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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

Overexpression of KLF2 expression inhibits HepG2 cell proliferation and improves apoptosis. a The mRNA level of KLF2 in HepG2 and Hep3B cells transfected with pCMV-Tag2B-KLF2 or empty vector was detected by qPCR. b, c MTT assays and colony-forming assays were used to determine the cell viability for pCMV-Tag2B-KLF2-transfected or empty vector-transfected HepG2 and Hep3B cells. Values represent the mean ± s.d. from three independent experiments. d Apoptosis was determined by flow cytometry. UL necrotic cells, UR terminal apoptotic cells, LR early apoptotic cells. *P < 0.05, **P < 0.01
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Fig6: Overexpression of KLF2 expression inhibits HepG2 cell proliferation and improves apoptosis. a The mRNA level of KLF2 in HepG2 and Hep3B cells transfected with pCMV-Tag2B-KLF2 or empty vector was detected by qPCR. b, c MTT assays and colony-forming assays were used to determine the cell viability for pCMV-Tag2B-KLF2-transfected or empty vector-transfected HepG2 and Hep3B cells. Values represent the mean ± s.d. from three independent experiments. d Apoptosis was determined by flow cytometry. UL necrotic cells, UR terminal apoptotic cells, LR early apoptotic cells. *P < 0.05, **P < 0.01

Mentions: To determine whether KLF2 involved in ANRIL mediated increased HCC cell proliferation, we up-regulated 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 up-regulated in pCMV-Tag2B-KLF2-transfected HCC cells when compared with control cells (Fig. 6a). Furthermore, MTT assays and colony formation assay revealed that KLF2 overexpression inhibited HCC cell growth (Fig. 6b, c), and flow cytometric analysis indicated that increased KLF2 expression induced cell apoptosis. These data suggest that KLF2 partly involved in HCC cell proliferation and apoptosis.Fig. 6


Long non-coding RNA ANRIL is upregulated in hepatocellular carcinoma and regulates cell proliferation by epigenetic silencing of KLF2
Overexpression of KLF2 expression inhibits HepG2 cell proliferation and improves apoptosis. a The mRNA level of KLF2 in HepG2 and Hep3B cells transfected with pCMV-Tag2B-KLF2 or empty vector was detected by qPCR. b, c MTT assays and colony-forming assays were used to determine the cell viability for pCMV-Tag2B-KLF2-transfected or empty vector-transfected HepG2 and Hep3B cells. Values represent the mean ± s.d. from three independent experiments. d Apoptosis was determined by flow cytometry. UL necrotic cells, UR terminal apoptotic cells, LR early apoptotic cells. *P < 0.05, **P < 0.01
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Fig6: Overexpression of KLF2 expression inhibits HepG2 cell proliferation and improves apoptosis. a The mRNA level of KLF2 in HepG2 and Hep3B cells transfected with pCMV-Tag2B-KLF2 or empty vector was detected by qPCR. b, c MTT assays and colony-forming assays were used to determine the cell viability for pCMV-Tag2B-KLF2-transfected or empty vector-transfected HepG2 and Hep3B cells. Values represent the mean ± s.d. from three independent experiments. d Apoptosis was determined by flow cytometry. UL necrotic cells, UR terminal apoptotic cells, LR early apoptotic cells. *P < 0.05, **P < 0.01
Mentions: To determine whether KLF2 involved in ANRIL mediated increased HCC cell proliferation, we up-regulated 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 up-regulated in pCMV-Tag2B-KLF2-transfected HCC cells when compared with control cells (Fig. 6a). Furthermore, MTT assays and colony formation assay revealed that KLF2 overexpression inhibited HCC cell growth (Fig. 6b, c), and flow cytometric analysis indicated that increased KLF2 expression induced cell apoptosis. These data suggest that KLF2 partly involved in HCC cell proliferation and apoptosis.Fig. 6

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