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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.


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Effects of down-regulation of ANRIL on tumor growth in vivo. a Tumors from mice 16 days after injection of HepG2 cells stably transfected with sh-ANRIL or empty vector. b The tumor volume was calculated every 4 days after injection of HepG2 cells stably transfected with sh-ANRIL or empty vector. Points, mean (n = 5); bars indicate s.d. c Tumor weights are represented as means of tumor weights ± s.d. d qPCR analysis of ANRIL expression in tumor tissues formed from HepG2/sh-ANRIL, HepG2/empty vector. e Tumors developed from sh-ANRIL-transfected HepG2 cells showed lower Ki-67 protein levels than tumors developed by control cells. Left: H & E staining; right: immunostaining. *P < 0.05, **P < 0.01
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Fig4: Effects of down-regulation of ANRIL on tumor growth in vivo. a Tumors from mice 16 days after injection of HepG2 cells stably transfected with sh-ANRIL or empty vector. b The tumor volume was calculated every 4 days after injection of HepG2 cells stably transfected with sh-ANRIL or empty vector. Points, mean (n = 5); bars indicate s.d. c Tumor weights are represented as means of tumor weights ± s.d. d qPCR analysis of ANRIL expression in tumor tissues formed from HepG2/sh-ANRIL, HepG2/empty vector. e Tumors developed from sh-ANRIL-transfected HepG2 cells showed lower Ki-67 protein levels than tumors developed by control cells. Left: H & E staining; right: immunostaining. *P < 0.05, **P < 0.01

Mentions: To further determine whether ANRIL affects tumorigenesis, we injected HepG2 cells transfected with either empty vector or sh-ANRIL into male nude mice. In consistent with in vitro results, tumor growth in the sh-ANRIL group was obviously slower than that in the empty vector group (Fig. 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) ((Fig. 4b). qRT-PCR analysis was performed to detect the average expression of ANRIL in tumor tissues selected from mice (Fig. 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 that in tumors formed from sh-ANRIL as detected by immunohistochemistry (IHC) analysis (Fig. 4d). These data further supported the role of ANRIL in HCC cell growth and proliferation.Fig. 4


Long non-coding RNA ANRIL is upregulated in hepatocellular carcinoma and regulates cell proliferation by epigenetic silencing of KLF2
Effects of down-regulation of ANRIL on tumor growth in vivo. a Tumors from mice 16 days after injection of HepG2 cells stably transfected with sh-ANRIL or empty vector. b The tumor volume was calculated every 4 days after injection of HepG2 cells stably transfected with sh-ANRIL or empty vector. Points, mean (n = 5); bars indicate s.d. c Tumor weights are represented as means of tumor weights ± s.d. d qPCR analysis of ANRIL expression in tumor tissues formed from HepG2/sh-ANRIL, HepG2/empty vector. e Tumors developed from sh-ANRIL-transfected HepG2 cells showed lower Ki-67 protein levels than tumors developed by control cells. Left: H & E staining; right: immunostaining. *P < 0.05, **P < 0.01
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Fig4: Effects of down-regulation of ANRIL on tumor growth in vivo. a Tumors from mice 16 days after injection of HepG2 cells stably transfected with sh-ANRIL or empty vector. b The tumor volume was calculated every 4 days after injection of HepG2 cells stably transfected with sh-ANRIL or empty vector. Points, mean (n = 5); bars indicate s.d. c Tumor weights are represented as means of tumor weights ± s.d. d qPCR analysis of ANRIL expression in tumor tissues formed from HepG2/sh-ANRIL, HepG2/empty vector. e Tumors developed from sh-ANRIL-transfected HepG2 cells showed lower Ki-67 protein levels than tumors developed by control cells. Left: H & E staining; right: immunostaining. *P < 0.05, **P < 0.01
Mentions: To further determine whether ANRIL affects tumorigenesis, we injected HepG2 cells transfected with either empty vector or sh-ANRIL into male nude mice. In consistent with in vitro results, tumor growth in the sh-ANRIL group was obviously slower than that in the empty vector group (Fig. 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) ((Fig. 4b). qRT-PCR analysis was performed to detect the average expression of ANRIL in tumor tissues selected from mice (Fig. 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 that in tumors formed from sh-ANRIL as detected by immunohistochemistry (IHC) analysis (Fig. 4d). These data further supported the role of ANRIL in HCC cell growth and proliferation.Fig. 4

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