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Mir-184 post-transcriptionally regulates SOX7 expression and promotes cell proliferation in human hepatocellular carcinoma.

Wu GG, Li WH, He WG, Jiang N, Zhang GX, Chen W, Yang HF, Liu QL, Huang YN, Zhang L, Zhang T, Zeng XC - PLoS ONE (2014)

Bottom Line: Recently, microRNAs (miRNAs) have been suggested to be closely associated with tumorigenesis.Overexpression of miR-184 in HCC cells increased cell proliferation, tumorigenicity, and cell cycle progression, whereas inhibition of miR-184 reduced cell proliferation, tumorigenicity, and cell cycle progression.Ectopic expression of miR-184 led to downregulation of the SOX7 protein, resulting in upregulation of c-Myc, Cyclin D1, and phosphorylation of Rb.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Zengcheng People's Hospital (BoJi-Affiliated Hospital of Sun Yat-Sen University), Zengcheng, China.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common human malignancies and the third leading cause of cancer mortality worldwide. The development and progression of HCC is a complicated process, involving the deregulation of multiple genes that are essential to cell biological processes. Recently, microRNAs (miRNAs) have been suggested to be closely associated with tumorigenesis. Our study showed that miR-184 is upregulated in HCC cell lines and tissues. Overexpression of miR-184 in HCC cells increased cell proliferation, tumorigenicity, and cell cycle progression, whereas inhibition of miR-184 reduced cell proliferation, tumorigenicity, and cell cycle progression. Additionally, we identified SOX7 as a direct target of miR-184. Ectopic expression of miR-184 led to downregulation of the SOX7 protein, resulting in upregulation of c-Myc, Cyclin D1, and phosphorylation of Rb. Our findings suggested that miR-184 represents a potential onco-miR and plays an important role in HCC progression by suppressing SOX7 expression.

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MiR-184 directly targets the 3′-UTR of SOX7 mRNA.A. Schematic representation of the mature miR-184 sequence, miR-184 target site in the 3′-UTR of SOX7 mRNA and a 3′-UTR mutant of SOX7 mRNA containing three altered nucleotides in the putative target site (SOX7-3′UTR-mut). B. The expression levels of SOX7 protein in HCC cells overexpressing miR-184 or transfected with miR-184 inhibitor, compared with control cells, by western blotting 48 hours after transfection; α-Tubulin served as the loading control. C. Luciferase assay of pGL3-SOX7-3′UTR or pGL3-SOX7-3′UTR-mut reporter cotransfected with different amounts (10, 50 nM) of miR-184 mimic in indicated cells, or different amounts (50, 100 nM) of miR-184 inhibitor, compared with negative control (NC). D. Real-time PCR analysis of the mRNA expression of genes, MYC, CyclinD1, LEF1 and TCF, in indicated HCC cells. E. Expression of c-Myc, CyclinD1, phosphorylated Rb, and Rb protein, as measured by western blotting in indicated HCC cells; α-Tubulin served as the loading control. Each bar represents the mean ± SD of three independent experiments. *P<0.05.
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pone-0088796-g004: MiR-184 directly targets the 3′-UTR of SOX7 mRNA.A. Schematic representation of the mature miR-184 sequence, miR-184 target site in the 3′-UTR of SOX7 mRNA and a 3′-UTR mutant of SOX7 mRNA containing three altered nucleotides in the putative target site (SOX7-3′UTR-mut). B. The expression levels of SOX7 protein in HCC cells overexpressing miR-184 or transfected with miR-184 inhibitor, compared with control cells, by western blotting 48 hours after transfection; α-Tubulin served as the loading control. C. Luciferase assay of pGL3-SOX7-3′UTR or pGL3-SOX7-3′UTR-mut reporter cotransfected with different amounts (10, 50 nM) of miR-184 mimic in indicated cells, or different amounts (50, 100 nM) of miR-184 inhibitor, compared with negative control (NC). D. Real-time PCR analysis of the mRNA expression of genes, MYC, CyclinD1, LEF1 and TCF, in indicated HCC cells. E. Expression of c-Myc, CyclinD1, phosphorylated Rb, and Rb protein, as measured by western blotting in indicated HCC cells; α-Tubulin served as the loading control. Each bar represents the mean ± SD of three independent experiments. *P<0.05.

Mentions: To explore the molecular mechanism of miR-184 function in HCC cells, we used publicly available algorithms (TargetScan, Pictar and miRANDA, which are public compilation of databases and web portals and servers used for microRNAs and their targets) to predict the target(s) of miR-184 in humans. The results indicated that SOX7 was one of the potential targets of miR-184 (Figure 4A). SOX7 was previously reported as a tumor suppressor in various cancers [19]–[23]. As predicted, western blotting revealed that SOX7 expression decreased in HepG3 and Huh7 cells, compared with normal cells THLE3, and further decreased in HepG3 and Huh7 cells overexpressing miR-184 and increased in cells transfected with the miR-184 inhibitor (Figure 4B, Figure S2B). To examine whether miR-184 mediated-SOX7 downregulation was effected via the 3′-UTR of SOX7, the SOX7-3′-UTR fragment, containing miR-184 binding site, was subcloned into a pGL3 luciferase reporter vector. The results of the luciferase reporter assay showed that ectopic expression of miR-184 decreased, and suppression of miR-184 increased, the luciferase activity of the SOX7 3′-UTR-luciferase reporter. By contrast, a SOX7 3′-UTR -luciferase reporter with a mutant miR-184 binding site seed sequence was not inhibited by ectopic expression of miR-184 (Figure 4C). Considering it was previously reported that SOX7 could remarkably reduce Wnt/β-catenin signaling activity and the expression of its downstream genes [21], [27], [28], we further examined the expression of some Wnt/β-catenin signaling related genes, Cyclin D1, MYC, LEF1, and TCF. The results showed that the mRNA of Cyclin D1, MYC, LEF1, and TCF were significantly upregulated by ectopic miR-184, whereas they were downregulated by inhibition of miR-184 (Figure 4D). Moreover, the expression of c-Myc and Cyclin D1 proteins were upregulated, and phosphorylated Rb was increased in miR-184 overexpressing cells compared with the negative control cells (Figure 4E). By contrast, the expression of c-Myc and Cyclin D1 were downregulated, and Rb phosphorylation was decreased, in cells transfected with the miR-184 inhibitor (Figure 4E). Furthermore, we examined the expression level of these Wnt/β-catenin signaling related genes in HCC tissues. The result showed that Cyclin D1, MYC, and LEF1 were upregulated in HCC tissues and the expression levels were positively correlated with the expression of miR-184 (Figure S3). Collectively, our results suggested that SOX7 is a direct target of miR-184.


Mir-184 post-transcriptionally regulates SOX7 expression and promotes cell proliferation in human hepatocellular carcinoma.

Wu GG, Li WH, He WG, Jiang N, Zhang GX, Chen W, Yang HF, Liu QL, Huang YN, Zhang L, Zhang T, Zeng XC - PLoS ONE (2014)

MiR-184 directly targets the 3′-UTR of SOX7 mRNA.A. Schematic representation of the mature miR-184 sequence, miR-184 target site in the 3′-UTR of SOX7 mRNA and a 3′-UTR mutant of SOX7 mRNA containing three altered nucleotides in the putative target site (SOX7-3′UTR-mut). B. The expression levels of SOX7 protein in HCC cells overexpressing miR-184 or transfected with miR-184 inhibitor, compared with control cells, by western blotting 48 hours after transfection; α-Tubulin served as the loading control. C. Luciferase assay of pGL3-SOX7-3′UTR or pGL3-SOX7-3′UTR-mut reporter cotransfected with different amounts (10, 50 nM) of miR-184 mimic in indicated cells, or different amounts (50, 100 nM) of miR-184 inhibitor, compared with negative control (NC). D. Real-time PCR analysis of the mRNA expression of genes, MYC, CyclinD1, LEF1 and TCF, in indicated HCC cells. E. Expression of c-Myc, CyclinD1, phosphorylated Rb, and Rb protein, as measured by western blotting in indicated HCC cells; α-Tubulin served as the loading control. Each bar represents the mean ± SD of three independent experiments. *P<0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928300&req=5

pone-0088796-g004: MiR-184 directly targets the 3′-UTR of SOX7 mRNA.A. Schematic representation of the mature miR-184 sequence, miR-184 target site in the 3′-UTR of SOX7 mRNA and a 3′-UTR mutant of SOX7 mRNA containing three altered nucleotides in the putative target site (SOX7-3′UTR-mut). B. The expression levels of SOX7 protein in HCC cells overexpressing miR-184 or transfected with miR-184 inhibitor, compared with control cells, by western blotting 48 hours after transfection; α-Tubulin served as the loading control. C. Luciferase assay of pGL3-SOX7-3′UTR or pGL3-SOX7-3′UTR-mut reporter cotransfected with different amounts (10, 50 nM) of miR-184 mimic in indicated cells, or different amounts (50, 100 nM) of miR-184 inhibitor, compared with negative control (NC). D. Real-time PCR analysis of the mRNA expression of genes, MYC, CyclinD1, LEF1 and TCF, in indicated HCC cells. E. Expression of c-Myc, CyclinD1, phosphorylated Rb, and Rb protein, as measured by western blotting in indicated HCC cells; α-Tubulin served as the loading control. Each bar represents the mean ± SD of three independent experiments. *P<0.05.
Mentions: To explore the molecular mechanism of miR-184 function in HCC cells, we used publicly available algorithms (TargetScan, Pictar and miRANDA, which are public compilation of databases and web portals and servers used for microRNAs and their targets) to predict the target(s) of miR-184 in humans. The results indicated that SOX7 was one of the potential targets of miR-184 (Figure 4A). SOX7 was previously reported as a tumor suppressor in various cancers [19]–[23]. As predicted, western blotting revealed that SOX7 expression decreased in HepG3 and Huh7 cells, compared with normal cells THLE3, and further decreased in HepG3 and Huh7 cells overexpressing miR-184 and increased in cells transfected with the miR-184 inhibitor (Figure 4B, Figure S2B). To examine whether miR-184 mediated-SOX7 downregulation was effected via the 3′-UTR of SOX7, the SOX7-3′-UTR fragment, containing miR-184 binding site, was subcloned into a pGL3 luciferase reporter vector. The results of the luciferase reporter assay showed that ectopic expression of miR-184 decreased, and suppression of miR-184 increased, the luciferase activity of the SOX7 3′-UTR-luciferase reporter. By contrast, a SOX7 3′-UTR -luciferase reporter with a mutant miR-184 binding site seed sequence was not inhibited by ectopic expression of miR-184 (Figure 4C). Considering it was previously reported that SOX7 could remarkably reduce Wnt/β-catenin signaling activity and the expression of its downstream genes [21], [27], [28], we further examined the expression of some Wnt/β-catenin signaling related genes, Cyclin D1, MYC, LEF1, and TCF. The results showed that the mRNA of Cyclin D1, MYC, LEF1, and TCF were significantly upregulated by ectopic miR-184, whereas they were downregulated by inhibition of miR-184 (Figure 4D). Moreover, the expression of c-Myc and Cyclin D1 proteins were upregulated, and phosphorylated Rb was increased in miR-184 overexpressing cells compared with the negative control cells (Figure 4E). By contrast, the expression of c-Myc and Cyclin D1 were downregulated, and Rb phosphorylation was decreased, in cells transfected with the miR-184 inhibitor (Figure 4E). Furthermore, we examined the expression level of these Wnt/β-catenin signaling related genes in HCC tissues. The result showed that Cyclin D1, MYC, and LEF1 were upregulated in HCC tissues and the expression levels were positively correlated with the expression of miR-184 (Figure S3). Collectively, our results suggested that SOX7 is a direct target of miR-184.

Bottom Line: Recently, microRNAs (miRNAs) have been suggested to be closely associated with tumorigenesis.Overexpression of miR-184 in HCC cells increased cell proliferation, tumorigenicity, and cell cycle progression, whereas inhibition of miR-184 reduced cell proliferation, tumorigenicity, and cell cycle progression.Ectopic expression of miR-184 led to downregulation of the SOX7 protein, resulting in upregulation of c-Myc, Cyclin D1, and phosphorylation of Rb.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Zengcheng People's Hospital (BoJi-Affiliated Hospital of Sun Yat-Sen University), Zengcheng, China.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common human malignancies and the third leading cause of cancer mortality worldwide. The development and progression of HCC is a complicated process, involving the deregulation of multiple genes that are essential to cell biological processes. Recently, microRNAs (miRNAs) have been suggested to be closely associated with tumorigenesis. Our study showed that miR-184 is upregulated in HCC cell lines and tissues. Overexpression of miR-184 in HCC cells increased cell proliferation, tumorigenicity, and cell cycle progression, whereas inhibition of miR-184 reduced cell proliferation, tumorigenicity, and cell cycle progression. Additionally, we identified SOX7 as a direct target of miR-184. Ectopic expression of miR-184 led to downregulation of the SOX7 protein, resulting in upregulation of c-Myc, Cyclin D1, and phosphorylation of Rb. Our findings suggested that miR-184 represents a potential onco-miR and plays an important role in HCC progression by suppressing SOX7 expression.

Show MeSH
Related in: MedlinePlus