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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.


ANRIL negatively regulates expression of KLF2 by rescue assays. a, b Colony-forming assays were used to determine the cell viability for HepG2 cells transfected with si-NC and si-ANRIL and co-transfected with siANRIL and si-KLF2. Values represent the mean ± s.d. from three independent experiments. c MTT assays were used to determine the cell viability for HepG2 cells transfected with si-NC and si-ANRIL and co-transfected with siANRIL and si-KLF2. Values represent the mean ± s.d. from three independent experiments. d, e The levels of KLF2 protein levels were determined by Western blotting when HepG2 cells transfected with si-NC and si-ANRIL and co-transfected with siANRIL and si-KLF2. *P < 0.05, **P < 0.01
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Fig7: ANRIL negatively regulates expression of KLF2 by rescue assays. a, b Colony-forming assays were used to determine the cell viability for HepG2 cells transfected with si-NC and si-ANRIL and co-transfected with siANRIL and si-KLF2. Values represent the mean ± s.d. from three independent experiments. c MTT assays were used to determine the cell viability for HepG2 cells transfected with si-NC and si-ANRIL and co-transfected with siANRIL and si-KLF2. Values represent the mean ± s.d. from three independent experiments. d, e The levels of KLF2 protein levels were determined by Western blotting when HepG2 cells transfected with si-NC and si-ANRIL and co-transfected with siANRIL and si-KLF2. *P < 0.05, **P < 0.01

Mentions: 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 assay indicated that co-transfection could partially rescue si-ANRIL-impaired proliferation in HepG2 cells (Fig. 7a, b). Western blotting showed the same results (Fig. 7c).Fig. 7


Long non-coding RNA ANRIL is upregulated in hepatocellular carcinoma and regulates cell proliferation by epigenetic silencing of KLF2
ANRIL negatively regulates expression of KLF2 by rescue assays. a, b Colony-forming assays were used to determine the cell viability for HepG2 cells transfected with si-NC and si-ANRIL and co-transfected with siANRIL and si-KLF2. Values represent the mean ± s.d. from three independent experiments. c MTT assays were used to determine the cell viability for HepG2 cells transfected with si-NC and si-ANRIL and co-transfected with siANRIL and si-KLF2. Values represent the mean ± s.d. from three independent experiments. d, e The levels of KLF2 protein levels were determined by Western blotting when HepG2 cells transfected with si-NC and si-ANRIL and co-transfected with siANRIL and si-KLF2. *P < 0.05, **P < 0.01
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Related In: Results  -  Collection

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Fig7: ANRIL negatively regulates expression of KLF2 by rescue assays. a, b Colony-forming assays were used to determine the cell viability for HepG2 cells transfected with si-NC and si-ANRIL and co-transfected with siANRIL and si-KLF2. Values represent the mean ± s.d. from three independent experiments. c MTT assays were used to determine the cell viability for HepG2 cells transfected with si-NC and si-ANRIL and co-transfected with siANRIL and si-KLF2. Values represent the mean ± s.d. from three independent experiments. d, e The levels of KLF2 protein levels were determined by Western blotting when HepG2 cells transfected with si-NC and si-ANRIL and co-transfected with siANRIL and si-KLF2. *P < 0.05, **P < 0.01
Mentions: 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 assay indicated that co-transfection could partially rescue si-ANRIL-impaired proliferation in HepG2 cells (Fig. 7a, b). Western blotting showed the same results (Fig. 7c).Fig. 7

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.