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

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.

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).

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).

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).

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.

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.

To determine whether KLF2 is involved in ANRIL-mediated 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.

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).