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MicroRNA-1269 promotes proliferation in human hepatocellular carcinoma via downregulation of FOXO1.

Yang XW, Shen GZ, Cao LQ, Jiang XF, Peng HP, Shen G, Chen D, Xue P - BMC Cancer (2014)

Bottom Line: Furthermore, we demonstrated that FOXO1 was a direct target of miR-1269.Suppression of FOXO1 by miR-1269 was associated with dysregulation of p21, cyclin D1, phosphorylated Rb and Ki67 expression, thereby playing an essential role in the growth of HCC cells.Our study indicated that overexpression of miR-1269 promotes cell proliferation in HCC through directly suppressing FOXO1, and functions as an oncomiR in HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Guangzhou, Medical University, Guangzhou 510260, China. drxueping@medmail.com.cn.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies and a major cause of cancer-related mortality in the world. MicroRNAs (miRNAs) are small, noncoding RNAs that play essential roles in various stages during cancer progression. The aim of the current study was to elucidate the role of miR-1269 in the pathogenesis of HCC.

Methods: The expression of miR-1269 in HCC cells and tissues were determined by Real-time PCR analysis. Cell viability, colony formation and anchorage-independent growth ability assays were performed to examine cell proliferative capacity and tumorigenicity. Flow cytometry analysis was conducted to determine cell cycle progression. The expression of p21, CyclinD1, phosphorylated Rb, Rb and FOXO1 were examined by Western blotting analysis. Luciferase assay was used to determine whether FOXO1 is the direct target of miR-1269.

Results: miR-1269 was upregulated in HCC cells and tissues. Ectopic miR-1269 expression promoted, but inhibition of miR-1269 reduced, proliferation, tumorigenicity and cell cycle progression of HCC cells. Furthermore, we demonstrated that FOXO1 was a direct target of miR-1269. Suppression of FOXO1 by miR-1269 was associated with dysregulation of p21, cyclin D1, phosphorylated Rb and Ki67 expression, thereby playing an essential role in the growth of HCC cells.

Conclusions: Our study indicated that overexpression of miR-1269 promotes cell proliferation in HCC through directly suppressing FOXO1, and functions as an oncomiR in HCC.

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Related in: MedlinePlus

FOXO1 is a direct target of miR-1269. A. Sequence alignment of miR-1269, miR-1269 mutant (miR-1269-mut), and putative FOXO1-3’UTR. B. Real-time PCR analysis of miR-1269 and western blotting analysis of FOXO1 in 8 paired cancerous tissues (T) and their adjacent noncancerous hepatic tissues (N). The quantification of western blotting bands is performed by OD value determined by Quantity One 4.6.2 software. C. Western blotting analysis of expression levels of FOXO1 and phosphorylated FOXO1 in the indicated cells. α-Tubulin served as the loading control. D. The FOXO1 luciferase reporter activity in the indicated cells transfected with miR-1269 mimic, or miR-1269-mut, miR-1269 inhibitor, or negative control. E. Real-time PCR analysis of mRNA expression of p21, Cyclin D1 and Rb in the indicated HCC cell lines. F. Western blotting analysis of p21, cyclin D1, Ki67, p-Rb, and Rb in the 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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Fig4: FOXO1 is a direct target of miR-1269. A. Sequence alignment of miR-1269, miR-1269 mutant (miR-1269-mut), and putative FOXO1-3’UTR. B. Real-time PCR analysis of miR-1269 and western blotting analysis of FOXO1 in 8 paired cancerous tissues (T) and their adjacent noncancerous hepatic tissues (N). The quantification of western blotting bands is performed by OD value determined by Quantity One 4.6.2 software. C. Western blotting analysis of expression levels of FOXO1 and phosphorylated FOXO1 in the indicated cells. α-Tubulin served as the loading control. D. The FOXO1 luciferase reporter activity in the indicated cells transfected with miR-1269 mimic, or miR-1269-mut, miR-1269 inhibitor, or negative control. E. Real-time PCR analysis of mRNA expression of p21, Cyclin D1 and Rb in the indicated HCC cell lines. F. Western blotting analysis of p21, cyclin D1, Ki67, p-Rb, and Rb in the indicated HCC cells. α-Tubulin served as the loading control. Each bar represents the mean ± SD of three independent experiments. * P < 0.05.

Mentions: It is known that miRNAs function by negatively regulating mRNAs via targeting their 3’UTRs. Thus, we used publicly available algorithms (TargetScan 6.2) to predict the potential targets of miR-1269. As shown in Figure 4A, there are three miR-1269 binding sites in the FOXO1 mRNA 3’UTR, including one conserved and two poorly conserved binding sites. Importantly, western blotting analysis showed that FOXO1 expression was downregulated in the eight pairs of HCC tissue, compared with the adjacent noncancerous hepatic tissues, and statistical analysis revealed that miR-1269 levels inversely correlated with the expression of FOXO1 in the clinical HCC samples (Figure 4B), suggesting that miR-1269 might play role in regulation of FOXO1 in HCC. Indeed, FOXO1 expression was found to be downregulated in the miR-1269-overexpressing BEL-7404 and Huh7 cells but upregulated in the cells transfected with miR-1269 inhibitor. In addition, overexpressing miR-1269 decreased the expression of p-FOXO1, but we did not found significant alterations of FOXO1 in miR-1269-inhibited cells (Figure 4C). To further confirm the direct regulation of FOXO1 by miR-1269, the pGL3-FOXO1-3’-UTR-luciferase reporter, containing the three putative miR-1269 binding sites, was constructed. The luciferase assay results showed that ectopic overexpression of miR-1269 decreased, but inhibition of miR-1269, increased luciferase activity of the pGL3-FOXO1-3’-UTR reporter (Figure 4D). Meanwhile, mutant miR-1269 failed to show an inhibitory effect on luciferase expression driven by the pGL3-FOXO1 -3’-UTR-luciferase reporter (Figure 4D). Collectively, these results suggest that miR-1269 directly targets FOXO1 in HCC cells.Figure 4


MicroRNA-1269 promotes proliferation in human hepatocellular carcinoma via downregulation of FOXO1.

Yang XW, Shen GZ, Cao LQ, Jiang XF, Peng HP, Shen G, Chen D, Xue P - BMC Cancer (2014)

FOXO1 is a direct target of miR-1269. A. Sequence alignment of miR-1269, miR-1269 mutant (miR-1269-mut), and putative FOXO1-3’UTR. B. Real-time PCR analysis of miR-1269 and western blotting analysis of FOXO1 in 8 paired cancerous tissues (T) and their adjacent noncancerous hepatic tissues (N). The quantification of western blotting bands is performed by OD value determined by Quantity One 4.6.2 software. C. Western blotting analysis of expression levels of FOXO1 and phosphorylated FOXO1 in the indicated cells. α-Tubulin served as the loading control. D. The FOXO1 luciferase reporter activity in the indicated cells transfected with miR-1269 mimic, or miR-1269-mut, miR-1269 inhibitor, or negative control. E. Real-time PCR analysis of mRNA expression of p21, Cyclin D1 and Rb in the indicated HCC cell lines. F. Western blotting analysis of p21, cyclin D1, Ki67, p-Rb, and Rb in the 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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Fig4: FOXO1 is a direct target of miR-1269. A. Sequence alignment of miR-1269, miR-1269 mutant (miR-1269-mut), and putative FOXO1-3’UTR. B. Real-time PCR analysis of miR-1269 and western blotting analysis of FOXO1 in 8 paired cancerous tissues (T) and their adjacent noncancerous hepatic tissues (N). The quantification of western blotting bands is performed by OD value determined by Quantity One 4.6.2 software. C. Western blotting analysis of expression levels of FOXO1 and phosphorylated FOXO1 in the indicated cells. α-Tubulin served as the loading control. D. The FOXO1 luciferase reporter activity in the indicated cells transfected with miR-1269 mimic, or miR-1269-mut, miR-1269 inhibitor, or negative control. E. Real-time PCR analysis of mRNA expression of p21, Cyclin D1 and Rb in the indicated HCC cell lines. F. Western blotting analysis of p21, cyclin D1, Ki67, p-Rb, and Rb in the indicated HCC cells. α-Tubulin served as the loading control. Each bar represents the mean ± SD of three independent experiments. * P < 0.05.
Mentions: It is known that miRNAs function by negatively regulating mRNAs via targeting their 3’UTRs. Thus, we used publicly available algorithms (TargetScan 6.2) to predict the potential targets of miR-1269. As shown in Figure 4A, there are three miR-1269 binding sites in the FOXO1 mRNA 3’UTR, including one conserved and two poorly conserved binding sites. Importantly, western blotting analysis showed that FOXO1 expression was downregulated in the eight pairs of HCC tissue, compared with the adjacent noncancerous hepatic tissues, and statistical analysis revealed that miR-1269 levels inversely correlated with the expression of FOXO1 in the clinical HCC samples (Figure 4B), suggesting that miR-1269 might play role in regulation of FOXO1 in HCC. Indeed, FOXO1 expression was found to be downregulated in the miR-1269-overexpressing BEL-7404 and Huh7 cells but upregulated in the cells transfected with miR-1269 inhibitor. In addition, overexpressing miR-1269 decreased the expression of p-FOXO1, but we did not found significant alterations of FOXO1 in miR-1269-inhibited cells (Figure 4C). To further confirm the direct regulation of FOXO1 by miR-1269, the pGL3-FOXO1-3’-UTR-luciferase reporter, containing the three putative miR-1269 binding sites, was constructed. The luciferase assay results showed that ectopic overexpression of miR-1269 decreased, but inhibition of miR-1269, increased luciferase activity of the pGL3-FOXO1-3’-UTR reporter (Figure 4D). Meanwhile, mutant miR-1269 failed to show an inhibitory effect on luciferase expression driven by the pGL3-FOXO1 -3’-UTR-luciferase reporter (Figure 4D). Collectively, these results suggest that miR-1269 directly targets FOXO1 in HCC cells.Figure 4

Bottom Line: Furthermore, we demonstrated that FOXO1 was a direct target of miR-1269.Suppression of FOXO1 by miR-1269 was associated with dysregulation of p21, cyclin D1, phosphorylated Rb and Ki67 expression, thereby playing an essential role in the growth of HCC cells.Our study indicated that overexpression of miR-1269 promotes cell proliferation in HCC through directly suppressing FOXO1, and functions as an oncomiR in HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Guangzhou, Medical University, Guangzhou 510260, China. drxueping@medmail.com.cn.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies and a major cause of cancer-related mortality in the world. MicroRNAs (miRNAs) are small, noncoding RNAs that play essential roles in various stages during cancer progression. The aim of the current study was to elucidate the role of miR-1269 in the pathogenesis of HCC.

Methods: The expression of miR-1269 in HCC cells and tissues were determined by Real-time PCR analysis. Cell viability, colony formation and anchorage-independent growth ability assays were performed to examine cell proliferative capacity and tumorigenicity. Flow cytometry analysis was conducted to determine cell cycle progression. The expression of p21, CyclinD1, phosphorylated Rb, Rb and FOXO1 were examined by Western blotting analysis. Luciferase assay was used to determine whether FOXO1 is the direct target of miR-1269.

Results: miR-1269 was upregulated in HCC cells and tissues. Ectopic miR-1269 expression promoted, but inhibition of miR-1269 reduced, proliferation, tumorigenicity and cell cycle progression of HCC cells. Furthermore, we demonstrated that FOXO1 was a direct target of miR-1269. Suppression of FOXO1 by miR-1269 was associated with dysregulation of p21, cyclin D1, phosphorylated Rb and Ki67 expression, thereby playing an essential role in the growth of HCC cells.

Conclusions: Our study indicated that overexpression of miR-1269 promotes cell proliferation in HCC through directly suppressing FOXO1, and functions as an oncomiR in HCC.

Show MeSH
Related in: MedlinePlus