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MiR-506 suppresses proliferation of hepatoma cells through targeting YAP mRNA 3'UTR.

Wang Y, Cui M, Sun BD, Liu FB, Zhang XD, Ye LH - Acta Pharmacol. Sin. (2014)

Bottom Line: Bioinformatics analysis revealed that YAP mRNA might be one of the targets of miR-506, and miR-506 in HCC tissues was found to be negatively correlated with YAP (r=-0.605).Furthermore, miR-506 significantly inhibited the proliferation of HepG2 and H7402 cells, while anti-miR-506 enhanced the cell proliferation, which was blocked by YAP siRNA.MiR-506 suppresses the proliferation of hepatoma cells by targeting YAP mRNA.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071, China.

ABSTRACT

Aim: MiR-506 is a miRNA involved in carcinogenesis of several kinds of cancer. In this study, we explored whether miR-506 played a critical role in hepatocellular carcinoma (HCC).

Methods: Twenty HCC and adjacent normal liver tissue samples were collected. Human hepatoma cell lines HepG2 and H7402 were used for in vitro studies. The expression of miR-506 and transcriptional co-activator YAP was examined using qRT-PCR. Western blot analysis was used to measure the expression of YAP and its target genes. Luciferase reporter gene assay was used to identify YAP as a target gene of miR-506. MTT and EdU assays were carried out for functional analysis.

Results: The expression of miR-506 was significantly lower in HCC than in adjacent normal liver tissues. Bioinformatics analysis revealed that YAP mRNA might be one of the targets of miR-506, and miR-506 in HCC tissues was found to be negatively correlated with YAP (r=-0.605). In both HepG2 and H7402 cells, miR-506 dose-dependently down-regulated YAP and its target genes c-Myc and the connective tissue growth factor (CTGF). Luciferase reporter gene assays demonstrated that miR-506 targeted the wild type 3'UTR of YAP mRNA, but not a 3'UTR with a mutant seed site. Furthermore, miR-506 significantly inhibited the proliferation of HepG2 and H7402 cells, while anti-miR-506 enhanced the cell proliferation, which was blocked by YAP siRNA.

Conclusion: MiR-506 suppresses the proliferation of hepatoma cells by targeting YAP mRNA.

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MiR-506 restrains the expression of YAP through direct targeting of YAP mRNA 3′UTR. (A and B) Model shows the binding site of miR-506 in the YAP mRNA 3′UTR by bioinformatics prediction. Schematic diagram shows the generated mutant site in the YAP 3′UTR seed region binding to miR-506 and the insertion sites of wild type YAP 3′UTR (or mutant) downstream of the luciferase reporter gene in pGL3-Control vector. (C) The effect of miR-506 on pGL3-YAP and pGL3-YAP-MUT reporter plasmids in HepG2 and H7402 cells was measured by luciferase reporter gene assays. (D) The effect of anti-miR-506 on pGL3-YAP and pGL3-YAP-MUT reporter plasmids in HepG2 and H7402 cells was measured by luciferase reporter gene assays. Statistically significant differences are indicated: bP<0.05, cP<0.01; Student's t test.
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fig3: MiR-506 restrains the expression of YAP through direct targeting of YAP mRNA 3′UTR. (A and B) Model shows the binding site of miR-506 in the YAP mRNA 3′UTR by bioinformatics prediction. Schematic diagram shows the generated mutant site in the YAP 3′UTR seed region binding to miR-506 and the insertion sites of wild type YAP 3′UTR (or mutant) downstream of the luciferase reporter gene in pGL3-Control vector. (C) The effect of miR-506 on pGL3-YAP and pGL3-YAP-MUT reporter plasmids in HepG2 and H7402 cells was measured by luciferase reporter gene assays. (D) The effect of anti-miR-506 on pGL3-YAP and pGL3-YAP-MUT reporter plasmids in HepG2 and H7402 cells was measured by luciferase reporter gene assays. Statistically significant differences are indicated: bP<0.05, cP<0.01; Student's t test.

Mentions: To gain insight into the mechanism by which miR-506 inhibits YAP, we identified the miR-506 binding site in the YAP mRNA 3′UTR (Figure 3A) and constructed pGL3-YAP and pGL3-YAP-MUT plasmids (Figure 3B). Our data showed that the co-transfection of miR-506 significantly suppressed the firefly luciferase activity of pGL3-YAP but failed to influence the luciferase activity of pGL3-YAP-MUT in HepG2 and H7402 cells (Figure 3C). Furthermore, inhibition of endogenous miR-506 in the cells with anti-miR-506 resulted in increased firefly luciferase activity of the wild-type reporter but the mutant reporter (Figure 3D). Thus, our data indicate that miR-506 can attenuate the expression of YAP through direct targeting of its mRNA 3′UTR in hepatoma cells.


MiR-506 suppresses proliferation of hepatoma cells through targeting YAP mRNA 3'UTR.

Wang Y, Cui M, Sun BD, Liu FB, Zhang XD, Ye LH - Acta Pharmacol. Sin. (2014)

MiR-506 restrains the expression of YAP through direct targeting of YAP mRNA 3′UTR. (A and B) Model shows the binding site of miR-506 in the YAP mRNA 3′UTR by bioinformatics prediction. Schematic diagram shows the generated mutant site in the YAP 3′UTR seed region binding to miR-506 and the insertion sites of wild type YAP 3′UTR (or mutant) downstream of the luciferase reporter gene in pGL3-Control vector. (C) The effect of miR-506 on pGL3-YAP and pGL3-YAP-MUT reporter plasmids in HepG2 and H7402 cells was measured by luciferase reporter gene assays. (D) The effect of anti-miR-506 on pGL3-YAP and pGL3-YAP-MUT reporter plasmids in HepG2 and H7402 cells was measured by luciferase reporter gene assays. Statistically significant differences are indicated: bP<0.05, cP<0.01; Student's t test.
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getmorefigures.php?uid=PMC4155531&req=5

fig3: MiR-506 restrains the expression of YAP through direct targeting of YAP mRNA 3′UTR. (A and B) Model shows the binding site of miR-506 in the YAP mRNA 3′UTR by bioinformatics prediction. Schematic diagram shows the generated mutant site in the YAP 3′UTR seed region binding to miR-506 and the insertion sites of wild type YAP 3′UTR (or mutant) downstream of the luciferase reporter gene in pGL3-Control vector. (C) The effect of miR-506 on pGL3-YAP and pGL3-YAP-MUT reporter plasmids in HepG2 and H7402 cells was measured by luciferase reporter gene assays. (D) The effect of anti-miR-506 on pGL3-YAP and pGL3-YAP-MUT reporter plasmids in HepG2 and H7402 cells was measured by luciferase reporter gene assays. Statistically significant differences are indicated: bP<0.05, cP<0.01; Student's t test.
Mentions: To gain insight into the mechanism by which miR-506 inhibits YAP, we identified the miR-506 binding site in the YAP mRNA 3′UTR (Figure 3A) and constructed pGL3-YAP and pGL3-YAP-MUT plasmids (Figure 3B). Our data showed that the co-transfection of miR-506 significantly suppressed the firefly luciferase activity of pGL3-YAP but failed to influence the luciferase activity of pGL3-YAP-MUT in HepG2 and H7402 cells (Figure 3C). Furthermore, inhibition of endogenous miR-506 in the cells with anti-miR-506 resulted in increased firefly luciferase activity of the wild-type reporter but the mutant reporter (Figure 3D). Thus, our data indicate that miR-506 can attenuate the expression of YAP through direct targeting of its mRNA 3′UTR in hepatoma cells.

Bottom Line: Bioinformatics analysis revealed that YAP mRNA might be one of the targets of miR-506, and miR-506 in HCC tissues was found to be negatively correlated with YAP (r=-0.605).Furthermore, miR-506 significantly inhibited the proliferation of HepG2 and H7402 cells, while anti-miR-506 enhanced the cell proliferation, which was blocked by YAP siRNA.MiR-506 suppresses the proliferation of hepatoma cells by targeting YAP mRNA.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071, China.

ABSTRACT

Aim: MiR-506 is a miRNA involved in carcinogenesis of several kinds of cancer. In this study, we explored whether miR-506 played a critical role in hepatocellular carcinoma (HCC).

Methods: Twenty HCC and adjacent normal liver tissue samples were collected. Human hepatoma cell lines HepG2 and H7402 were used for in vitro studies. The expression of miR-506 and transcriptional co-activator YAP was examined using qRT-PCR. Western blot analysis was used to measure the expression of YAP and its target genes. Luciferase reporter gene assay was used to identify YAP as a target gene of miR-506. MTT and EdU assays were carried out for functional analysis.

Results: The expression of miR-506 was significantly lower in HCC than in adjacent normal liver tissues. Bioinformatics analysis revealed that YAP mRNA might be one of the targets of miR-506, and miR-506 in HCC tissues was found to be negatively correlated with YAP (r=-0.605). In both HepG2 and H7402 cells, miR-506 dose-dependently down-regulated YAP and its target genes c-Myc and the connective tissue growth factor (CTGF). Luciferase reporter gene assays demonstrated that miR-506 targeted the wild type 3'UTR of YAP mRNA, but not a 3'UTR with a mutant seed site. Furthermore, miR-506 significantly inhibited the proliferation of HepG2 and H7402 cells, while anti-miR-506 enhanced the cell proliferation, which was blocked by YAP siRNA.

Conclusion: MiR-506 suppresses the proliferation of hepatoma cells by targeting YAP mRNA.

Show MeSH
Related in: MedlinePlus