Research Article Periostin Facilitates the Epithelial ...signaling pathway. Playing a pivotal role...

Research ArticlePeriostin Facilitates the Epithelial-Mesenchymal Transition ofEndometrial Epithelial Cells through ILK-Akt Signaling Pathway

Qiao-mei Zheng Jing-jing Lu Jing Zhao Xuan Wei Lu Wang and Pei-shu Liu

Department of Obstetrics and Gynecology Qilu Hospital of Shandong University 107Wenhua Xi Road Jinan Shandong 250012 China

Correspondence should be addressed to Pei-shu Liu peishu liu126com

Received 22 November 2015 Revised 2 February 2016 Accepted 15 February 2016

Academic Editor Koichiro Wada

Copyright copy 2016 Qiao-mei Zheng et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Although periostin was confirmed to facilitate the pathogenesis of endometriosis by enhancing the migration invasion andadhesion of human endometrial stromal cells (ESCs) its effect on the endometrial epithelial cells (EECs) is still unknown Thecurrent study aimed to determine whether periostin enhanced the epithelial-mesenchymal transition (EMT) of EECs EECs wereisolated from 12 women with endometriosis The migration and invasion abilities of EECs were evaluated by transwell assaysExpressions of proteins were detected by western blot After treatment with periostin the migration and invasion abilities of EECswere enhanced Additionally E-cadherin and keratin were downregulated while N-cadherin and vimentin were upregulated inEECs Simultaneously levels of ILK p-Akt slug and Zeb1 were all upregulated in EECs After silencing the expression of ILK inEECs levels of p-Akt slug Zeb1 N-cadherin and vimentin were downregulated while E-cadherin and keratin were upregulatedAlthough periostin weakened the above effects in EECs after silencing the expression of ILK it failed to induce the EMT ofEECs Thus periostin enhanced invasion and migration abilities of EECs and facilitated the EMT of EECs through ILK-Aktsignaling pathway Playing a pivotal role in the pathogenesis of endometriosis periostin may be a new clinical therapy target forendometriosis

1 Introduction

Endometriosis defined as the presence of endometrial andstromal cells at extrauterine locations is a persistent gyneco-logical problemwhich can result in dysmenorrhea infertilityand decreased quality of life [1] It affects about 10 of womenof reproductive age [2] Nearly two-thirds of adolescentswith dysmenorrhea or chronic pelvic pain have laparoscopicevidence of endometriosis [3] However there is no radicalcure other than surgery for endometriosis due to its unclearpathogenesis To date the most widely accepted theory is theretrograde refluxhypothesis which suggests that endometrialtissues can regurgitate into pelvic cavity duringmenstruationand develop into endometriosis [4] However it is knownthat cells will die when they detach from extracellular matrix(ECM) or adhere to inappropriate location namely anoikis[5] Epithelial-mesenchymal transition (EMT) amain featureassociated with anoikis resistance plays vital roles in tumorprogression and metastatic colonization [6]

EMT is a crucial event in embryogenesis and tumormetastasis characterized by epithelial cells losing epithelialmarkers and acquiring mesenchymal markers During EMTepithelial cells lose cell polarity and are converted into mes-enchymal cells endowing cells with invasive and metastaticproperties Downregulation of E-cadherin a cell adhesionmolecule expressed in epithelial cells is a crucial molecularfeature of EMT [7] Transcription factors including snailslug Zeb1 and Twist can initiate EMT via repressing theexpression of E-cadherin [8 9] Previous studies point outthat EMTplays essential roles in themetastasis of tumors [10ndash12] Although endometriosis is a benign disease it behavesmalignantly by penetrating and developing elsewhere likecancer metastasis Additionally emerging evidences indicatethat EMT plays a significant part in the initial formation ofendometriosis [13 14]

Periostin is a secretory extracellular matrix protein whichis widely expressed in bone tooth heart uterus and breastAbnormally high levels of periostin have been reported in

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 9842619 8 pageshttpdxdoiorg10115520169842619

2 BioMed Research International

breast cancer ovarian cancer and hepatocellular carcinoma[15ndash17] Existing evidence suggests that periostin can facil-itate tumor metastasis through inducing EMT [18 19] Asa ligand for integrins periostin mainly promoted cancercells invasion and metastasis via integrin pathways [20 21]Integrin-linked kinase (ILK) a key role in the integrinpathway directly phosphorylated its downstream target suchas Akt resulting in the EMT process [22]

In our previous study we observed significantly higherexpression of periostin in the ectopic and eutopic endo-metrium of endometriosis [23] Additionally we demon-strated that periostin facilitated endometriosis by enhanc-ing the migration adhesion and invasion of endometrialstromal cells (ESCs) [24] But the effect of periostin on theEECs is still unknown Given that endometriosis behavesmalignantly by penetrating and developing elsewhere liketumor metastasis we herein hypothesize that periostin mayfacilitate endometriosis by inducing the EMT of EECsThe current study was undertaken to determine whetherperiostin enhances the EMT of EECs as well as exploring themechanism through which periostin favored EMT in EECs

2 Materials and Methods

21 Sample Collection and Cell Culture Eutopic endome-trium tissues were obtained from 12 women (23ndash41 yearsold menstrual cycle proliferative phase) with endometriosisAll of the participants were at reproductive age had regularmenstruation and received no hormonal therapy at least 6months before the study Endometriosis was visually diag-nosed during the laparoscopy for ovarian cysts and thenascertained by pathological examination All the participantswere from the Department of Obstetrics and GynecologyQilu Hospital of Shandong University from July 2014 to May2015 Informed consent was obtained from all participantsprior to surgeryThe Institutional Review Board of ShandongUniversity approved the study

After collection tissues were immediately washed withPBS to remove blood mucous and debris The EECs wereisolated following the digestion of type IV collagenase(5mgmL) Cell suspension was filtrated through a sterilestainless steel wire mesh (100 120583m) to remove undigestedtissues ESCs were removed by passing a 40120583m sieve Thenthe 40 120583m mesh was washed thoroughly upside down withmedium to get the EECs The medium was collected andcentrifuged at 1000 rpm for 7 minutes After removingthe supernatant cells were then cultured at 37∘C and 5carbon dioxide in Dulbecco modified Eagle medium F-12(DMEMF12 Sigma-Aldrich St Louis Missouri) contain-ing 10 fetal bovine serum (FBS Gibco Australia) and1 antibiotic The purity of EECs was evaluated by cellimmunofluorescence using mouse antihuman keratin (1 50Cell Signaling Technology Danvers Massachusetts) and rab-bit antihuman vimentin (1 50 Cell Signaling Technology)The purity of EECs was over 95 which was shown as theproportion of epithelial cells in 5 randomly selected pictures(200x magnification) These cells were used for the followingexperiments

22 Transwell Migration and Invasion Assays Cells weretreated with periostin (20 ngmL and 40 ngmL) when reach-ing 80 to 90 confluence After treatment for 48 h cellswere digested for migration and invasion assays as previouslydescribed [25] Pictures of stained cells were taken by theOlympus IX51 inverted microscope Cells were counted infive random fields (200x) of each chamber The average cellnumbers of three duplicate assays for each experimentalcondition were used for statistical analysis

23 Silencing of the ILK Gene in EECs The small-interferingRNA sequences targeting human ILK (siRNA-ILK) weredesigned by GenePharma Company (Shanghai China) Cellswere seeded in 6-well plate without antibiotics treated for12 hours and transfected with blank sequence or siRNA-ILK (50 nmolL) using lipofectamine 2000 (Invitrogen LifeTechnologies) when cell confluence gets 50 to 60 After 48hours of transfection cells were digested for the ensuing cellexperiments The siRNA-ILK sequences are listed in Table 1

24 Western Blot Total protein was extracted from sam-ples for western blot as described previously [25] Pri-mary antibodies used for immunodetection were anti-ILK anti-p-Akt anti-E-cadherin anti-N-cadherin anti-vimentin anti-keratin anti-slug and anti-Zeb1 as well asanti-glyceraldehyde-3-phosphate dehydrogenase (GAPDHCell Signaling Technology Danvers Massachusetts USA)Secondary antibodies were anti-rabbit and anti-mouse IgGperoxidase conjugate (Zhongshan Jinqiao BiotechnologyCoLtd Beijing China) GAPDH was used as a loading controlThe results were quantified by densitometry using ImageJsoftware (NIH Bethesda MD USA)

25 Statistical Analysis GraphPad Prism Version 501(GraphPad Software San Diego California USA) wasused for statistical analysis Data were shown as mean plusmnSEM Studentrsquos 119905-test and one-way ANOVA analysis wererespectively conducted to analyze the differences betweengroups and among groups 119875 value lt 005 was consideredstatistically significant

3 Results

31 The Purity of EECs The purity of EECs was956 plusmn 35 as confirmed by cell immunofluorescencewhich was judged by blue fluorescence for cell nucleus greenfluorescence for keratin and red fluorescence for vimentin(Figure 1)

32 Periostin Enhanced the Migration and Invasion Abilities ofEECs In our previous study we detected the concentrationof periostin in peritoneal washing fluids of patients withand without endometriosis which was 4832 ngmL and2229 ngmL respectively (data not published yet) Afterbeing treated with periostin (20 ngmL and 40 ngmL resp)for 48 h cells were digested formigration and invasion assaysAs shown in Figure 2 the migration and invasion abilitiesof EECs were enhanced by the treatment with periostinespecially for the treatment with 40 ngmL periostin

BioMed Research International 3

Table 1 The sequences of siRNA-ILK

Number Sense (51015840-31015840) Antisense (51015840-31015840)ILK-homo-412 UGG ACA CCG UGA UAU UGU ATT UAC AAU AUC ACG GUG UCC ATTILK-homo-755 CAG CUU AAC UUC CUG ACG ATT UCG UCA GGA AGU UAA GCU GTTILK-homo-1486 GAC CCA AAU UUG ACA UGA UTT AUC AUG UCA AAU UUG GGU CTTNegative control UUC UCC GAA CGU GUC ACG UTT ACG UGA C AC GUU CGG AGA ATT

Vimentin Merge

DAPI Keratin

Figure 1 Identification of purity of EECs Representative staining of cell nucleus is shown in the first image green immunofluorescenceshows the expression of cytokeratin lack of red fluorescence represents negative immunoreactivity for vimentin first three images aremergedtogether as shown in the last picture

33 Periostin Facilitated the EMT and Upregulated the Expres-sion of ILK and p-Akt in EECs To investigate whetherperiostin facilitated the EMT of EECs hallmarks of EMT (E-cadherin N-cadherin keratin and vimentin) were detectedby western blot As depicted in Figures 3(a) and 3(b) thelevels of E-cadherin and keratin were decreased in EECswhen treated with periostin though only the 40 ngmLtreatment was statistically significant On the contrary theN-cadherin and vimentin expression were markedly increasedin the EECs particularly in the 40 ngmL treatment Further-more the ILK expression and p-Akt expression were bothupregulated by periostin in EECs (Figures 3(c) and 3(d))As critical transcription factors of EMT levels of slug andZeb1 were also markedly upregulated by periostin in EECs

(Figures 3(c) and 3(d))However only the 40 ngmLperiostingroup was statistically significant

34 Periostin Facilitated the EMT of EECs through the ILK-Akt Pathway The expression of ILK was obviously decreasedafter the transfection of 3 siRNA-ILKs in EECs especiallythe transfection of ILK-homo-755 So ILK-homo-755 wasused in the remaining tests After being transfected with thesiRNA-ILK ILK p-Akt slug and Zeb1 expressions were alldownregulated significantly in EECs which can be weakenedby the addition of periostin (40 ngmL) (Figures 4(a) and4(b)) Additionally E-cadherin and keratin were upregulatedwhile N-cadherin and vimentin were downregulated in EECsafter receiving ILK silencing which can also be weakened by


0 20 40

Mig

ratio

nIn

vasio

n

Periostin (ngmL)

(a)

lowast

Con Periostin(40ngmL)

Periostin(20ngmL)

Fold

chan

ge

Invasion25

20

15

10

05

00

lowastlowast

(b)

Migration


Periostin(20ngmL)

Fold

chan

ge

3

2

1

0

lowastlowast

lowastlowastlowast

(c)

Figure 2 The influence of periostin on the migration and invasion abilities of EECs lowast119875 lt 005 lowastlowast119875 lt 0005 and lowastlowastlowast119875 lt 0001

the addition of periostin (Figures 4(c) and 4(d)) Althoughthe above effects were weakened by periostin periostin failedto induce the EMT of EECs receiving ILK silencing whencompared to normal EECs or EECs receiving blank siRNAsequence (Figure 4) Thus periostin facilitated the EMT ofEECs through the ILK-Akt pathway

4 Discussion

Emerging evidences suggest that periostin is overexpressedin various types of human cancers and further results inaccelerating migration and invasion abilities of tumor cells[26] Although endometriosis shares many characteristicswith tumors little is known about the role of periostinin endometriosis Our previous study showed significantlyhigher expression of periostin in the eutopic and ectopicendometrium of endometriosis [23] In line with this

we demonstrated that periostin facilitated the progress ofendometriosis by enhancing the adhesion migration andinvasion of ESCs [24] However the effect of periostin onthe EECs of endometriosis is still unknown In the presentstudy we assessed the effect of periostin on themigration andinvasion abilities of EECs of endometriosis and demonstratedthat periostin indeed enhanced the migration and invasionabilities of EECs

Periostin was shown to be not only a newmarker of EMTbut also an inducer of this program [27] Hu et al pointed outthat periostin was an important mediator of TGF-120573-inducedEMT in prostate cancer cells and its overexpression pro-moted cell proliferation invasion and migration of prostatecancer [28] Upregulated periostin significantly promotedthe EMT of adamantinomatous craniopharyngioma cells byactivating Akt signaling pathway [29] Silencing of periostininhibited nicotine-mediated cell growth and EMT in lung


0 20 40

Periostin (ngmL)

GAPDH

Vimentin

N-cad

Keratin

E-cad

(a)

3

4

2

1

0

E-cad Keratin N-cad Vimentin

lowast

lowast

lowast

lowastlowastlowast

lowastlowast

ConPeriostin (20ngmL)Periostin (40ngmL)

(b)

0 20 40

Periostin (ngmL)

Akt

Slug

Zeb1

GAPDH

p-Akt

ILK

(c)

3

2

1

0

lowast

lowastlowast

lowast


ILK p-Akt Slug Zeb1

(d)

Figure 3 Effects of periostin on EMT-related markers ILK and p-Akt in EECs lowast119875 lt 005 lowastlowast119875 lt 0005 and lowastlowastlowast119875 lt 0001

cancer cells [30] Apart from tumors EMT also plays asignificant part in the initial formation of endometriosis [1314]Thus we propose that periostinmay facilitate the EMT ofEECs in the pathogenesis of endometriosis As expected E-cadherin and keratin were downregulated while N-cadherinand vimentin were upregulated in EECs after being treatedwith periostin indicating that periostin induced the EMTof EECs The results were in line with previous evidencesthat E-cadherin was downregulated while N-cadherin andvimentin were upregulated in endometriosis [31ndash33] EMT-inducing transcription factors (EMT-TFs) are reported toprominently initiate the EMT by repressing the expressionof E-cadherin directly or indirectly [8] Periostin upregulatedsnail expression in prostate cancer cells but downregulatedTwist expression in bladder cancer cells [18] Silencing ofperiostin decreased cell invasion and snail expression in lung

cancer cells [30] So we examined the effect of periostinon the expression of EMT-TFs in EECs and verified thatlevels of slug and Zeb1 were both increased after the additionof periostin Therefore periostin played crucial roles in thepathogenesis of endometriosis by facilitating the EMT ofEECs

Accumulating evidences suggest that periostin is a lig-and for integrin and mainly facilitates the EMT processvia integrin pathway [34 35] Yan and Shao demonstratedthat transduction of periostin into nonmetastatic 293T cellsinduced cell invasion and metastasis via EMT and the roleof periostin in EMT required integrin signaling pathway[19] ILK an essential role in the integrin pathway is aserine-threonine kinase that can directly phosphorylate itsdownstream targets to mediate cell-ECM and intracellularprocesses [36] Besides that ILK promotes the migration


p-Akt

Slug

Zeb1

Akt

GAPDH

NCminus minus minus +minus + minus minus

minus minus + +siRNA-ILK

Periostin

ILK

(a)

siRNA-ILKsiRNA-ILK + periostinNC

Con

ILK p-Akt Slug Zeb1

12

10

08

06

04

02

00

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(b)

E-cad

Keratin

N-cad

Vimentin

GAPDH



Periostin

(c)


Con


20

15

10

05

00

lowastlowast

lowast

lowastlowastlowastlowast

lowastlowast

(d)

Figure 4 Effects of siRNA-ILK and periostin on ILK p-Akt and EMT-related markers in EECs lowast119875 lt 005 lowastlowast119875 lt 0005 and lowastlowastlowast119875 lt 0001

and invasion of cancer cells by facilitating the EMT process[37 38] In hepatocellular carcinoma ILK activated Aktthrough phosphorylating Akt at Ser473 resulting in EMT ofliver epithelial cells and radioresistance and chemoresistanceof hepatocellular carcinoma cells [22] Silencing of the ILKdecreased the phosphorylation of Akt and prevented themigration of thyroid cancer cells [39] Additionally periostinoverexpression induced theEMTof adamantinomatous cran-iopharyngioma cells by activatingAkt signaling pathway [29]Consistent with this our previous study demonstrated thatperiostin enhanced the adhesion migration and invasion ofESCs through ILK-Akt pathway in endometriosis [24] In thepresent study the ILK-Akt pathway was activated in EECsafter the treatment with periostin Silencing the expressionof ILK decreased the phosphorylation rate of Akt in theEECs leading to upregulation of epithelial markers of EECsMoreover periostin failed to induce the EMT of EECs after

silencing the expression of ILK From the above ILK-Aktpathway was activated in EECs and periostin facilitated theEMT of EECs through the ILK-Akt pathway

Recently studies have been focused on the therapeuticpotential by targeting periostin in different diseases Inglioma Let-7f inhibited the cell proliferation migrationand invasion by repressing the expression of periostin[40] Quercetin suppressed the production and function ofperiostin in human nasal epithelial cells and resulted inimprovement of clinical conditions of allergic rhinitis [41]Given its critical role in the pathogenesis of endometriosisperiostin may be a promising therapy target for endometrio-sis Drugs repressing the expression or inhibiting the functionof periostin may suppress the progress and recurrence ofendometriosis However the therapeutic effect of targetingperiostin on endometriosis remains to be investigated in thefuture


5 Conclusion

Our results suggest that periostin facilitated the EMTof EECsthrough ILK-Akt pathways Playing critically key roles inthe EMT process during the pathogenesis of endometrio-sis periostin may be a new clinical therapy target forendometriosis

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This study was supported by grants from the NationalNatural Science Foundation of China (NSFC) (81370696 and81101984) as well as the Science andTechnologyDevelopmentPlanning of Shandong (2013GGE27031)

References

[1] C L Hughes W G Foster and S K Agarwal ldquoThe impactof endometriosis across the lifespan of women foreseeableresearch and therapeutic prospectsrdquo BioMed Research Interna-tional vol 2015 Article ID 158490 8 pages 2015

[2] P Vigano F Parazzini E Somigliana and P VercellinildquoEndometriosis epidemiology and aetiological factorsrdquo BestPractice amp Research Clinical Obstetrics amp Gynaecology vol 18no 2 pp 177ndash200 2004

[3] E B Janssen A C M Rijkers K Hoppenbrouwers CMeuleman and T M DrsquoHooghe ldquoPrevalence of endometriosisdiagnosed by laparoscopy in adolescents with dysmenorrhea orchronic pelvic pain a systematic reviewrdquo Human ReproductionUpdate vol 19 no 5 pp 570ndash582 2013

[4] J A Sampson ldquoPeritoneal endometriosis due to the menstrualdissemination of endometrial tissue into the peritoneal cavityrdquoAmerican Journal of Obstetrics and Gynecology vol 14 no 4 pp422ndash469 1927

[5] SM Frisch andH Francis ldquoDisruption of epithelial cell-matrixinteractions induces apoptosisrdquo Journal of Cell Biology vol 124no 4 pp 619ndash626 1994

[6] P Paoli E Giannoni and P Chiarugi ldquoAnoikis molecularpathways and its role in cancer progressionrdquo Biochimica etBiophysica Acta (BBA)mdashMolecular Cell Research vol 1833 no12 pp 3481ndash3498 2013

[7] M A Huber N Kraut and H Beug ldquoMolecular requirementsfor epithelialndashmesenchymal transition during tumor progres-sionrdquoCurrent Opinion in Cell Biology vol 17 no 5 pp 548ndash5582005

[8] A Puisieux T Brabletz and J Caramel ldquoOncogenic roles ofEMT-inducing transcription factorsrdquo Nature Cell Biology vol16 no 6 pp 488ndash494 2014

[9] M Tania M A Khan and J Fu ldquoEpithelial to mesenchymaltransition inducing transcription factors andmetastatic cancerrdquoTumor Biology vol 35 no 8 pp 7335ndash7342 2014

[10] T E Anwar and C G Kleer ldquoTissue-based identification ofstem cells and epithelial-to-mesenchymal transition in breastcancerrdquo Human Pathology vol 44 no 8 pp 1457ndash1464 2013

[11] C Mirantes I Espinosa I Ferrer X Dolcet J Prat and XMatias-Guiu ldquoEpithelial-to-mesenchymal transition and stem

cells in endometrial cancerrdquo Human Pathology vol 44 no 10pp 1973ndash1981 2013

[12] D Vergara B Merlot J-P Lucot et al ldquoEpithelialndashmesenchymal transition in ovarian cancerrdquo Cancer Letters vol291 no 1 pp 59ndash66 2010

[13] J Bartley A Julicher B Hotz S Mechsner and H HotzldquoEpithelial to mesenchymal transition (EMT) seems to beregulated differently in endometriosis and the endometriumrdquoArchives of Gynecology and Obstetrics vol 289 no 4 pp 871ndash881 2014

[14] S Matsuzaki and C Darcha ldquoEpithelial to mesenchymaltransition-like and mesenchymal to epithelial transition-likeprocesses might be involved in the pathogenesis of pelvicendometriosisrdquoHuman Reproduction vol 27 no 3 pp 712ndash7212012

[15] R S Ismail R L Baldwin J Fang et al ldquoDifferential geneexpression between normal and tumor-derived ovarian epithe-lial cellsrdquo Cancer Research vol 60 no 23 pp 6744ndash6749 2000

[16] X Wang J Liu Z Wang et al ldquoPeriostin contributes to theacquisition of multipotent stem cell-like properties in humanmammary epithelial cells and breast cancer cellsrdquo PLoS ONEvol 8 no 8 Article ID e72962 2013

[17] Y Lv W Wang W-D Jia et al ldquoHigh-level expression ofperiostin is closely related to metastatic potential and poorprognosis of hepatocellular carcinomardquo Medical Oncology vol30 no 1 article 385 2013

[18] C J Kim K Sakamoto Y Tambe and H Inoue ldquoOppositeregulation of epithelial-to-mesenchymal transition and cellinvasiveness by periostin between prostate and bladder cancercellsrdquo International Journal of Oncology vol 38 no 6 pp 1759ndash1766 2011

[19] W Yan and R Shao ldquoTransduction of a mesenchyme-specificgene periostin into 293T cells induces cell invasive activitythrough epithelial-mesenchymal transformationrdquo The Journalof Biological Chemistry vol 281 no 28 pp 19700ndash19708 2006

[20] G Ouyang M Liu K Ruan G Song Y Mao and S BaoldquoUpregulated expression of periostin by hypoxia in non-small-cell lung cancer cells promotes cell survival via the AktPKBpathwayrdquo Cancer Letters vol 281 no 2 pp 213ndash219 2009

[21] K Utispan J Sonongbua P Thuwajit et al ldquoPeriostin acti-vates integrin 12057251205731 through a PI3KAKT-dependent pathwayin invasion of cholangiocarcinomardquo International Journal ofOncology vol 41 no 3 pp 1110ndash1118 2012

[22] X Jiang J Wang K Zhang S Tang C Ren and Y ChenldquoThe role of CD29-ILK-Akt signaling-mediated epithelialndashmesenchymal transition of liver epithelial cells and chemore-sistance and radioresistance in hepatocellular carcinoma cellsrdquoMedical Oncology vol 32 no 5 article 141 2015

[23] L Shen P Liu P Zhang X Zhang and J Cui ldquoCharacterizationof periostin expression in human endometrium and endometri-otic lesionsrdquo Gynecological Endocrinology vol 28 no 10 pp815ndash818 2012

[24] X Xu Q Zheng Z Zhang X Zhang R Liu and PLiu ldquoPeriostin enhances migration invasion and adhesionof human endometrial stromal cells through integrin-linkedkinase 1Akt signaling pathwayrdquo Reproductive Sciences vol 22no 9 pp 1098ndash1106 2015

[25] R Liu J Zheng C Li et al ldquoCelecoxib induces epithelial-mesenchymal transition in epithelial ovarian cancer cells viaregulating ZEB1 expressionrdquoArchives of Gynecology and Obstet-rics vol 291 no 6 pp 1361ndash1369 2015


[26] K Ratajczak-Wielgomas and P Dziegiel ldquoThe role of periostinin neoplastic processesrdquo Folia Histochemica et Cytobiologicavol 53 no 2 pp 120ndash132 2015

[27] L Morra and H Moch ldquoPeriostin expression and epithelial-mesenchymal transition in cancer a review and an updaterdquoVirchows Archiv vol 459 no 5 pp 465ndash475 2011

[28] Q Hu S Tong X Zhao et al ldquoPeriostin mediates TGF-120573-induced epithelial mesenchymal transition in prostate cancercellsrdquo Cellular Physiology and Biochemistry vol 36 no 2 pp799ndash809 2015

[29] M Chen S Zheng Y Liu J Shi and S Qi ldquoPeriostin activatespathways involved in epithelialndashmesenchymal transition inadamantinomatous craniopharyngiomardquo Journal of the Neuro-logical Sciences vol 360 pp 49ndash54 2016

[30] S-Q Wu Y-E Lv B-H Lin et al ldquoSilencing of periostininhibits nicotine-mediated tumor cell growth and epithelial-mesenchymal transition in lung cancer cellsrdquo MolecularMedicine Reports vol 7 no 3 pp 875ndash880 2013

[31] R Gaetje S Kotzian G Herrmann R Baumann and AStarzinski-Powitz ldquoNonmalignant epithelial cells potentiallyinvasive in human endometriosis lack the tumor suppressormolecule E-cadherinrdquo The American Journal of Pathology vol150 no 2 pp 461ndash467 1997

[32] K M Eyster A L Boles J D Brannian and K A HansenldquoDNA microarray analysis of gene expression markers ofendometriosisrdquo Fertility and Sterility vol 77 no 1 pp 38ndash422002

[33] H Zhang Y Niu J Feng H Guo X Ye and H Cui ldquoUse ofproteomic analysis of endometriosis to identify different pro-tein expression in patients with endometriosis versus normalcontrolsrdquo Fertility and Sterility vol 86 no 2 pp 274ndash282 2006

[34] S Kim H Y Kang E-H Nam et al ldquoTMPRSS4 induces inva-sion and epithelial-mesenchymal transition through upregula-tion of integrin 1205725 and its signaling pathwaysrdquo Carcinogenesisvol 31 no 4 pp 597ndash606 2010

[35] P P Shah M Y Fong and S S Kakar ldquoPTTG induces EMTthrough integrin 120572V1205733-focal adhesion kinase signaling in lungcancer cellsrdquo Oncogene vol 31 no 26 pp 3124ndash3135 2012

[36] G Hannigan A A Troussard and S Dedhar ldquoIntegrin-linkedkinase a cancer therapeutic target unique among its ILKrdquoNature Reviews Cancer vol 5 no 1 pp 51ndash63 2005

[37] D Chen Y Zhang X Zhang et al ldquoOverexpression of integrin-linked kinase correlates with malignant phenotype in non-small cell lung cancer and promotes lung cancer cell invasionandmigration via regulating epithelial-mesenchymal transition(EMT)-related genesrdquoActaHistochemica vol 115 no 2 pp 128ndash136 2013

[38] Z Yan H Yin R Wang et al ldquoOverexpression of integrin-linked kinase (ILK) promotes migration and invasion of col-orectal cancer cells by inducing epithelial-mesenchymal tran-sition via NF-120581B signalingrdquo Acta Histochemica vol 116 no 3pp 527ndash533 2014

[39] L A Shirley S McCarty M Yang et al ldquoIntegrin-linked kinaseaffects signaling pathways and migration in thyroid cancer cellsand is a potential therapeutic targetrdquo Surgery vol 159 no 1 pp163ndash171 2016

[40] S Yan X Han H Xue et al ldquoLet-7f inhibits glioma cellproliferation migration and invasion by targeting periostinrdquoJournal of Cellular Biochemistry vol 116 no 8 pp 1680ndash16922015

[41] S Irie M Kashiwabara A Yamada and K Asano ldquoSuppressiveactivity of quercetin on periostin functions in vitrordquo InVivo vol30 no 1 pp 17ndash25 2016

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


breast cancer ovarian cancer and hepatocellular carcinoma[15ndash17] Existing evidence suggests that periostin can facil-itate tumor metastasis through inducing EMT [18 19] Asa ligand for integrins periostin mainly promoted cancercells invasion and metastasis via integrin pathways [20 21]Integrin-linked kinase (ILK) a key role in the integrinpathway directly phosphorylated its downstream target suchas Akt resulting in the EMT process [22]

In our previous study we observed significantly higherexpression of periostin in the ectopic and eutopic endo-metrium of endometriosis [23] Additionally we demon-strated that periostin facilitated endometriosis by enhanc-ing the migration adhesion and invasion of endometrialstromal cells (ESCs) [24] But the effect of periostin on theEECs is still unknown Given that endometriosis behavesmalignantly by penetrating and developing elsewhere liketumor metastasis we herein hypothesize that periostin mayfacilitate endometriosis by inducing the EMT of EECsThe current study was undertaken to determine whetherperiostin enhances the EMT of EECs as well as exploring themechanism through which periostin favored EMT in EECs

2 Materials and Methods

21 Sample Collection and Cell Culture Eutopic endome-trium tissues were obtained from 12 women (23ndash41 yearsold menstrual cycle proliferative phase) with endometriosisAll of the participants were at reproductive age had regularmenstruation and received no hormonal therapy at least 6months before the study Endometriosis was visually diag-nosed during the laparoscopy for ovarian cysts and thenascertained by pathological examination All the participantswere from the Department of Obstetrics and GynecologyQilu Hospital of Shandong University from July 2014 to May2015 Informed consent was obtained from all participantsprior to surgeryThe Institutional Review Board of ShandongUniversity approved the study

After collection tissues were immediately washed withPBS to remove blood mucous and debris The EECs wereisolated following the digestion of type IV collagenase(5mgmL) Cell suspension was filtrated through a sterilestainless steel wire mesh (100 120583m) to remove undigestedtissues ESCs were removed by passing a 40120583m sieve Thenthe 40 120583m mesh was washed thoroughly upside down withmedium to get the EECs The medium was collected andcentrifuged at 1000 rpm for 7 minutes After removingthe supernatant cells were then cultured at 37∘C and 5carbon dioxide in Dulbecco modified Eagle medium F-12(DMEMF12 Sigma-Aldrich St Louis Missouri) contain-ing 10 fetal bovine serum (FBS Gibco Australia) and1 antibiotic The purity of EECs was evaluated by cellimmunofluorescence using mouse antihuman keratin (1 50Cell Signaling Technology Danvers Massachusetts) and rab-bit antihuman vimentin (1 50 Cell Signaling Technology)The purity of EECs was over 95 which was shown as theproportion of epithelial cells in 5 randomly selected pictures(200x magnification) These cells were used for the followingexperiments

22 Transwell Migration and Invasion Assays Cells weretreated with periostin (20 ngmL and 40 ngmL) when reach-ing 80 to 90 confluence After treatment for 48 h cellswere digested for migration and invasion assays as previouslydescribed [25] Pictures of stained cells were taken by theOlympus IX51 inverted microscope Cells were counted infive random fields (200x) of each chamber The average cellnumbers of three duplicate assays for each experimentalcondition were used for statistical analysis

23 Silencing of the ILK Gene in EECs The small-interferingRNA sequences targeting human ILK (siRNA-ILK) weredesigned by GenePharma Company (Shanghai China) Cellswere seeded in 6-well plate without antibiotics treated for12 hours and transfected with blank sequence or siRNA-ILK (50 nmolL) using lipofectamine 2000 (Invitrogen LifeTechnologies) when cell confluence gets 50 to 60 After 48hours of transfection cells were digested for the ensuing cellexperiments The siRNA-ILK sequences are listed in Table 1

24 Western Blot Total protein was extracted from sam-ples for western blot as described previously [25] Pri-mary antibodies used for immunodetection were anti-ILK anti-p-Akt anti-E-cadherin anti-N-cadherin anti-vimentin anti-keratin anti-slug and anti-Zeb1 as well asanti-glyceraldehyde-3-phosphate dehydrogenase (GAPDHCell Signaling Technology Danvers Massachusetts USA)Secondary antibodies were anti-rabbit and anti-mouse IgGperoxidase conjugate (Zhongshan Jinqiao BiotechnologyCoLtd Beijing China) GAPDH was used as a loading controlThe results were quantified by densitometry using ImageJsoftware (NIH Bethesda MD USA)

25 Statistical Analysis GraphPad Prism Version 501(GraphPad Software San Diego California USA) wasused for statistical analysis Data were shown as mean plusmnSEM Studentrsquos 119905-test and one-way ANOVA analysis wererespectively conducted to analyze the differences betweengroups and among groups 119875 value lt 005 was consideredstatistically significant

3 Results

31 The Purity of EECs The purity of EECs was956 plusmn 35 as confirmed by cell immunofluorescencewhich was judged by blue fluorescence for cell nucleus greenfluorescence for keratin and red fluorescence for vimentin(Figure 1)

32 Periostin Enhanced the Migration and Invasion Abilities ofEECs In our previous study we detected the concentrationof periostin in peritoneal washing fluids of patients withand without endometriosis which was 4832 ngmL and2229 ngmL respectively (data not published yet) Afterbeing treated with periostin (20 ngmL and 40 ngmL resp)for 48 h cells were digested formigration and invasion assaysAs shown in Figure 2 the migration and invasion abilitiesof EECs were enhanced by the treatment with periostinespecially for the treatment with 40 ngmL periostin




Vimentin Merge

DAPI Keratin






0 20 40

Mig

ratio

nIn

vasio

n

Periostin (ngmL)

(a)

lowast


Periostin(20ngmL)

Fold

chan

ge

Invasion25

20

15

10

05

00

lowastlowast

(b)

Migration


Periostin(20ngmL)

Fold

chan

ge

3

2

1

0

lowastlowast

lowastlowastlowast

(c)



4 Discussion





0 20 40

Periostin (ngmL)

GAPDH

Vimentin

N-cad

Keratin

E-cad

(a)

3

4

2

1

0


lowast

lowast

lowast

lowastlowastlowast

lowastlowast


(b)

0 20 40

Periostin (ngmL)

Akt

Slug

Zeb1

GAPDH

p-Akt

ILK

(c)

3

2

1

0

lowast

lowastlowast

lowast


ILK p-Akt Slug Zeb1

(d)






p-Akt

Slug

Zeb1

Akt

GAPDH



Periostin

ILK

(a)


Con

ILK p-Akt Slug Zeb1

12

10

08

06

04

02

00

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(b)

E-cad

Keratin

N-cad

Vimentin

GAPDH



Periostin

(c)


Con


20

15

10

05

00

lowastlowast

lowast


lowastlowast

(d)






5 Conclusion


Competing Interests


Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Vimentin Merge

DAPI Keratin






0 20 40

Mig

ratio

nIn

vasio

n

Periostin (ngmL)

(a)

lowast


Periostin(20ngmL)

Fold

chan

ge

Invasion25

20

15

10

05

00

lowastlowast

(b)

Migration


Periostin(20ngmL)

Fold

chan

ge

3

2

1

0

lowastlowast

lowastlowastlowast

(c)



4 Discussion





0 20 40

Periostin (ngmL)

GAPDH

Vimentin

N-cad

Keratin

E-cad

(a)

3

4

2

1

0


lowast

lowast

lowast

lowastlowastlowast

lowastlowast


(b)

0 20 40

Periostin (ngmL)

Akt

Slug

Zeb1

GAPDH

p-Akt

ILK

(c)

3

2

1

0

lowast

lowastlowast

lowast


ILK p-Akt Slug Zeb1

(d)






p-Akt

Slug

Zeb1

Akt

GAPDH



Periostin

ILK

(a)


Con

ILK p-Akt Slug Zeb1

12

10

08

06

04

02

00

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(b)

E-cad

Keratin

N-cad

Vimentin

GAPDH



Periostin

(c)


Con


20

15

10

05

00

lowastlowast

lowast


lowastlowast

(d)






5 Conclusion


Competing Interests


Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 20 40

Mig

ratio

nIn

vasio

n

Periostin (ngmL)

(a)

lowast


Periostin(20ngmL)

Fold

chan

ge

Invasion25

20

15

10

05

00

lowastlowast

(b)

Migration


Periostin(20ngmL)

Fold

chan

ge

3

2

1

0

lowastlowast

lowastlowastlowast

(c)



4 Discussion





0 20 40

Periostin (ngmL)

GAPDH

Vimentin

N-cad

Keratin

E-cad

(a)

3

4

2

1

0


lowast

lowast

lowast

lowastlowastlowast

lowastlowast


(b)

0 20 40

Periostin (ngmL)

Akt

Slug

Zeb1

GAPDH

p-Akt

ILK

(c)

3

2

1

0

lowast

lowastlowast

lowast


ILK p-Akt Slug Zeb1

(d)






p-Akt

Slug

Zeb1

Akt

GAPDH



Periostin

ILK

(a)


Con

ILK p-Akt Slug Zeb1

12

10

08

06

04

02

00

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(b)

E-cad

Keratin

N-cad

Vimentin

GAPDH



Periostin

(c)


Con


20

15

10

05

00

lowastlowast

lowast


lowastlowast

(d)






5 Conclusion


Competing Interests


Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 20 40

Periostin (ngmL)

GAPDH

Vimentin

N-cad

Keratin

E-cad

(a)

3

4

2

1

0


lowast

lowast

lowast

lowastlowastlowast

lowastlowast


(b)

0 20 40

Periostin (ngmL)

Akt

Slug

Zeb1

GAPDH

p-Akt

ILK

(c)

3

2

1

0

lowast

lowastlowast

lowast


ILK p-Akt Slug Zeb1

(d)






p-Akt

Slug

Zeb1

Akt

GAPDH



Periostin

ILK

(a)


Con

ILK p-Akt Slug Zeb1

12

10

08

06

04

02

00

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(b)

E-cad

Keratin

N-cad

Vimentin

GAPDH



Periostin

(c)


Con


20

15

10

05

00

lowastlowast

lowast


lowastlowast

(d)






5 Conclusion


Competing Interests


Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















p-Akt

Slug

Zeb1

Akt

GAPDH



Periostin

ILK

(a)


Con

ILK p-Akt Slug Zeb1

12

10

08

06

04

02

00

lowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(b)

E-cad

Keratin

N-cad

Vimentin

GAPDH



Periostin

(c)


Con


20

15

10

05

00

lowastlowast

lowast


lowastlowast

(d)






5 Conclusion


Competing Interests


Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5 Conclusion


Competing Interests


Acknowledgments


References

















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Periostin Facilitates the Epithelial ...signaling pathway. Playing a pivotal role...

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Transcript of Research Article Periostin Facilitates the Epithelial ...signaling pathway. Playing a pivotal role...