Long non-coding RNA ANRIL is upregulated in hepatocellular carcinoma and regulates cell apoptosis by epigenetic silencing of KLF2

Background Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death, especially in China. And the mechanism of its progression remains poorly understood. Growing evidence indicates that long non-coding RNAs (lncRNAs) are found to be dysregulated in many cancers, including HCC. ANRIL, a lncRNA co-clustered mainly with p14/ARF has been reported to be dysregulated in gastric cancer, esophageal squamous cell carcinoma, and lung cancer. However, its clinical significance and potential role in HCC are still not documented. Methods and results In this study, expression of ANRIL was analyzed in 77 HCC tissues and matched normal tissues by using quantitative polymerase chain reaction (qRT-PCR). ANRIL expression was upregulated in HCC tissues, and the higher expression of ANRIL was significantly correlated with tumor size and Barcelona Clinic Liver Cancer (BCLC) stage. Moreover, taking advantage of loss-of-function experiments in HCC cells, we found that knockdown of ANRIL expression could impair cell proliferation and invasion and induce cell apoptosis both in vitro and in vivo. We also found that ANRIL could epigenetically repress Kruppel-like factor 2 (KLF2) transcription in HCC cells by binding with PRC2 and recruiting it to the KLF2 promoter region. We also found that SP1 could regulate the expression of ANRIL. Conclusion Our results suggest that lncRNA ANRIL, as a growth regulator, may serve as a new biomarker and target for therapy in HCC. Electronic supplementary material The online version of this article (doi:10.1186/s13045-015-0146-0) contains supplementary material, which is available to authorized users.


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.
lncRNAs are non-protein-coding transcripts with a length greater than 200 nucleotides. Accumulating evidence showed that lncRNAs participated in cancer cell biological processes, such as cell growth, cell metastasis, cell differentiation, and fate decision [2][3][4]. Additionally, many studies demonstrate that lncRNAs play a critical role in tumorigenesis, and their misexpression confers tumor initiation, cancer cell growth, and metastasis [5][6][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 ANRIL (CDKN2B antisense RNA 1) is transcribed from the INK4b-ARF-INK4a gene cluster in the opposite direction, which has been identified as a shared genetic susceptibility locus associated with coronary disease, intracranial aneurysm, type 2 diabetes, and also cancers [10,11]. Moreover, ANRIL could be induced by the 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 remain unclear. Here we investigate the relationship between ANRIL and HCC. We found that ANRIL was upregulated in HCC tissues than in corresponding non-tumor tissues and its upregulation 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 epigenetic silencing of Kruppel-like factor 2 (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.

Results
ANRIL is upregulated in hepatocellular carcinoma tissues and is associated with tumor size and BCLC stage ANRIL expression was significantly upregulated in 75.32% (58 of 77, fold ≥1.0) of tumor tissues compared with normal counterparts (P < 0.01) ( Figure 1A,B). To understand the significance of ANRIL overexpression in HCC, we investigated the potential associations between ANRIL expression and patients' clinicopathological features. Clinicopathological features of HCC patients are shown in Table 1. Noticeably, high ANRIL expression was significantly correlated with tumor size (P < 0.01) and advanced BCLC stage (P < 0.01). However, ANRIL expression was not associated with other parameters such as drinking state (P = 0.932), age (P = 0.850), gender (P = 0.608), AFP (P = 0.713), HBV (P = 0.713), and cirrhosis (P = 0.319) in HCC.

ANRIL is upregulated in HCC cell lines and could be activated by transcript factor SP1
To investigate the functional role of ANRIL in HCC cells, quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of ANRIL in three HCC cell lines. As shown in Figure 1C, three cell lines (HepG2, HepG3B, MHCC-97H) expressed high levels of ANRIL compared with the normal hepatic epithelium cell line (L02). A previous study indicated that ANRIL expression could be activated by E2F1. In this study, we performed bioinformatics analysis and found that there are 13 SP1 binding sites in the ANRIL promoter region (as shown in Table 2), which suggest that SP1 could also regulate ANRIL transcription in HCC cells. Chromatin immunoprecipitation (ChIP) assay showed that SP1 could directly bind to ANRIL promoter regions (1,081 bp) to silence ANRIL transcription. In addition, overexpression of SP1 in HCC cells could upregulate ANRIL expression, while knockdown of SP1 in HCC cells could downregulate ANRIL expression (as shown in Figure 1D,E,F,G,H,I,J,K).

Knockdown of ANRIL inhibits HCC cell proliferation and induces cell apoptosis in vitro
To investigate the potential role of ANRIL on HCC cell proliferation, ANRIL siRNA was transfected into HepG2 and HepG3B cells. To ensure the efficiency of interference and avoid off-target effects, we used a validated effective interference target sequence of ANRIL, according to Kotake's study [12]. qRT-PCR assays revealed that ANRIL expression was significantly reduced after transfection with si-ANRIL ( Figure 2A). Then, MTT assay showed that knockdown of ANRIL expression significantly inhibited cell proliferation both in HepG2 and HepG3B cells compared with control cells ( Figure 2B). Similarly, the result of colony formation assay revealed that clonogenic survival was significantly decreased following inhibition of ANRIL both in HepG2 and Hep3B cell lines ( Figure 2C). Next, flow cytometry analysis was performed to further examine the effect of ANRIL on proliferation of HCC cells by altering cell cycle progression or apoptosis. The results revealed that the cell cycle progression of HepG2/si-ANRIL and Hep3B/si-ANRIL cells was significantly stalled at the G1-G0 phase compared with cells transfected with si-NC ( Figure 2D). In addition, knockdown of ANRIL could obviously induce cell apoptosis ( Figure 2E).

Effect of ANRIL on HCC cell migration and invasion
Migration and invasion are significant aspects of cancer progression, which involve the dissolution of extracellular matrix proteins and the migration of tumor cells into contiguous tissues. To investigate whether ANRIL had a direct functional role in cell invasion in HCC, we performed transwell assays. The results showed that inhibition of ANRIL could significantly impair HCC cell migration and invasion ability when compared with control cells (Figure 3).

ANRIL promotes HCC cell proliferation in vivo
To further determine whether ANRIL affects tumorigenesis, we injected HepG2 cells transfected with either empty vector or sh-ANRIL into male nude mice. Consistent with in vitro results, tumor growth in the sh-ANRIL group was obviously slower than that in the empty vector group ( Figure 4A). Up to 16 days after injection, the average tumor weight in the sh-ANRIL group (0.260 ± 0.107 g) was significantly lower than that in the control group (0.442 ± 0.716 g) (P < 0.01) ( Figure 4B). qRT-PCR analysis was performed to detect the average expression of ANRIL in tumor tissues selected from mice ( Figure 4C). Results demonstrated that the average expression levels of ANRIL in the sh-ANRIL group were lower than those in the empty group. Moreover, we found that the tumors developed from empty vector-transfected cells showed a stronger Ki-67 expression than tumors formed from sh-ANRIL as detected by immunohistochemistry (IHC) analysis ( Figure 4D). These data further supported the role of ANRIL in HCC cell growth and proliferation.

ANRIL negatively regulates expression of KLF2
As previously reported, ANRIL could suppress p15 and p21 expression by binding with PRC2. In the present study, to investigate whether there are some other target genes that may be regulated by ANRIL, we performed co-expression analysis by using GSE45435 data from GEO datasets. The results showed that KLF2 may be a new target of ANRIL in HCC (as shown in Figure 5A). We also analyzed the KLF2 gene expression in HCC by using GSE 56140. It showed that KLF2 was downregulated in HCC (as shown in Figure 5B). And we further found that knockdown of ANRIL expression could upregulate both KLF2 mRNA and protein expression levels in HCC cells ( Figure 5C, D, E). Moreover, knockdown of EZH2 or SUZ12 could also upregulate KLF2 mRNA and protein expression levels in HCC cells ( Figure 5F,G,H,I,J,K). We examined the ANRIL expression levels in the HCC cell cytoplasm and nucleus distribution, and the results showed that ANRIL expression is more located in the nucleus (seen in Figure 5L,M). In addition, the results of RNA immunoprecipitation (RIP) assays revealed that ANRIL could directly bind with PRC2 in HCC cells (seen in Figure 5N,O). And ChIP assays were performed to determine whether EZH2 could directly bind to KLF2 promoter regions to silence KLF2 transcription. The results showed that EZH2 can directly bind to KLF2 promoter regions (616 bp), while knockdown of ANRIL expression decreased its binding ability (seen in Figure 5P,Q). Then, qRT-PCR analysis was performed to detect the average expression of KLF2 in tumor tissues from mice ( Figure 5R). Results demonstrated that the average expression levels of KLF2 in the sh-ANRIL group were higher than those in the empty group. Finally, we found that the tumors developed from sh-ANRIL-transfected cells showed a stronger KLF2 expression than tumors formed from the empty vector as detected by IHC analysis ( Figure 5S). These data indicated that KLF2 was a new ANRIL target gene in HCC, and its expression can be silenced by EZH2 which is recruited by ANRIL to the KLF2 promoter region and mediated H3K27 trimethylation modification.

Overexpression of KLF2 impairs HCC cell proliferation and induces cell apoptosis
To determine whether KLF2 is involved in ANRILmediated increase in HCC cell proliferation, we upregulated KLF2 expression in HCC cells by transfecting with a FLAG-tagged KLF2 expression vector using the pCMV-Tag2B vector (Stratagene, Santa Clara, CA, USA). The qRT-PCR results showed that KLF2 expression is significantly upregulated in pCMV-Tag2B-KLF2-transfected HCC cells when compared with control cells ( Figure 6A). Furthermore, MTT assays and colony formation assay revealed that KLF2 overexpression inhibited HCC cell growth ( Figure 6B,C), and flow cytometry analysis indicated that increased KLF2 expression induced cell apoptosis. These data suggest that KLF2 was partly involved in HCC cell proliferation and apoptosis.

ANRIL negatively regulates expression of KLF2 by rescue assays
Rescue assays were performed to determine whether ANRIL regulates HCC cell proliferation via repressing KLF2 expression. HepG2 cells were co-transfected with si-ANRIL and si-KLF2. The results of MTT and colony formation assays indicated that co-transfection could partially rescue si-ANRIL-impaired proliferation in HepG2 cells ( Figure 7A,B). Western blot showed the same results ( Figure 7C).

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 participates in cancer progression and predicts patients' outcome [18][19][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 upregulated [21,22]. A number of lncRNAs, such as MVIH and URHC, have been reported to be involved in HCC development and progression [23,24]. In this study, we found another lncRNA, ANRIL, whose expression is significantly upregulated 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); for example, c-myc could activate HOTAIR transcription, and PVT-1 expression can be regulated by p53 [25,26]. ANRIL expression has been reported to be regulated by a key TF, E2F1 [13,27]; however, in this study, we performed bioinformatics analysis and found that SP1 could also regulate ANRIL transcription in HCC cells. The 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 upregulate ANRIL expression, while knockdown of SP1 in HCC cells could downregulate 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 suggest 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 CBX7 (chromobox homolog 7), a subunit of the polycomb repressive complex 1 (PRC1), as a 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 and EZH2 and SUZ12 expression indeed both upregulated KLF2 expression levels in HCC cells. In addition, ChIP assays also demonstrated that EZH2 could directly bind to the 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 [28]. They have been identified as suppressors or activators of different genes in a cell type and Thirteen putative sites were predicted with these settings (90%) in sequence named gi 568815589: 21992791-21994791. Comment: This type of analysis has a high sensitivity but abysmal selectivity. In other words, while true function will be detected in most cases, most predictions will correspond to sites bound in vitro but with no function in vivo. A number of additional constraints of the analysis can improve the prediction; phylogenetic footprinting is the most common. We recommend using the ConSite service, which uses the JASPAR datasets. The review Nat Rev Genet. 2004 Apr;5(4):276-87 gives a comprehensive overview of transcription binding site prediction [34]. promoter-dependent manner [29]. KLF2 is one of the critical members due to its tumor suppressor function in tumors [30,31]. Moreover, a previous study showed that EZH2 could directly bind to the KLF2 promoter and silence of KLF2 expression results in blocking the tumor suppressor features of KLF2, which is partly mediated by p21 [32]. 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 assays 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 downregulation 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 upregulated 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 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.

RNA extraction and qRT-PCR analysis
The total RNA was extracted from tissues or cells with TRIzol reagent (  collection were carried out on an 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, ANRIL siRNA, and to avoid off-target effects and ensure the efficiency of interference, we used an indeed effective interference target sequence of ANRIL, according to a previous study [12]. EZH2 siRNA 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 in 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 using Cell Proliferation Reagent Kit I (MTT) (Roche, Basel, Switzerland). The transfected cells were plated in 96-well plates (3,000 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 being transfected with si-ANRIL or respective control, and 48 h after being transfected with

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 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 system (FACScan®, BD Biosciences) equipped with 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.

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 × 10 4 cells in serum-free medium were placed into the upper chamber of an insert (8-μm pore size, Millipore). For the invasion assays, 1 × 10 5 cells in serum-free medium were placed into the upper chamber of an insert coated with Matrigel (Sigma-Aldrich). A 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 to or invaded the membrane were fixed with methanol, stained with 0.1% crystal violet, and 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.

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 the EZ-ChIP Kit according to the manufacturer's instruction (Millipore,