Background
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death globally. Half of these deaths were estimated to occur in China [
1]. The prognosis of patients with HCC remains poor despite the therapeutic advances in HCC treatment recently. Therefore, a great challenge lies ahead in the understanding of the molecular mechanisms of hepatocarcinogenesis and the identification of the new biomarkers for HCC that will supply an arm for improving diagnosis and management of human HCC.
Long non-coding RNAs (LncRNAs) are non-protein coding transcripts with a length greater than 200 nucleotides. Accumulating evidence showed that lncRNAs participated in cancer cells biological processes, such as cell growth, cell metastasis, cell differentiation, and fate decision [
2‐
4]. Additionally, many studies demonstrate that lncRNAs play a critical role in tumorigenesis, and their misexpression confers tumor initiation and cancer cell growth and metastasis [
5‐
7]. For example, lncRNA HOTAIR is dysregulated in many cancers [
8,
9]. Moreover, it could promote the invasion-metastasis cascade in cancer cells by binding to PRC2 [
8]. In a word, there has been a heavy focus on the ways that lncRNAs contribute to cancer development. However, their aberrant expression and functional roles in HCC development are still not well-documented.
Among them, lncRNA CDKN2B antisense RNA 1 (ANRIL) is transcribed from the INK4b-ARF-INK4a gene cluster in the opposite direction, which has been identified as a genetic susceptibility locus shared associated by coronary disease, intracranial aneurysm, type 2 diabetes, and also cancers [
10,
11]. Moreover, ANRIL could be induced by ATM-E2F1 signaling pathway and is required for the silencing of p15INK4B by recruiting PRC2 [
12,
13]. In our previous study, we found that ANRIL was overexpressed and played an important role in gastric carcinogenesis and NSCLC development [
14,
15]. However, the functional role and underlying mechanism of ANRIL in HCC remains unclear. Here we investigate the relationship between ANRIL and HCC. We found that ANRIL was up-regulated in HCC tissues than that in corresponding non-tumor tissues, and its up-regulation is related with tumor size and Barcelona Clinic Liver Cancer (BCLC) stage. Moreover, ANRIL could regulate cell growth both in vitro and in vivo via epigenetically silencing KLF2 by binding to PRC2. We also found that Sp1 could regulate the expression of ANRIL. Our results suggest that Sp1-induced ANRIL can regulate KLF2 expression in the epigenetic level and facilitate the development of lncRNA-directed diagnostics and therapeutics of HCC.
Discussion
In recent years, the discovery of lncRNAs, which have emerged as a new and crucial layer of gene regulators, has dramatically altered our understanding of the biology of complex diseases including cancers [
16,
17]. A large number of studies have shown that dysregulated expression of lncRNAs participate in cancer progression and predict patients’ outcome [
18‐
20]. For example, GAS5 can promote the apoptosis of prostate cancer cells and its levels decline as prostate cancer cells acquire castrate-resistance, so that enhancing GAS5 expression may improve the effectiveness of chemotherapies [
6]. In HCC, HULC was the first reported lncRNA that is specifically up-regulated [
21,
22]. A number of lncRNAs, such as MVIH and URHC, have been reported to be involved in HCC development and progression [
21,
23]. In this study, we found another lncRNA ANRIL whose expression is significantly up-regulated in HCC tissues compared with normal tissues. Moreover, increased ANRIL expression was correlated with HCC tumor size and BCLC stage, which suggests that ANRIL may play a key role in HCC development and progression.
Recently, several studies indicated that lncRNA expression could also be regulated by some transcript factors (TF), such as c-myc which could activate HOTAIR transcription, and PVT-1 expression can be regulated by p53 [
24,
25]. ANRIL expression has been reported to be regulated by a key TF E2F1 [
13,
26]; however, in this study, we performed bioinformatics analysis and found that SP1 could also regulate ANRIL transcription in HCC cells. ChIP assay also showed that SP1 could directly bind to ANRIL promoter regions to silence ANRIL transcription. In addition, overexpression of SP1 in HCC cells could up-regulate ANRIL expression, while knockdown of SP1 in HCC cells could down-regulate ANRIL expression. These data showed that ANRIL expression could also be regulated by SP1 in HCC cells, which suggests that one lncRNA may be simultaneously regulated by multiple different transcript factors.
As is known, lncRNAs participated in cancer cells’ biological function, and we found that knockdown of ANRIL could impair HCC cell proliferation and invasion and induce cell apoptosis both in vitro and in vivo. These data suggests that lncRNA ANRIL contributes to HCC development via regulation of cell proliferation and apoptosis. A completely different mode of action is executed by the lncRNA ANRIL to block the activity of tumor suppressor genes. For example, ANRIL interacts with SUZ12 (a subunit of the PRC2) and recruits the complex to repress the expression of p15 (INK4B), a well-known tumor suppressor gene [
13]. A similar study identified chromobox homolog 7 (CBX7), a subunit of the polycomb repressive complex 1 (PRC1) as molecular interaction partner of ANRIL, which results in the recruitment of PRC1 to the p16(INK4A)/p14(ARF) locus and silencing of this gene locus by H3K27 trimethylation [
10]. However, we found that ANRIL could bind with both EZH2 and SUZ12 in HCC cells. Furthermore, bioinformatics analysis indicated that KLF2 could be a new ANRIL downstream target, and knockdown of ANRIL, EZH2 and SUZ12 expression indeed both up-regulated KLF2 expression levels in HCC cells. In addition, ChIP assays also demonstrated that EZH2 could directly bind to KLF2 promoter region and inhibition of ANRIL decreased its binding ability. Our results indicated that ANRIL could repress KLF2 transcription by binding with EZH2 and SUZ12 and recruitment of PRC2 to the KLF2 gene locus in HCC cells.
The Kruppel-like factor (KLF) family which consists of a set of transcription factors that have been identified in diverse organisms functions in cell differentiation and proliferation [
27]. They have been identified as suppressors or activators of different genes in a cell type and promoter-dependent manner [
28]. KLF2 is one of the critical members due to its tumor suppressor function in tumors [
29,
30]. Moreover, previous study showed that EZH2 could directly bind to KLF2 promoter and silence of KLF2 expression result in blocking the tumor-suppressor features of KLF2, which is partly mediated by p21 [
31]. Our data also showed that ANRIL could take part in HCC cell proliferation by silencing KLF2 transcription, and KLF2 overexpression further led to the decreased HCC cell proliferation and increased cell apoptosis. Furthermore, we performed rescue assays to determine whether ANRIL regulates HCC cell proliferation via repressing KLF2 expression. The results of MTT and colony formation assay indicated that co-transfection could partially rescue si-ANRIL-impaired proliferation in HepG2 cells. These data indicate that ANRIL promotes HCC cell proliferation through the down-regulation of KLF2 expression. Our results suggested that lncRNA, especially ANRIL, may influence the same cell biological function via regulating different target genes depending on different cancer cells.
Conclusion
In summary, the expression of ANRIL was significantly up-regulated in HCC tissues and cells, suggesting that its overexpression may be an important factor for HCC progression. We showed that ANRIL may regulate the proliferation ability of HCC cells partially through silencing of the KLF2 by binding with PRC2, which suggested that lncRNAs contribute to different cancer cells’ biological function through regulating different genes. Further insights into the functional and clinical implications of ANRIL and its targets, which are identified as KLF2, may contribute to the understanding of HCC pathogenesis and facilitate the development of lncRNA-directed diagnostics and therapeutics against this disease.
Materials and methods
Patient data and tissue samples
A total of 77 fresh HCC tissue samples and matched normal adjacent tissue samples were selected from patients who underwent resection of HCC at Huai’an First People’s Hospital, Nanjing Medical University (Huai’an, China). The HCC diagnosis was histopathologically confirmed. None of the patients received preoperative therapy. Data from all subjects were obtained from medical records, pathology reports, and personal interviews with the subjects. The collected data included gender, age, drinking state, the history of HBV and cirrhosis, and HCC features (e.g., tumor size, stage). HCC clinical stage was determined according to the BCLC staging classification based on the article by Bruix et al. [
32]. The clinical information for all of the samples is detailed in Table
1. Fresh samples were snap-frozen in liquid nitrogen immediately after resection and stored at −80 °C. Matched non-tumor specimens were obtained from a part of the resected specimen that was farthest from the tumor.
Ethical approval of the study protocol
This study was conducted according to the principles expressed in the Declaration of Helsinki. Tissue specimen collections were made with full informed consent of all patients following institutional ethical guidelines that were reviewed and approved by Huai’an First People’s Hospital, Nanjing Medical University (Huai’an, China).
Cell culture
Human HCC cell lines (HepG2, Hep3B, MHCC-97H) and one normal hepatic epithelial cell line (L02, control) were provided by Dr Beicheng Sun from the Department of Hepatopancreatobiliary, First Affiliated Hospital, Nanjing Medical University (Nanjing City, Jiangsu Province, People’s Republic of China). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO-BRL) medium supplemented with 10 % fetal bovine serum (FBS) at 37 °C in 5 % CO2.
RNA extraction and qRT-PCR analysis
The total RNA was extracted from tissues or cells with TRIzol reagent (Invitrogen, Grand Island, NY, USA), according to the manufacturer’s protocol. One microgram total RNA was reverse transcribed in a final volume of 20 μL under standard conditions using PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China; RR047A). After the RT reaction, 1 μL of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) following the manufacturer’s protocol. The results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The qRT-PCR and data collection were carried out on ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA), and results were analyzed and expressed relative to threshold cycle (CT) values, and then converted to fold changes. All primer sequences are summarized in Additional file
1: Table S1.
Transfection of cell lines
HCC cell lines were transfected with specific siRNA oligonucleotides. To avoid off-target effects and ensure the efficiency of interference, we used an indeed effective interference target sequence of ANRIL, according to the previous study [
12]. EZH2 and SUZ12 siRNA were purchased from Realgene (Nanjing, China). Non-specific siRNA (si-NC) and si-ANRIL were purchased from Invitrogen. Typically, cells were seeded at six-well plates and then transfected the next day with specific siRNA (100 nM) and control siRNA (100 nM) by using Lipofectamine RNAi MAX, according to the manufacturer’s protocol (Invitrogen). EGFP-SP1 was purchased from Add gene. Plasmid vectors (EGFP-SP1, sh-ANRIL pCMV-Tag2B-FLAG-KLF2 and empty vector) for transfection were prepared using DNA Midiprep or Midiprep kits (Qiagen, Hilden, Germany) and transfected into HepG2 and Hep3B cells.
Cell proliferation assays
Cell proliferation was monitored by Cell Proliferation Reagent Kit I (MTT) (Roche, Basel, Switzerland). The transfected cells were plated in 96-well plates (3000 cells/well). Cell proliferation was determined every 24 h following the manufacturer’s protocol. For the colony formation assay, 500 transfected cells were placed into each well of a six-well plate and maintained in DMEM containing 10 % FBS for 12 days, replacing the medium every 4 days. Colonies were fixed with methanol and stained with 0.1 % crystal violet (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 15 min. The colony formation was determined by counting the number of stained colonies. Triplicate wells were measured in each treatment group.
Flow cytometry for cell cycle analysis
HepG2 or Hep3B cells for cell cycle analysis were collected 24 h after transfected with si-ANRIL or respective control and 48 h after transfected with pCMV-Tag2B-KLF2 or empty vector. Then cells were stained with propidium iodide (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.
Flow cytometry for cell apoptosis analysis
HepG2 or Hep3B cells transfected with si-ANRIL, pCMV-Tag2B-KLF2, or respective control were harvested 48 h and then collected. After the double staining with FITC-Annexin V and PI was done using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s protocol, 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 was compared to control transfectant from each experiment.
Cell migration and invasion assays
HepG2 or Hep3B cells transfected with si-ANRIL or respective control were harvested 48 h and then collected. For the migration assays, 5 × 104 cells in serum-free medium were placed into the upper chamber of an insert (8 μm pore size, Millipore). For the invasion assays, 1× 105 cells in serum-free medium were placed into the upper chamber of an insert coated with Matrigel (Sigma-Aldrich). Medium containing 10 % FBS was added to the lower chamber. After incubation for 24 h, we removed the cells remaining on the upper membrane with cotton wool. Cells that had migrated or invaded through the membrane were fixed with methanol, stained with 0.1 % crystal violet, imaged, and counted using an IX71 inverted microscope (Olympus, Tokyo, Japan). Experiments were repeated three times.
Xenograft study
HepG2 cells were transfected with sh-ANRIL or Scramble using Lipofectamine 2000 (Invitrogen). Forty-eight hours later, cells were collected and injected into either side of the posterior flank of the male BALB/c nude mice (4–5 weeks old). Mice were purchased from Shanghai Experimental Animal Center of the Chinese Academy of Sciences. The tumor volumes and weights were measured every 4 days in mice from the control (five mice) or sh-ANRIL (five mice) groups, and tumor volumes were calculated by using the equation V = 0.5 × D × d
2 (V, volume; D, longitudinal diameter; d, latitudinal diameter). Sixteen days after injection, the mice were killed and tumors were collected for further study (weight measure, RNA extraction, and IHC). This study was carried out strictly in 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 Nanjing Medical University.
Immunohistochemistry
Tumors from mice were immunostained for HE, ki-67, and KLF2. The signal was amplified and visualized with 3′-diaminobenzidine chromogen, followed by counterstaining with hematoxylin. Expression was considered to be positive when 50 % or more tumor cells were stained. Anti-ki-67 (1:50) and anti-KLF2 (1:50) were purchased from R&D company.
Western blot assay
Cells were lysed by using mammalian protein extraction reagent RIPA (Beyotime, Haimen, China) supplemented with protease inhibitors cocktail (Roche). Fifty micrograms of the protein extractions were separated by 10 % SDS-PAGE transferred to 0.22-mm nitrocellulose (NC) membranes (Sigma-Aldrich) and incubated with specific antibodies. The autoradiograms were quantified by densitometry (Quantity One software, Bio-Rad, Hercules, CA, USA). Anti-KLF2 was purchased from Sigma (1:1000). Results were normalized to the expression GAPDH (mouse anti-GAPDH) (Sigma (1:1000)).
Subcellular fractionation location
The separation of the nuclear and cytosolic fractions of HCC cell lines was performed according to the protocol of the PARIS Kit (Life Technologies, Carlsbad, CA, USA).
Chromatin immunoprecipitation assays
The ChIP assays were performed by using EZ-ChIP KIT according to the manufacturer’s instruction (Millipore, Billerica, MA, USA). HepG2 and Hep3B cells were treated with formaldehyde and incubated for 10 min to generate DNA-protein cross-links. Cell lysates were then sonicated to generate chromatin fragments of 200–300 bp and immunoprecipitated with EZH2, SUZ12, and H3K27me3-specific antibody (CST) or IgG as control. Precipitated chromatin DNA was recovered and analyzed by qRT-PCR.
RNA immunoprecipitation
RIP experiments were performed by using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the protocol. Antibody for RIP assays of EZH2 and SUZ12 was purchased from Millipore.
Statistical analysis
All statistical analyses were performed by 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 χ
2 test. Two-sided P values were calculated, and differences were considered to be statistically significant at P < 0.05. Kendall’s Tau-b and Pearson correlation analyses were used to investigate the correlation between ANRIL and KLF2 expressions.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (81172140, 81272532), Jiangsu Province Clinical Science and Technology projects (Clinical Research Center, BL2012008), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine, JX10231801). We are very grateful to Dr Beicheng Sun for providing the HCC cell lines and L02 cell line.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
M-dH designed this study, detected the cells’ biological function test, conducted the qRT-PCR assays, carried out the Western blotting assays, established the animal model, performed RIP and ChIP assays, done the statistical analysis, performed the immunohistochemistry assays, and drafted the manuscript. W-mC and FQ provided the tissue samples and the clinical data. RX participated in the design of the study and administrated the data analysis. MS, TX, LY, E-bZ, and WD helped to acquire the experimental data. Y-qS conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.