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

View Article: PubMed Central - PubMed

ABSTRACT

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. CDKN2B antisense RNA1 (ANRIL), a lncRNA, coclustered 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 is 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 real-time polymerase chain reaction (qRT-PCR). ANRIL expression was up-regulated 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 KLF2 transcription in HCC cells by binding with PRC2 and recruiting it to 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/s13045-015-0153-1) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Effects of knockdown of ANRIL on HCC cell viability and apoptosis in vitro. a The ANRIL expression level was determined by qPCR when HepG2 and Hep3B cells transfected with si-ANRIL. b MTT assays were used to determine the cell viability for si-ANRIL-transfected HepG2 and Hep3B cells. Values represented the mean ± s.d. from three independent experiments. c Colony-forming assays were conducted to determine the proliferation of si-ANRIL-transfected HepG2 and Hep3B cells. d Flow cytometry assays were performed to analyze the cell cycle progression when HCC cells transfected with si-ANRIL 24 h later. The bar chart represented the percentage of cells in G0/G1, S, or G2/M phase, as indicated. e Flow cytometry assays were performed to analyze the cell apoptosis when HCC cells transfected with si-ANRIL 48 h later. *P < 0.05, **P < 0.01
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Fig2: Effects of knockdown of ANRIL on HCC cell viability and apoptosis in vitro. a The ANRIL expression level was determined by qPCR when HepG2 and Hep3B cells transfected with si-ANRIL. b MTT assays were used to determine the cell viability for si-ANRIL-transfected HepG2 and Hep3B cells. Values represented the mean ± s.d. from three independent experiments. c Colony-forming assays were conducted to determine the proliferation of si-ANRIL-transfected HepG2 and Hep3B cells. d Flow cytometry assays were performed to analyze the cell cycle progression when HCC cells transfected with si-ANRIL 24 h later. The bar chart represented the percentage of cells in G0/G1, S, or G2/M phase, as indicated. e Flow cytometry assays were performed to analyze the cell apoptosis when HCC cells transfected with si-ANRIL 48 h later. *P < 0.05, **P < 0.01

Mentions: 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 (Fig. 2a). Then MTT assay showed that knockdown of ANRIL expression significantly inhibited cell proliferation both in HepG2 and HepG3B cells compared with control cells (Fig. 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 (Fig. 2c). Next, flow cytometric analysis was performed to further examine whether 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 (Fig. 2d). In addition, knockdown of ANRIL could obviously induce cell apoptosis (Fig. 2e).Fig. 2


Long non-coding RNA ANRIL is upregulated in hepatocellular carcinoma and regulates cell proliferation by epigenetic silencing of KLF2
Effects of knockdown of ANRIL on HCC cell viability and apoptosis in vitro. a The ANRIL expression level was determined by qPCR when HepG2 and Hep3B cells transfected with si-ANRIL. b MTT assays were used to determine the cell viability for si-ANRIL-transfected HepG2 and Hep3B cells. Values represented the mean ± s.d. from three independent experiments. c Colony-forming assays were conducted to determine the proliferation of si-ANRIL-transfected HepG2 and Hep3B cells. d Flow cytometry assays were performed to analyze the cell cycle progression when HCC cells transfected with si-ANRIL 24 h later. The bar chart represented the percentage of cells in G0/G1, S, or G2/M phase, as indicated. e Flow cytometry assays were performed to analyze the cell apoptosis when HCC cells transfected with si-ANRIL 48 h later. *P < 0.05, **P < 0.01
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Fig2: Effects of knockdown of ANRIL on HCC cell viability and apoptosis in vitro. a The ANRIL expression level was determined by qPCR when HepG2 and Hep3B cells transfected with si-ANRIL. b MTT assays were used to determine the cell viability for si-ANRIL-transfected HepG2 and Hep3B cells. Values represented the mean ± s.d. from three independent experiments. c Colony-forming assays were conducted to determine the proliferation of si-ANRIL-transfected HepG2 and Hep3B cells. d Flow cytometry assays were performed to analyze the cell cycle progression when HCC cells transfected with si-ANRIL 24 h later. The bar chart represented the percentage of cells in G0/G1, S, or G2/M phase, as indicated. e Flow cytometry assays were performed to analyze the cell apoptosis when HCC cells transfected with si-ANRIL 48 h later. *P < 0.05, **P < 0.01
Mentions: 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 (Fig. 2a). Then MTT assay showed that knockdown of ANRIL expression significantly inhibited cell proliferation both in HepG2 and HepG3B cells compared with control cells (Fig. 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 (Fig. 2c). Next, flow cytometric analysis was performed to further examine whether 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 (Fig. 2d). In addition, knockdown of ANRIL could obviously induce cell apoptosis (Fig. 2e).Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

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. CDKN2B antisense RNA1 (ANRIL), a lncRNA, coclustered 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 is 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 real-time polymerase chain reaction (qRT-PCR). ANRIL expression was up-regulated 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 KLF2 transcription in HCC cells by binding with PRC2 and recruiting it to 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/s13045-015-0153-1) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus