-99areveals two novel oncogenic proteins E2F2 and EMR2 andrepresses stemness in lung cancerrdquo Cell Death amp Disease vol8 no 10 p e3141 2017

[21] T Tao Q Shen J Luo Y Xu and W Liang ldquoMicroRNA-125a regulates cell proliferation via directly targeting E2F2 inosteosarcomardquoCellular Physiology andBiochemistry vol 43 no2 pp 768ndash774 2017

[22] H K Kim J-Y Hwang J K Hyun et al ldquoExpression ofa peroxisome proliferator-activated receptor 1205741 splice variantthat was identified in human lung cancers suppresses celldeath induced by cisplatin and oxidative stressrdquo Clinical CancerResearch vol 13 no 9 pp 2577ndash2583 2007

[23] M Monami I Dicembrini and E Mannucci ldquoThiazolidine-diones and cancer results of a meta-analysis of randomizedclinical trialsrdquo Acta Diabetologica vol 51 no 1 pp 91ndash101 2014

[24] S Chang and H Hu ldquoAssociation of thiazolidinediones withgastric cancer in type 2 diabetes mellitus a population-basedcasendashcontrol studyrdquo BMC Cancer vol 13 no 1 article no 4202013

[25] M T Bazelier F de Vries P Vestergaard H Leufkens and MDe Bruin ldquoUse of thiazolidinediones and risk of bladder cancerdisease or drugsrdquo Current Drug Safety vol 8 no 5 pp 364ndash370 2013

[26] VVellaM LNicolosi S GiulianoM BellomoA Belfiore andR Malaguarnera ldquoPPAR-120574 agonists as antineoplastic agents incancers with dysregulated IGF axisrdquo Frontiers in Endocrinologyvol 8 article no 31 2017

[27] V Costa D Foti F Paonessa et al ldquoThe insulin receptorA new anticancer target for peroxisome proliferator-activatedreceptor-120574 (PPAR120574) and thiazolidinedione- PPAR120574 agonistsrdquoEndocrine-Related Cancer vol 15 no 1 pp 325ndash335 2008

[28] S Wakino U Kintscher S Kim F Yin W A Hsueh and RE Law ldquoPeroxisome proliferator-activated receptor 120574 ligandsinhibit retinoblastoma phosphorylation andG1 997888rarr S transitionin vascular smooth muscle cellsrdquo e Journal of BiologicalChemistry vol 275 no 29 pp 22435ndash22441 2000

[29] S Altiok M Xu and B M Spiegelman ldquoPPAR120574 induces cellcycle withdrawal Inhibition of E2fDP DNA-binding activityvia down-regulation of PP2ArdquoGenesampDevelopment vol 11 no15 pp 1987ndash1998 1997

[30] L Wu C Timmers B Malti et al ldquoThe E2F1-3 transcriptionfactors are essential for cellular proliferationrdquo Nature vol 414no 6862 pp 457ndash462 2001

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

8 PPAR Research

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE1(a)

E2F2

PPAR-

GAPDH

0 5 10 20Rog(M)

CNE2(b)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE1

minus

minus

minus

minus

(c)

E2F2

PPAR-

GAPDH

Rog

(20M)

GW9662(100nM)

+ +

+ +

CNE2

minus minus

minus minus

(d)Figure 4 E2F2 and PPAR-120574 expression in CNE1 and CNE2 nasopharyngeal carcinoma cells a13er treatment with PPAR-120574 ligand (a b) Westernblotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cells after treatment with dose-dependentPPAR-120574 agonist Rog (c d) Western blotting was performed to detect the expression of E2F2 PPAR-120574 and GAPDH in CNE1 and CNE2 cellsafter treatment with PPAR-120574 agonist (rosiglitazone Rog) and PPAR-120574 antagonist (GW9662)

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

None of the authors has conflicts of interest with thissubmission

Acknowledgments

This paper was supported by grants from the First AffiliatedHospital of Shihezi University School of Medicine (noZZZC201828A)

References

[1] M Altun A Fandi O Dupuis E Cvitkovic Z Krajina and FEschwege ldquoUndifferentiated nasopharyngeal cancer (UCNT)current diagnostic and therapeutic aspectsrdquo International Jour-nal of Radiation Oncology ∙ Biology ∙ Physics vol 32 no 3 pp859ndash877 1995

[2] M L K Chua J T S Wee E P Hui and A T C ChanldquoNasopharyngeal carcinomardquoeLancet vol 387 no 10022 pp1012ndash1024 2016

[3] D K Dimova and N J Dyson ldquoThe E2F transcriptional net-work old acquaintances with new facesrdquo Oncogene vol 24 no17 pp 2810ndash2826 2005

[4] J DeGregori and D G Johnson ldquoDistinct and overlappingroles for E2F family members in transcription proliferation andapoptosisrdquo Current Molecular Medicine vol 6 no 7 pp 739ndash748 2006

[5] Q Dong P Meng T Wang et al ldquoMicroRNA let-7a inhibitsproliferation of human prostate cancer cells in vitro and in vivoby targeting E2F2 and CCND2rdquo PLoS ONE vol 5 no 4 ArticleID e10147 2010

[6] S Hayami M Yoshimatsu A Veerakumarasivam et al ldquoOver-expression of the JmjC histone demethylase KDM5B in humancarcinogenesis involvement in the proliferation of cancer cellsthrough the E2FRB pathwayrdquoMolecular Cancer vol 9 no 1 p59 2010

[7] Y Gao D Han L Sun et al ldquoPPAR120572 regulates the proliferationof human glioma cells through miR-214 and E2F2rdquo BioMedResearch International vol 2018 Article ID 3842753 10 pages2018

[8] H-X Liu Y Fang YHu F J Gonzalez J Fang andY-J YWanldquoPPAR120573 regulates liver regeneration bymodulating Akt and E2fsignalingrdquo PLoS ONE vol 8 no 6 Article ID e65644 2013

[9] Y Komatsu I Ito M Wayama et al ldquoPPAR120574 ligands suppressthe feedback loop between E2F2 and cyclin-E1rdquo Biochemicaland Biophysical Research Communications vol 370 no 1 pp145ndash148 2008

[10] Y Zhang O S Dallner T Nakadai et al ldquoA noncanonicalPPAR120574RXR120572-binding sequence regulates leptin expression inresponse to changes in adipose tissue massrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 115 no 26 pp E6039ndashE6047 2018

PPAR Research 9

[11] D Aleman-Gonzalez-Duhart F Tamay-Cach S Alvarez-Almazan and J E Mendieta-Wejebe ldquoCurrent advances in thebiochemical and physiological aspects of the treatment of type 2diabetes mellitus with thiazolidinedionesrdquo PPAR Research vol2016 Article ID 7614270 10 pages 2016

[12] S H Kim H S Park M J Hong H J Hur D Y Kwon andMS Kim ldquoCaffeic acid phenethyl ester improves metabolic syn-drome by activating ppar-gamma and inducing adipose tissueremodeling in diet-induced obese micerdquoMolecular Nutrition ampFood Research vol 62 Article ID e1700701 2018

[13] M Kukida M Mogi H Kan-no et al ldquoAT2 receptor stimula-tion inhibits phosphate-induced vascular calcificationrdquo KidneyInternational vol 95 no 1 pp 138ndash148 2019

[14] L Michalik B Desvergne and W Wahli ldquoPeroxisome-prolif-erator-activated receptors and cancers complex storiesrdquoNatureReviews Cancer vol 4 no 1 pp 61ndash70 2004

[15] M Senba X Zhong M Senba and H Itakura ldquoEBV andnasopharyngeal carcinomardquo e Lancet vol 343 no 8905 p1104 1994

[16] Y Li J Huang D Yang et al ldquoExpression patterns of E2Ftranscription factors and their potential prognostic roles inbreast cancerrdquoOncology Letters vol 15 pp 9216ndash9230 2018

[17] M N Gnanapragasam and J J Bieker ldquoOrchestration of lateevents in erythropoiesis by KLF1EKLFrdquo Current Opinion inHematology vol 24 no 3 pp 183ndash190 2017

[18] C Sun Q Zhou W Hu et al ldquoTranscriptional E2F1258 aspotential targets and transcriptional E2F367 as new biomark-ers for the prognosis of human lung carcinomardquo AGING vol10 no 5 pp 973ndash987 2018

[19] L Chen X Huang and X Chen ldquoMiR-365 suppresses cholan-giocarcinoma cell proliferation and induces apoptosis by target-ing E2F2rdquoOncology Research Featuring Preclinical and ClinicalCancer erapeutics vol 26 no 9 pp 1375ndash1382 2018

[20] A Feliciano Y Garcia-Mayea L Jubierre et al ldquomiR-99areveals two novel oncogenic proteins E2F2 and EMR2 andrepresses stemness in lung cancerrdquo Cell Death amp Disease vol8 no 10 p e3141 2017

[21] T Tao Q Shen J Luo Y Xu and W Liang ldquoMicroRNA-125a regulates cell proliferation via directly targeting E2F2 inosteosarcomardquoCellular Physiology andBiochemistry vol 43 no2 pp 768ndash774 2017

[22] H K Kim J-Y Hwang J K Hyun et al ldquoExpression ofa peroxisome proliferator-activated receptor 1205741 splice variantthat was identified in human lung cancers suppresses celldeath induced by cisplatin and oxidative stressrdquo Clinical CancerResearch vol 13 no 9 pp 2577ndash2583 2007

[23] M Monami I Dicembrini and E Mannucci ldquoThiazolidine-diones and cancer results of a meta-analysis of randomizedclinical trialsrdquo Acta Diabetologica vol 51 no 1 pp 91ndash101 2014

[24] S Chang and H Hu ldquoAssociation of thiazolidinediones withgastric cancer in type 2 diabetes mellitus a population-basedcasendashcontrol studyrdquo BMC Cancer vol 13 no 1 article no 4202013

[25] M T Bazelier F de Vries P Vestergaard H Leufkens and MDe Bruin ldquoUse of thiazolidinediones and risk of bladder cancerdisease or drugsrdquo Current Drug Safety vol 8 no 5 pp 364ndash370 2013

[26] VVellaM LNicolosi S GiulianoM BellomoA Belfiore andR Malaguarnera ldquoPPAR-120574 agonists as antineoplastic agents incancers with dysregulated IGF axisrdquo Frontiers in Endocrinologyvol 8 article no 31 2017

[27] V Costa D Foti F Paonessa et al ldquoThe insulin receptorA new anticancer target for peroxisome proliferator-activatedreceptor-120574 (PPAR120574) and thiazolidinedione- PPAR120574 agonistsrdquoEndocrine-Related Cancer vol 15 no 1 pp 325ndash335 2008

[28] S Wakino U Kintscher S Kim F Yin W A Hsueh and RE Law ldquoPeroxisome proliferator-activated receptor 120574 ligandsinhibit retinoblastoma phosphorylation andG1 997888rarr S transitionin vascular smooth muscle cellsrdquo e Journal of BiologicalChemistry vol 275 no 29 pp 22435ndash22441 2000

[29] S Altiok M Xu and B M Spiegelman ldquoPPAR120574 induces cellcycle withdrawal Inhibition of E2fDP DNA-binding activityvia down-regulation of PP2ArdquoGenesampDevelopment vol 11 no15 pp 1987ndash1998 1997

[30] L Wu C Timmers B Malti et al ldquoThe E2F1-3 transcriptionfactors are essential for cellular proliferationrdquo Nature vol 414no 6862 pp 457ndash462 2001

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

PPAR Research 9

[11] D Aleman-Gonzalez-Duhart F Tamay-Cach S Alvarez-Almazan and J E Mendieta-Wejebe ldquoCurrent advances in thebiochemical and physiological aspects of the treatment of type 2diabetes mellitus with thiazolidinedionesrdquo PPAR Research vol2016 Article ID 7614270 10 pages 2016

[12] S H Kim H S Park M J Hong H J Hur D Y Kwon andMS Kim ldquoCaffeic acid phenethyl ester improves metabolic syn-drome by activating ppar-gamma and inducing adipose tissueremodeling in diet-induced obese micerdquoMolecular Nutrition ampFood Research vol 62 Article ID e1700701 2018

[13] M Kukida M Mogi H Kan-no et al ldquoAT2 receptor stimula-tion inhibits phosphate-induced vascular calcificationrdquo KidneyInternational vol 95 no 1 pp 138ndash148 2019

[14] L Michalik B Desvergne and W Wahli ldquoPeroxisome-prolif-erator-activated receptors and cancers complex storiesrdquoNatureReviews Cancer vol 4 no 1 pp 61ndash70 2004

[15] M Senba X Zhong M Senba and H Itakura ldquoEBV andnasopharyngeal carcinomardquo e Lancet vol 343 no 8905 p1104 1994

[16] Y Li J Huang D Yang et al ldquoExpression patterns of E2Ftranscription factors and their potential prognostic roles inbreast cancerrdquoOncology Letters vol 15 pp 9216ndash9230 2018

[17] M N Gnanapragasam and J J Bieker ldquoOrchestration of lateevents in erythropoiesis by KLF1EKLFrdquo Current Opinion inHematology vol 24 no 3 pp 183ndash190 2017

[18] C Sun Q Zhou W Hu et al ldquoTranscriptional E2F1258 aspotential targets and transcriptional E2F367 as new biomark-ers for the prognosis of human lung carcinomardquo AGING vol10 no 5 pp 973ndash987 2018

[19] L Chen X Huang and X Chen ldquoMiR-365 suppresses cholan-giocarcinoma cell proliferation and induces apoptosis by target-ing E2F2rdquoOncology Research Featuring Preclinical and ClinicalCancer erapeutics vol 26 no 9 pp 1375ndash1382 2018

[20] A Feliciano Y Garcia-Mayea L Jubierre et al ldquomiR-99areveals two novel oncogenic proteins E2F2 and EMR2 andrepresses stemness in lung cancerrdquo Cell Death amp Disease vol8 no 10 p e3141 2017

[21] T Tao Q Shen J Luo Y Xu and W Liang ldquoMicroRNA-125a regulates cell proliferation via directly targeting E2F2 inosteosarcomardquoCellular Physiology andBiochemistry vol 43 no2 pp 768ndash774 2017

[22] H K Kim J-Y Hwang J K Hyun et al ldquoExpression ofa peroxisome proliferator-activated receptor 1205741 splice variantthat was identified in human lung cancers suppresses celldeath induced by cisplatin and oxidative stressrdquo Clinical CancerResearch vol 13 no 9 pp 2577ndash2583 2007

[23] M Monami I Dicembrini and E Mannucci ldquoThiazolidine-diones and cancer results of a meta-analysis of randomizedclinical trialsrdquo Acta Diabetologica vol 51 no 1 pp 91ndash101 2014

[24] S Chang and H Hu ldquoAssociation of thiazolidinediones withgastric cancer in type 2 diabetes mellitus a population-basedcasendashcontrol studyrdquo BMC Cancer vol 13 no 1 article no 4202013

[25] M T Bazelier F de Vries P Vestergaard H Leufkens and MDe Bruin ldquoUse of thiazolidinediones and risk of bladder cancerdisease or drugsrdquo Current Drug Safety vol 8 no 5 pp 364ndash370 2013

[26] VVellaM LNicolosi S GiulianoM BellomoA Belfiore andR Malaguarnera ldquoPPAR-120574 agonists as antineoplastic agents incancers with dysregulated IGF axisrdquo Frontiers in Endocrinologyvol 8 article no 31 2017

[27] V Costa D Foti F Paonessa et al ldquoThe insulin receptorA new anticancer target for peroxisome proliferator-activatedreceptor-120574 (PPAR120574) and thiazolidinedione- PPAR120574 agonistsrdquoEndocrine-Related Cancer vol 15 no 1 pp 325ndash335 2008

[28] S Wakino U Kintscher S Kim F Yin W A Hsueh and RE Law ldquoPeroxisome proliferator-activated receptor 120574 ligandsinhibit retinoblastoma phosphorylation andG1 997888rarr S transitionin vascular smooth muscle cellsrdquo e Journal of BiologicalChemistry vol 275 no 29 pp 22435ndash22441 2000

[29] S Altiok M Xu and B M Spiegelman ldquoPPAR120574 induces cellcycle withdrawal Inhibition of E2fDP DNA-binding activityvia down-regulation of PP2ArdquoGenesampDevelopment vol 11 no15 pp 1987ndash1998 1997

[30] L Wu C Timmers B Malti et al ldquoThe E2F1-3 transcriptionfactors are essential for cellular proliferationrdquo Nature vol 414no 6862 pp 457ndash462 2001

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom