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MicroRNA-1246 enhances migration and invasion through CADM1 in hepatocellular carcinoma.

Sun Z, Meng C, Wang S, Zhou N, Guan M, Bai C, Lu S, Han Q, Zhao RC - BMC Cancer (2014)

Bottom Line: The aberrant expression of microRNAs has been demonstrated to play a crucial role in the initiation and progression of hepatocarcinoma. miR-1246 expression in High invasive ability cell line than significantly higher than that in low invasive ability cell line.Inhibition of miR-1246 effectively reduced migration and invasion of hepatocellular carcinoma cell lines.Our study showed that miR-1246, by down-regulation CADM1, enhances migration and invasion in HCC cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, People's Republic of China. hanqinhanqin@126.com.

ABSTRACT

Background: The aberrant expression of microRNAs has been demonstrated to play a crucial role in the initiation and progression of hepatocarcinoma. miR-1246 expression in High invasive ability cell line than significantly higher than that in low invasive ability cell line.

Methods: Transwell chambers (8-uM pore size; Costar) were used in the in vitro migration and invison anssay. Dual luciferase reporter gene construct and Dual luciferase reporter assay to identify the target of miR-1246. CADM1 expression was evaluated by immunohistochemistric staining. The clinical manifestations, treatments and survival were collected for statistical analysis.

Results: Inhibition of miR-1246 effectively reduced migration and invasion of hepatocellular carcinoma cell lines. Bioinformatics and luciferase reporter assay revealed that miR-1246 specifically targeted the 3'-UTR of Cell adhesion molecule 1 and regulated its expression. Down-regulation of CADM1 enhanced migration and invasion of HCC cell lines. Furthermore, in tumor tissues obtained from liver cancer patients, the expression of miR-1246 was negatively correlated with CADM1 and the high expression of miR-1246 combined with low expression of CADM1 might serve as a risk factor for stage1 liver cancer patients.

Conclusions: Our study showed that miR-1246, by down-regulation CADM1, enhances migration and invasion in HCC cells.

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Related in: MedlinePlus

CADM1 was a direct target of miR-1246. (A) Putative binding site for miR-1246 in the 3′UTR of CADM1 was revealed by TargetScan. (B) The miR-1246 binding site on CADM1 3′UTR was confirmed by luciferase assay in 293 T cells after cotransfection with (i) a plasmid containing a fragment of CADM1 3′UTR that included either the wild type or mutant predicted miR-1246 binding site and (ii) the miR-1246 mimic or the mimic control. Data represent the mean ± SD of at least three independent experiments. *P < 0.01. (C) Western blot assay showed increased CADM1 expression in SMMC7721 cells after transfection with the miR-1246 inhibitor (800 nM). (D) Real time PCR showed that miR-1246 did not affect CADM1 mRNA expression.
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Fig3: CADM1 was a direct target of miR-1246. (A) Putative binding site for miR-1246 in the 3′UTR of CADM1 was revealed by TargetScan. (B) The miR-1246 binding site on CADM1 3′UTR was confirmed by luciferase assay in 293 T cells after cotransfection with (i) a plasmid containing a fragment of CADM1 3′UTR that included either the wild type or mutant predicted miR-1246 binding site and (ii) the miR-1246 mimic or the mimic control. Data represent the mean ± SD of at least three independent experiments. *P < 0.01. (C) Western blot assay showed increased CADM1 expression in SMMC7721 cells after transfection with the miR-1246 inhibitor (800 nM). (D) Real time PCR showed that miR-1246 did not affect CADM1 mRNA expression.

Mentions: To understand the mechanisms by which inhibition of miR-1246 reduced migration and invasion, we used bioinformatics analysis to identify miR-1246 targets. There was a conserved binding site of miR-1246 in the CADM1 3′UTR (Figure 3A). To test whether CADM1 is a target of miR-1246, we conducted a standard luciferase reporter assay in 293 T cells. 293 T cells were transfected with the luciferase construct CADM1-WT or CADM1-MT, along with the internal control vector pGL3 and either the miR-1246 mimic or the mimic control. The cells were harvested at 48 hours and analyzed for dual luciferase activity. The results showed that the renilla luciferase activity in CADM1-WT-transfected cells decreased by more than 40% in miR-1246 mimic-cotransfected cells compared with that in mimic control-cotransfected cells. In addition, site-directed mutation of the seed region offset the inhibitory effect of miR-1246 mimic (Figure 3B).Figure 3


MicroRNA-1246 enhances migration and invasion through CADM1 in hepatocellular carcinoma.

Sun Z, Meng C, Wang S, Zhou N, Guan M, Bai C, Lu S, Han Q, Zhao RC - BMC Cancer (2014)

CADM1 was a direct target of miR-1246. (A) Putative binding site for miR-1246 in the 3′UTR of CADM1 was revealed by TargetScan. (B) The miR-1246 binding site on CADM1 3′UTR was confirmed by luciferase assay in 293 T cells after cotransfection with (i) a plasmid containing a fragment of CADM1 3′UTR that included either the wild type or mutant predicted miR-1246 binding site and (ii) the miR-1246 mimic or the mimic control. Data represent the mean ± SD of at least three independent experiments. *P < 0.01. (C) Western blot assay showed increased CADM1 expression in SMMC7721 cells after transfection with the miR-1246 inhibitor (800 nM). (D) Real time PCR showed that miR-1246 did not affect CADM1 mRNA expression.
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Fig3: CADM1 was a direct target of miR-1246. (A) Putative binding site for miR-1246 in the 3′UTR of CADM1 was revealed by TargetScan. (B) The miR-1246 binding site on CADM1 3′UTR was confirmed by luciferase assay in 293 T cells after cotransfection with (i) a plasmid containing a fragment of CADM1 3′UTR that included either the wild type or mutant predicted miR-1246 binding site and (ii) the miR-1246 mimic or the mimic control. Data represent the mean ± SD of at least three independent experiments. *P < 0.01. (C) Western blot assay showed increased CADM1 expression in SMMC7721 cells after transfection with the miR-1246 inhibitor (800 nM). (D) Real time PCR showed that miR-1246 did not affect CADM1 mRNA expression.
Mentions: To understand the mechanisms by which inhibition of miR-1246 reduced migration and invasion, we used bioinformatics analysis to identify miR-1246 targets. There was a conserved binding site of miR-1246 in the CADM1 3′UTR (Figure 3A). To test whether CADM1 is a target of miR-1246, we conducted a standard luciferase reporter assay in 293 T cells. 293 T cells were transfected with the luciferase construct CADM1-WT or CADM1-MT, along with the internal control vector pGL3 and either the miR-1246 mimic or the mimic control. The cells were harvested at 48 hours and analyzed for dual luciferase activity. The results showed that the renilla luciferase activity in CADM1-WT-transfected cells decreased by more than 40% in miR-1246 mimic-cotransfected cells compared with that in mimic control-cotransfected cells. In addition, site-directed mutation of the seed region offset the inhibitory effect of miR-1246 mimic (Figure 3B).Figure 3

Bottom Line: The aberrant expression of microRNAs has been demonstrated to play a crucial role in the initiation and progression of hepatocarcinoma. miR-1246 expression in High invasive ability cell line than significantly higher than that in low invasive ability cell line.Inhibition of miR-1246 effectively reduced migration and invasion of hepatocellular carcinoma cell lines.Our study showed that miR-1246, by down-regulation CADM1, enhances migration and invasion in HCC cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, People's Republic of China. hanqinhanqin@126.com.

ABSTRACT

Background: The aberrant expression of microRNAs has been demonstrated to play a crucial role in the initiation and progression of hepatocarcinoma. miR-1246 expression in High invasive ability cell line than significantly higher than that in low invasive ability cell line.

Methods: Transwell chambers (8-uM pore size; Costar) were used in the in vitro migration and invison anssay. Dual luciferase reporter gene construct and Dual luciferase reporter assay to identify the target of miR-1246. CADM1 expression was evaluated by immunohistochemistric staining. The clinical manifestations, treatments and survival were collected for statistical analysis.

Results: Inhibition of miR-1246 effectively reduced migration and invasion of hepatocellular carcinoma cell lines. Bioinformatics and luciferase reporter assay revealed that miR-1246 specifically targeted the 3'-UTR of Cell adhesion molecule 1 and regulated its expression. Down-regulation of CADM1 enhanced migration and invasion of HCC cell lines. Furthermore, in tumor tissues obtained from liver cancer patients, the expression of miR-1246 was negatively correlated with CADM1 and the high expression of miR-1246 combined with low expression of CADM1 might serve as a risk factor for stage1 liver cancer patients.

Conclusions: Our study showed that miR-1246, by down-regulation CADM1, enhances migration and invasion in HCC cells.

Show MeSH
Related in: MedlinePlus