Long non coding RNA ANRIL promotes non small cell lung ......Dec 27, 2014 · 1 Long non-coding RNA...
Transcript of Long non coding RNA ANRIL promotes non small cell lung ......Dec 27, 2014 · 1 Long non-coding RNA...
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Long non-coding RNA ANRIL promotes non small cell lung cancer cells proliferation and
inhibits apoptosis by silencing KLF2 and P21 expression
Feng-qi Nie1#
, Ming Sun2#
, Jin-song Yang3#
, Min Xie2, Tong-peng Xu
1, Rui Xia
2, Yan-wen
Liu2, Xiang-hua Liu
2, Er-bao Zhang
2, Kai-hua Lu
1* and Yong-qian Shu
1*
1Department of Oncology, First Affiliated Hospital, Nanjing Medical University, Nanjing,
People’s Republic; 2Department of Biochemistry and Molecular Biology, Nanjing Medical
University, Nanjing, People’s Republic of China;3Department of Oncology, Nanjing First
Hospital, Nanjing Medical University, P. R. China.
*Corresponding author:
Kai-hua Lu, Department of Oncology, First Affiliated Hospital, Nanjing Medical University,
Nanjing, People’s Republic,Tel:+86-025 68136711 ; Fax: +86-025 68136711; E-mail:
Yong-qian Shu, Department of Oncology, First Affiliated Hospital, Nanjing Medical
University, Nanjing, People’s Republic, E-mail: [email protected]
#These authors contributed equally to this work and should be regarded as joint first
authors
Ruining title: ANRIL promotes proliferation in NSCLC
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant Support
This work was supported by grants from the National Natural Scientific Foundation of
China No. 81372397 to KH. Lu, 81301824 to XH. Liu, 81172140 and 81272532 to YQ Shu;
M. Sun was supported by a Jiangsu province ordinary university graduate student
research innovation project for 2013 (CXZZ13_0562, JX22013265).
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Abstract
Recent evidence highlight long non-coding RNAs (lncRNAs) as crucial regulators of
cancer biology, which contribute to essential cancer cell functions such as cell proliferation,
apoptosis and metastasis. In non small cell lung cancer (NSCLC), several lncRNAs
expression are misregulated and have been nominated as critical actors in NSCLC
tumorigenesis. LncRNA ANRIL was firstly found to be required for the PRC2 recruitment
to and silencing of p15INK4B
, which expression is induced by ATM-E2F1 signaling pathway.
Our previous study showed that ANRIL was significantly up-regulated in gastric cancer,
and it could promote cell proliferation and inhibit cell apoptosis by silencing of miR-99a
and miR-449a transcription. However, its clinical significance and potential role in NSCLC
is still not documented. In this study, we reported that ANRIL expression was increased in
NSCLC tissues and Its expression level was significantly correlated with TNM stages and
tumor size. Moreover, patients with high levels of ANRIL expression had a relatively poor
prognosis. In addition, taking advantage of loss of function experiments in NSCLC cells,
we found that knockdown of ANRIL expression could impair cell proliferation and induce
cell apoptosis both in vitro and vivo. Furthermore, we uncover that ANRIL could not
repress p15 expression in PC9 cells, but through silencing of KLF2 and P21 transcription.
Thus, we conclusively demonstrate that lncRNA ANRIL plays an key role in NSCLC
development by associating its expression with survival in NSCLC patients, providing
novel insights on the function of lncRNA-driven tumorigenesis.
Key words: NSCLC; ANRIL; cell proliferation; KLF2; P21
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Introduction
Lung cancer is the most common type of cancer and the primary cause of cancer
death worldwide (1). NSCLC accounts for 80% of all lung cancer cases, represents the
most prevalent class of this cancer type and includes several histological subtypes such
as squamous cell carcinoma (SCC), adenocarcinoma and large cell carcinoma (LCC) (2,
3). In spite of current advances in the treatments for NSCLC, including surgical therapy,
chemotherapy and molecular targeting therapy, the overall 5-year survival rate for NSCLC
patients have not been markedly improved over years and remains as low as 15% (4).
Therefore, a greater understanding of the molecular mechanisms involved in the
development, progression and spread of the NSCLC is essential for the developing of
specific diagnostic methods and designing of more individualized and effective
therapeutic strategies.
Recently, studies using the great advances in genomic technologies have revealed
the majority of the human genome is transcribed, whereas only 2% of the transcribed
genome codes for protein (5). Meanwhile, it is becoming increasingly apparent that the
large majority of genome is transcribed into non-coding RNAs (ncRNAs) including
microRNAs and long non-coding RNAs (lncRNAs) (6). The ENCODE Consortium has
elucidated the prevalence of thousands of human lncRNAs, but only very few of them
have been assigned with any biological function (7). To date, studies showed that miRNAs
play important roles in the post-transcriptional regulation of gene expression; however, the
lncRNAs counterpart of ncRNA is not well characterized (8). Although very few are
characterized in detail, LncRNAs are involved in a large range of biological processes,
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including modulation of apoptosis and invasion, reprogramming stem cell pluripotency,
and parental imprinting through the regulation of gene expression by chromatin
remodeling, histone protein modification, regulation of mRNA splicing and acting as
sponges for microRNAs (9-12).
In the past decade, lots of evidence have linked the dysregulation of lncRNAs with
diverse human diseases, in particular cancers (13-15). Therefore, identification of
cancer-associated lncRNAs and investigation of their molecular and biological functions
are important in understanding the molecular biology of NSCLC development and
progression. Our previous study showed that lncRNA ANRIL was significantly
up-regulated in gastric cancer, and increased ANRIL promoted gastric cancer cells
proliferation and inhibited apoptosis by epigenetic silencing of miR-99a and miR-449a
transcription (16). Moreover, ANRIL can bind to and recruits PRC2 to repress the
expression of p15INK4B locus, which resulted in increased cell proliferation (17, 18).
However, the ANRIL clinical significance and potential role in NSCLC development and
progression is still not documented.
In this study, we found that lncRNA ANRIL expression was increased in NSCLC
tissues compared with adjacent normal tissues. Its expression level was significantly
correlated with TNM stages and tumor size. Moreover, patients with higher level of ANRIL
expression had a relatively poor prognosis. Furthermore, we investigated the effects of
ANRIL expression on NSCLC cell phenotype in vitro and in vivo with loss of function study.
Moreover, we also showed that ANRIL could bind to PRC2 to repress KLF2 and P21
transcription, but not regulate P15INK4
expression in NSCLC PC9 cells, which indicated
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that ANRIL affected NSCLC cells proliferation and apoptosis partly via silencing of KLF2
and P21 transcription. This study advances our understanding of the role of lncRNAs,
such as a regulator of pathogenesis of NSCLC and facilitates the development of
lncRNA-directed diagnostics and therapeutics.
Materials and Methods
Tissue collection
We obtained 68 paired NSCLC and adjacent non-tumor lung tissues from patients who
underwent surgery at Jiangsu Province Hospital between 2010 and 2011, and were
diagnosed with NSCLC based on histopathological evaluation. Clinicopathological
characteristics, including tumor-node-metastasis (TNM) staging, were recorded. No local
or systemic treatment was conducted in these patients before surgery. All collected tissue
samples were immediately snap-frozen in liquid nitrogen and stored at –80°C until
required. Our study was approved by the Research Ethics Committee of Nanjing Medical
University, China. Written informed consent was obtained from all patients.
Cell lines
Five NSCLC adenocarcinoma cell lines (PC9, SPC-A1, NCI-H1975, H1299, and
H358), and one NSCLC squamous carcinomas cell lines (H520) were purchased from the
Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai,
China). A549, H1975, H1299 and H520 cells were cultured in RPMI 1640; 16HBE, PC9
and SPC-A1 cells were cultured in DMEM (GIBCO-BRL) medium supplemented with 10%
fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen,
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Carlsbad, CA, USA) at 37ºC/5% CO2. All cell lines were authenticated by short tandem
repeat DNA profiling.
RNA extraction and qPCR assays
Total RNA was isolated with Trizol reagent (Invitrogen) according to the
manufacturer’s instructions. Total RNA (500 ng) was reverse transcribed in a final volume
of 10 µl using random primers under standard conditions for the PrimeScript RT reagent
Kit (TaKaRa, Dalian, China). We used the SYBR Premix Ex Taq (TaKaRa, Dalian, China)
to determine ANRIL expression levels, following the manufacturer’s instructions. Results
were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). The specific primers used are shown in Supplementary table S1. The qPCR
assays were conducted on an ABI 7500, and data collected with this instrument. Our
qPCR results were analyzed and expressed relative to threshold cycle (CT) values, and
then converted to fold changes.
Cell transfection
Human ANRIL cDNA clone L6ChoCKO-2-E10 with functional region was provided by
the Functional Genomics Research Center, KRIBB, Korea. Plasmid vectors
(pCNS-ANRIL, sh-ANRIL and empty vector) for transfection were prepared using DNA
Midiprep or Midiprep kits (Qiagen, Hilden, Germany), and transfected into16HBE, SPC-A1,
H1299 or PC9cells. The si-ANRIL or si-NC were transfected into SPC-A1, H1299 or PC9
cells. SPC-A1, H1299 or PC9 cells were grown on six-well plates to confluency and
transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s
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instructions. At 48 h post-transfection, cells were harvested for qPCR or western blot
analysis.
Cell viability assays
Cell viability was monitored using a Cell Proliferation Reagent Kit I (MTT) (Roche
Applied Science). The SPC-A1, H1299, PC9 or A549 cells transfected with si-ANRIL
(3000 cells/well) were grown in 96-well plates. Cell viability was assessed every 24 h
following the manufacturer’s protocol. All experiments were performed in quadruplicate.
For each treatment group wells were assessed in triplicate.
Flow cytometry
SPC-A1, H1299 or PC9 cells transfected with si-ANRIL were harvested 48 hr after
transfection by trypsinization. After the double staining with FITC-Annexin V and
Propidium iodide (PI) was done using the FITC Annexin V Apoptosis Detection Kit (BD
Biosciences) according to the manufacturer’s recommendations, the cells were analyzed
with a flow cytometry (FACScan®; BD Biosciences) equipped with a CellQuest software
(BD Biosciences). Cells were discriminated into viable cells, dead cells, early apoptotic
cells, and apoptotic cells, and then the relative ratio of early apoptotic cells were
compared to control transfectant from each experiment. Cells for cell cycle analysis were
stained with PI using the CycleTEST™ PLUS DNA Reagent Kit (BD Biosciences)
following the protocol and analyzed by FACScan. The percentage of the cells in G0/G1, S,
and G2/M phase were counted and compared.
Tumor formation assay in a nude mouse model
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Female athymic BALB/c nude mice (4-weeks-old) were maintained under pathogen-free
conditions and manipulated according to protocols approved by the Shanghai Medical
Experimental Animal Care Commission. PC9 cells were stably transfected with sh-ANRIL
and empty vector and harvested from 6-well cell culture plates, washed with
phosphate-buffered saline (PBS), and re-suspended at a concentration of 1 × 108 cells/ml.
A total of 100 µL of suspended cells was subcutaneously injected into a single side of the
posterior flank of each mouse. Tumor growth was examined every 3 days, and tumor
volumes were calculated using the equation V = 0.5 × D × d2 (V, volume; D, longitudinal
diameter; d, latitudinal diameter). At 18 days post-injection, mice were euthanized, and
the subcutaneous growth of each tumor was examined. This study was carried out in strict
accordance with the recommendations in the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health. The protocol was approved by the Committee
on the Ethics of Animal Experiments of the Nanjing medical University.
RNA immunoprecipitation
For immunoprecipitation (IP) of endogenous PRC2 complexes from whole-cell extracts,
cells were lysed. The supernatants were incubated with protein A Sepharose beads
coated with antibodies that recognized EZH2, SNRNP70 or with control IgG (millipore) for
6hr at 4℃. After the beads were washed with wash buffer, the complexes were incubated
with 0.1% SDS/0.5 mg/ml Proteinase K (30 min at 55℃) to remove proteins, respectively.
The PRC2 isolated from the IP materials was further assessed by qPCR analysis (19).
Chromatin Immunoprecipitation
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PC9 cells were treated with formaldehyde and incubated for 10 mins to generate
DNA-protein cross-links. Cell lysates were then sonicated to generate chromatin
fragments of 200-300 bp and immunoprecipitated with EZH2 and H3K27me3-specific
antibody (CST) or IgG as control. Precipitated chromatin DNA was recovered and
analyzed by qPCR.
Western blot assay and antibodies
Cells protein lysates were separated by 10% SDS-polyacrylamide gel electrophoresis
(SDS-PAGE), transferred to 0.22μm NC membranes (Sigma)and incubated with specific
antibodies. ECL chromogenic substrate was used to were quantified by densitometry
(Quantity One software; Bio-Rad). GAPDH antibody was used as control, Anti-P21, CDK2,
CDK4, CDK6, P15 and PARP (1:1000) were purchased from Cell Signaling Technology,
Inc (CST); Anti-KLF2 were purchased from sigma.
Statistical analysis
All statistical analyses were performed using SPSS 17.0 software (IBM, Chicago, IL, USA).
The significance of differences between groups was estimated by the Student t-test,
Wilcoxon test or χ2 test. DFS and OS rates were calculated by the Kaplan–Meier method
with the log-rank test applied for comparison. The date of survival were evaluated by
univariate and multivariate Cox proportional hazards models. Variables with p < 0.05 in
univariate analysis were used in subsequent multivariate analysis on the basis of Cox
regression analyses. Kendall’s Tau-b and Pearson correlation analyses were used to
investigate the correlation between ANRIL and KLF2 expressions. Two-sided p-values
were calculated, and a probability level of 0.05 was chosen for statistical significance.
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Results
ANRIL expression was up-regulated and correlated with poor prognosis of NSCLC.
ANRIL expression levels were investigated in 68 paired NSCLC samples and
adjacent histologically normal tissues using qPCR assays. ANRIL expression was
significantly up-regulated (Fold change >1.5, P < 0.01) in 76% (52/68) of cancerous
tissues compared with normal tissues (Fig.1A); the ANRIL expression level in each patient
was shown in Supplementary table S2. Increased ANRIL expression levels in NSCLC
were significantly correlated with tumor size (p = 0.001), and advanced pathological stage
(p=0.007). However, ANRIL expression was not associated with other parameters such
as gender (p = 0.625) and age (p = 0.627) in NSCLC (Table1).
To investigate whether up-regulation of ANRIL is caused by DNA copy number
variation, we referred to array comparative genomic hybridization (aCGH) database in
GSE20393, where deposits 52 lung cancer copy number alteration data generated by
Agilent Human Genome CGH 244A Microarrays. We investigated 26 probes representing
region of ANRIL and extracted GLAD segmented copy number of these probes. The
results showed that there is no significant gain of DNA copy number in this region,
suggesting that up-regulation of ANRIL in lung cancer is not due to copy number variation
(Supplementary Fig. S1A).
Association of ANRIL expression with patients survival
Kaplan-Meier survival analysis was conducted to investigate the correlation between
ANRIL expression and NSCLC patient prognosis. According to relative ANRIL expression
in tumor tissues, the 68 NSCLC patients were classified into two groups: the high ANRIL
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group (n = 34, fold-change ≤ mean ratio); and the low ANRIL group (n = 34, fold-change
≥mean ratio) (Fig. 1B). With respect to progression-free survival (PFS), this was 35.3%
for the high ANRIL group, and 13.6% for the low ANRIL group. Median survival time for
the high ANRIL group was 31 months, and 14 months for the low ANRIL group (Fig. 1C).
The overall survival rate over 3 years for the high ANRIL group was 44.4%, and 20.8% for
the low ANRIL group. Median survival time for the high ANRIL group was 32 months, and
18 months for the low ANRIL group (Fig. 1D).
Univariate analysis identified three prognostic factors: lymph node metastasis; TNM
stage; and ANRIL expression level. Other clinicopathological features such as gender and
age were not statistically significant prognosis factors (Supplementary Table S3).
Multivariate analysis of the three prognosis factors confirmed that HR for ARAIL
expression is 3.509 (95%CI: 1.619-7.607) of progression-free survival, indicating that
ANRIL expression may serve as a potential independent prognostic value in NSCLC
(Supplementary Table S4).
Modulation of ANRIL expression in NSCLC cells
We next performed qPCR analysis to examine the expression of ANRIL in 6 human
NSCLC cell lines, including both adenocarcinoma and squamous carcinoma subtypes
(Fig. 2A). To investigate the functional effects of ANRIL in NSCLC cells, we modulated its
expression through RNA interference. QPCR analysis of ANRIL levels was performed 48
h post-transfection. ANRIL expression was knocked down by 74% in SPC-A1 cells, 75%
in H1299 cells and 94% in PC9 cells by si-ANRIL transfection when compared with control
cells (si-NC) (Fig. 2B).
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Knockdown of ANRIL impaired NSCLC cells proliferation and induced apoptosis
To assess the role of ANRIL in NSCLC, we investigated the effect of targeted
knockdown of ANRIL on cell proliferation. MTT assays revealed that cell growth was
inhibited in SPC-A1, H1299 and PC9 cells transient transfected with si-ANRIL compared
with controls (Fig. 2C). Meanwhile, knockdown of ANRIL expression could also inhibit
A549 cells (with relative low endogenous ANRIL expression level) proliferation
(Supplementary Fig. S1B). Colony formation assay results revealed that clonogenic
survival was inhibited following down-regulation of ANRIL in SPC-A1, H1299 and
PC9cells (Fig. 2D). However, over-expression of ANRIL in 16HBE cells showed no
significant effect on cell proliferation (Supplementary Fig. S1C).
To further examine whether the effect of knockdown ANRIL on proliferation of
NSCLC cells reflected cell cycle arrest, cell cycle progression was analyzed by flow
cytometry analysis. The results revealed that SPC-A1 and PC9 cells transfected with
si-ANRIL had an obvious cell cycle arrest at the G1/G0 phase and had a decreased G2/S
phase (Fig. 3A). To determine whether NSCLC cell proliferation was influenced by cell
apoptosis, we performed flow cytometry and Tunel staining analysis. The results showed
that NSCLC cells transfected with ANRIL siRNA promoted apoptosis in comparison with
that in control cells (Fig. 3B and 2C). These data indicate that ANRIL could promote the
proliferation phenotype of NSCLC cells.
Decreased ANRIL expression inhibits NSCLC cells migration
To investigate the effect of ANRIL knockdown on NSCLC cells migration, transwells
assays were performed. The results showed that decreased ANRIL expression levels
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impeded the migration of SPC-A1 and PC9 cells compared with controls (Supplementary
Fig. S1D).
Down-regulation of ANRIL inhibits NSCLC cells tumorigenesis in vivo.
To explore whether the level of ANRIL expression could affect tumorigenesis, PC9 cells
stably transfected with sh-ANRIL or empty vector were inoculated into female nude mice.
Eighteen days after the injection, the tumors formed in the sh-ANRIL group were
substantially smaller than those in the control group (Fig. 4A). Moreover, the mean tumor
weight at the end of the experiment was markedly lower in the sh-ANRIL group (0.62 ±
0.35 g) compared with the empty vector group (1.41± 0.57 g) (Fig. 4B). QPCR analysis
found that the levels of ANRIL expression in tumor tissues formed from sh-ANRIL cells
were lower than in tumors formed in the control group (Fig. 4C). Tumors formed from
sh-ANRIL-transfected PC9 cells exhibited decreased positive for Ki67 than those from
control cells (Fig. 4D). These findings indicate that knockdown of ANRIL inhibits tumor
growth in vivo.
ANRIL silences KLF2 and P21 transcription by binding with EZH2
Previously studies have indicated that ANRIL could silence p15INK4
transcription and
contribute to cancer cells proliferation (18). The results of qPCR showed that p15 and p16
expression was increased in SPCA1 and H1299 cells with transfection of si-ANRIL;
however, there was no significant difference of p15 expression in PC9 cells when
knockdown of ANRIL expression (Fig. 5A and Supplementary Fig. S1E). There are
evidence showed that EZH2 could regulate KLF2 and P21 expression (20, 21), and our
qPCR results also showed that inhibition of ANRIL expression led to increased KLF2 and
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P21 expression. Moreover, knockdown of EZH2 or SUZ12 could also up-regulate KLF2
and P21 expression (Fig. 5B). Meanwhile, the western blot assays showed the same
results (Fig. 5C), which indicated that KLF2 and P21 could be ANRIL novel targets in PC9
cells. Additionally, we found that ANRIL RNAs were mostly located in the nucleus (Fig.
5D).
To further investigate whether ANRIL repress KLF2 and P21 expression through
binding PRC2, we performed RIP analysis and the results showed that ANRIL could
directly bind with EZH2 in PC9 cells (Fig. 6A). Furthermore, the results of ChIP assays
showed that EZH2 could directly bind to KLF2 and P21 promoter region and mediate
H3K27me3 modification (Fig. 6B). However, knockdown of ANRIL reduced EZH2 binding
with KLF2 and P21 promoter (Fig. 6C). Finally, we detected the KLF2 expression in
NSCLC tissues, and found that there is an inverse relationship between ANRIL and KLF2
expression (Fig. 6D). These data suggested that ANRIL promotes NSCLC PC9 cells
proliferation is not dependent on regulation p15 expression, but also through silencing of
KLF2 and P21 transcription.
Silence of KLF2 is potentially involved in the oncogenic function of ANRIL.
To investigate whether KLF2 is involved in the ANRIL-induced increase in NSCLC
cell proliferation, we performed gain of function assays. The results of western blot
showed that KLF2 expression was significantly up-regulated in PC9 cells transfected with
pCDNA-KLF2 compared with control cells (Fig. 7A). Meanwhile, MTT and colony
formation assay results revealed that over-expression of KLF2 could impaired NSCLC
cells proliferation (Fig. 7B). Moreover, flow cytometry analysis indicated that increased
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KLF2 expression could induce NSCLC cells apoptosis (Fig. 7C). Furthermore, to
determine whether ANRIL regulate NSCLC cell proliferation via repressing KLF2
expression, rescue assays were performed. PC9 cells were co-transfected with si-ANRIL
and si-KLF2, and this was shown to rescue the decreased expression of ANRIL induced
by knockdown of KLF2 (Fig. 7D). The results of MTT and colony formation assay results
indicated that co-transfection could partially rescue si-ANRIL-impaired proliferation in PC
cells (Fig. 7E). These data indicate that ANRIL promotes NSCLC cell proliferation through
the down-regulation of KLF2 expression.
Discussion
Recently, numerous pieces of evidence show that many lncRNAs are characterized
and play important roles in cancer pathogenesis, suggesting that they could provide new
insights into the biology of this disease. For example, increased lncRNA HOTTIP is
associated with progression and predicts outcome in hepatocellular carcinoma patients by
regulating HOXA13 expression (22). However, the roles of lncRNAs in NSCLC are still not
well documented, and one of these lncRNAs is metastasis-associated lung
adenocarcinoma transcript 1 (MALAT1), which is a highly conserved nuclear lncRNA and
a predictive marker for metastasis development in lung cancer (23). In our previous
studies, we found that increased lncRNA HOTAIR promoted NSCLC cells invasion and
metastasis via regulating HOXA5 expression, and lncRNA BANCR over-expression could
impaired NSCLC cells proliferation and metastasis by affecting epithelial-mesenchymal
transition (24, 25).
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In this study, we demonstrated that the expression of another lncRNA, ANRIL, is
significantly up-regulated in NSCLC tissues. Specifically, increased ANRIL expression
appears to be a significant, independent predictive value for NSCLC patients. Moreover,
knockdown of ANRIL expression led to the significant inhibition of cell proliferation and the
promotion of apoptosis both in vitro and in vivo. These findings suggest that ANRIL plays
a direct role in the modulation of cell proliferation and NSCLC progression, and could be a
useful novel prognostic or progression marker for NSCLC. As more and more lncRNAs
are studied, many have been shown to function by binding with PRC2 and silencing
downstream target genes that involved in multiple cancers including NSCLC (26, 27).
ANRIL has been reported to involve in cancer cells proliferation by silencing p15INK4
expression. In this study, we found that ANRIL is mostly located in cell nucleus and could
directly bind with EZH2, an core subunit of PRC2, resulted in repressing KLF2 and P21
transcription. However, knockdown of ANRIL could not influence p15INK4
expression in
NSCLC PC9 cells, which indicated that ANRIL contributed to NSCLC cell proliferation is
not dependent on regulating p15INK4
, but also could through silencing KLF2 and P21
transcription.
The Kruppel-like factor (KLF) family transcription factors, with Cys2/His2 zinc-finger
domains, could function as suppressors or activators in a cell type and
promoter-dependent manner and involve in cell differentiation and proliferation (28, 29).
Some KLF members are emerging as tumor suppressor genes due to their roles in the
inhibition of proliferation, migration, and induction of apoptosis (30, 31). KLF2, as an
member of KLF family, is diminished in many cancers and possesses tumor-suppressor
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features such as inhibition of cell proliferation mediated by KRAS (32-34). Moreover, there
is evidence showed that EZH2 could silence KLF2 expression and block the
tumor-suppressor features of KLF2, which is partly mediated by p21 (21). Our results also
showed lncRNA ANRIL take part in NSCLC cells proliferation by epigenetic silencing
KLF2 and P21 transcription, and KLF2 inactivation further led to the decreased P21
expression. As more and more studies indicated that lncRNAs are often expressed in a
spatial- or temporal-specific pattern, and more cell and tissue -specific pattern. Our results
also showed that even in NSCLC cells, lncRNA ANRIL could regulate different target
genes in different cell lines, which suggested that lncRNA, especially ANRIL, can
influence the same cell biological function via regulating differnet target genes dependent
on different cell lines.
To date, although only a small number of lncRNAs have been well characterized, they
have been shown to regulate gene expression at various levels, including chromatin
modification and post-transcriptional processing (35, 36). Here, the possible other targets
and mechanism that underlie such regulatory behaviors still remain to be fully understood
despite our observation of ANRIL-induced NSCLC cell proliferation. In summary, the
expression of ANRIL was significantly increased in NSCLC tissues, suggesting that its
up-regulation may be a negative prognostic factor for NSCLC patients, indicative of poor
survival rates, and a higher risk for cancer metastasis. We showed that ANRIL possibly
regulates the proliferation ability of NSCLC cells, partially through its regulation of the
KLF2 and P21, which indicated that lncRNAs contribute to different cancer cells biological
function maybe through regulating different target genes. Our findings further the
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18
understanding of NSCLC pathogenesis, and facilitate the development of
lncRNA-directed diagnostics and therapeutics against cancers.
Acknowledgements
We are very grateful to professor Xiongbin Lu for providing the ANRIL over-expression
plamid.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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Table 1 Correlation between ANRIL expression and clinicopathological characteristics of
NSCLC patients
Characteristics ANRIL P
High No. cases
(34)
Low No. cases
(34)
Chi-squared
test P-value
Age(years) 0.627
≤65 17 19
>65 17 15
Gender 0.625
Male 18 20
Female 16 14
Histological subtype 0.324
Squamous cell
carcinoma
22 18
Adenocarcinoma 12 16
TNM Stage 0.007*
Ia + Ib 4 15
IIa + IIb 14 12
IIIa 16 7
Tumor size 0.001*
≤5cm 13 26
>5cm 21 8
Lymph node
metastasis
0.051
Negative 11 19
Positive 23 15
Smoking History 0.793
Smokers 23 24
Never Smokers 11 10
* Overall P<0.05
Figure legends
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21
Figure 1. Relative ANRIL expression in NSCLC tissues and its clinical significance.
(A) Relative expression of ANRIL in NSCLC tissues (n = 68) compared with
corresponding non-tumor tissues (n = 68). ANRIL expression was examined by qPCR and
normalized to GAPDH expression. Results were presented as the fold-change in tumor
tissues relative to normal tissues. (B) ANRIL expression was classified into two groups. (C,
D) Kaplan–Meier disease-free survival and overall survival curves according to ANRIL
expression levels. *P < 0.05, **P < 0.01.
Figure 2. Effects of knockdown of ANRIL on NSCLC cell viability and apoptosis in
vitro. (A) ANRIL expression levels of NSCLC cell lines (PC9, SPC-A1, NCI-H1975,
H1299, and H358 and H520) compared with that in normal human bronchial epithelial
cells (16HBE). (B) SPC-A1, H1299 and PC9 cells were transfected with si-ANRIL. (C)
MTT assays were used to determine the cell viability for si-ANRIL-transfected SPC-A1,
H1299 and PC9 cells. Values represented the mean ± s.d. from three independent
experiments. (D) Colony-forming assays were conducted to determine the proliferation of
si-ANRIL-transfected SPC-A1, H1299 and PC9 cells. Flow cytometry assays were
performed to analysis the cell cycle progression and apoptosis when NSCLC cells
transfected with si-ANRIL. *P<0.05, **P<0.01.
Figure 3. Effects of knockdown of ANRIL on NSCLC cell apoptosis in vitro. (A) The
bar chart represented the percentage of cells in G0/G1, S, or G2/M phase, as indicated.
(B) Apoptosis was determined by flow cytometry. UL, necrotic cells; UR, terminal
apoptotic cells; LR, early apoptotic cells. (C) Apoptosis was determined by Tunel staining.
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22
All experiments were performed in biological triplicates with three technical
replicates.*P<0.05, **P<0.01.
Figure 4. Effects of down-regulation of ANRIL on tumor growth in vivo. (A) The
tumor volume was calculated once every three days after injection of PC9 cells stably
transfected with sh-ANRIL or empty vector. Points, mean (n=7); bars indicate S.D. (B)
Tumor weights are represented as means of tumor weights ± s.d. (C) QPCR analysis of
ANRIL expression in tumor tissues formed from PC9/sh-ANRIL, PC9/empty vector. (D).
Tumors developed from sh-ANRIL transfected PC9 cells showed lower Ki67 protein levels
than tumors developed by control cells. Upper: H & E staining; Lower: immunostaining.
*P<0.05, **P<0.01 and N.S. not significant.
Figure 5. ANRIL could silence KLF2 and P21 expression. (A) The levels of p15INK4B
,
p21, and KLF2 mRNA were determined by qPCR when SPC-A1 and PC9 cells
transfected with si-ANRIL and results are expressed relative to the corresponding values
for control cells. (B,C) The levels of p21 and KLF2 mRNA and protein levels were
determined by qPCR when PC9 cells transfected with si-EZH2 or si-SUZ12. (D) ANRIL
expression levels in cell cytoplasm or Nucleus of NSCLC cell lines SPC-A1, PC9 and
H520 were detected by qPCR.
Figure 6. ANRIL could directly bind PRC2 and silence KLF2 and P21 transcription.
(A) RIP with rabbit monoclonal anti-EZH2, preimmune IgG or 10% input from PC9 cell
extracts. RNA levels in immunoprecipitates were determined by qPCR. Expression levels
of ANRIL RNA were presented as fold enrichment in EZH2 relative to IgG
immunoprecipitates; relative RNA levels of U1 snRNA in SNRNP70 relative to IgG
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23
immunoprecipitates were used as positive control. (B, C) ChIP–qPCR of EZH2 occupancy
and H3K27-3me binding in the KLF2 promoter in PC9 cells, and IgG as a negative control;
ChIP–qPCR of EZH2 occupancy and H3K27-3me binding in the KLF2 promoter in PC9
cells treated with ANRIL siRNA (48 h) or scrambled siRNA. (D) Analysis of the
relationship between ANRIL expression and KLF2 mRNA level (△Ct value) in 40 NSCLC
tissues. The mean values and s.d.s were calculated from triplicates of a representative
experiment.
Figure 7. Over-expression of KLF2 expression inhibit PC9 cells proliferation. PC9
cells were transfected with pCDNA-KLF2 or co-transfected with si-ANRIL and si-KLF2. (A)
The protein level of KLF2 in PC9 cell transfected with pCDNA-KLF2 was detected by
western blot. (B) MTT assays and colony-forming assays were used to determine the cell
viability for pCDNA-KLF2-transfected PC9 cells. Values represent the mean ± s.d. from
three independent experiments. (C) Apoptosis was determined by flow cytometry. UL,
necrotic cells; UR, terminal apoptotic cells; LR, early apoptotic cells. (D) The protein level
of KLF2 in PC9 cell co-transfected with si-ANRIL and si-KLF2 was detected by western
blot. (E) MTT assays and colony-forming assays were used to determine the cell viability
for si-ANRIL and si-KLF2 co-transfected PC9 cells. Values represent the mean ± s.d. from
three independent experiments. *P < 0.05 and **P < 0.01
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Published OnlineFirst December 12, 2014.Mol Cancer Ther Feng-qi Nie, Ming Sun, Jin-song Yang, et al. KLF2 and P21 expressioncancer cells proliferation and inhibits apoptosis by silencing Long noncoding RNA ANRIL promotes non small cell lung
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