MiR-221 Promotes Hepatocellular Carcinoma Cells Migration via Targeting PHF2 - PubMed (original) (raw)

MiR-221 Promotes Hepatocellular Carcinoma Cells Migration via Targeting PHF2

Yi Fu et al. Biomed Res Int. 2019.

Abstract

MicroRNAs (MiRNAs), which regulate the gene expression leading to translational inhibition or mRNA degradation, are involved in carcinogenesis and tumor progression. Previous studies have demonstrated that miR-221 was one of the most consistent overexpressed miRNAs in several types of cancer. However, the role of miR-221 in human liver cancer progression is not yet fully elucidated. Levels of miR-221 and plant homeodomain finger 2 (PHF2) expressions in human hepatocellular carcinoma (HCC) tissues and cell lines were detected using western blotting and quantitative real-time PCR (qRT-PCR). Cell migration was studied using the transwell assays. A dual-luciferase reporter system was used to validate the target gene of miR-221. The results indicated that miR-221 promoted HCC cell migration. By performing subsequent systematic bioinformatic analyses, we found PHF2 was the target gene of miR-221 and the direct binding relationship was further validated by dual-luciferase reporter assay. In addition, lower expression of PHF2 promoted HCC cell migration and linked to worse overall survival in HCC patients. Finally, the negative correlation between miR-221 and PHF2 expression levels in HCC specimens was further confirmed. Taken together, our findings implied that miR-221 could be a potential candidate for the therapeutics of HCC metastasis.

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Figures

Figure 1

Figure 1

miR-221 is upregulated in HCC tissues and cell lines and promotes the cell migration. (a) qRT-PCR results of miR-221 mRNA levels in HCC cell lines and human normal hepatocyte. (b) Quantitative PCR results of miR-221 mRNA levels in HCC tissues (T) and in adjacent noncancerous tissues (N). MiR-221 mRNA levels are higher in the HCC tissues (T) than in adjacent noncancerous tissues (N) (n=12). (c) miR-221 increases SMMC-7721 cells wound healing. Anti-miR-221 inhibits SMMC-7721 cells wound healing. Lines indicate the border of the healing wounds. (d) The cell migration of SMMC-7721 cells after transfection with miR-221 or anti-miR-221 and their negative control detected by transwell assays. Each bar represents the mean ± SD of three independent experiments. ∗∗P<0.01.

Figure 2

Figure 2

MiR-221 has effects on some key metastasis-related proteins. (a-b) qRT-PCR was used to detect E-cadherin and N-cadherin mRNA level in miR-221-overexpression and miR-221-knockdown SMMC-7721 cells. (c-d) Western blot was used to detect E-cadherin and N-cadherin protein level in miR-221-overexpression and miR-221-knockdown SMMC-7721 cells. Each bar represents the mean ± SD of three independent experiments. ∗∗P<0.01.

Figure 3

Figure 3

PHF2 is a direct target of miR-221. (a) miRNAs were computationally predicted using two independent miRNA databases. The PHF2 protein level was measured by western blot analysis in SMMC-7721 cells transfected with miR-221 or anti-miR-221. (b) qRT-PCR results of PHF2 mRNA levels after transfecting SMMC-7721 cells with miR-221 or anti-miR-221. (c) Schematic description of wild type (WT) and mutated 3′-UTR of the PHF2 mRNA. The WT and mutated 3′UTR sequences were inserted into luciferase reporter plasmids. In luciferase activity assays, miR-221 suppressed luciferase activity of the wild type but not mutant PHF2 3′-UTR constructs in SMMC-7721 cells. (d) Knockdown of PHF2 promotes the migration of SMMC-7721 cells. Overexpression of PHF2 inhibits the migration of SMMC-7721 cells. (e) Restoration of PHF2 rescues miR-221 mediated promotion of cell migration. Each bar represents the mean ± SD of three independent experiments. ∗∗P<0.01.

Figure 4

Figure 4

PHF2 is decreased in hepatocellular carcinoma tissues and associated with clinicopathological features in 60 hepatocellular cancer patients. (a) Representative photos of PHF2 expression patterns in hepatocellular tumors. The left panel depicts matched adjacent cancer tissues, whereas the right panel represents the hepatocellular carcinoma tissues (original magnification, ×100). The magnifying detail of the immunohistochemical analysis for each case can be shown in the bottom side (original magnification, ×200). (b) Compared with the adjacent cancer tissues, the overall expression level of PHF2 in the hepatocellular carcinoma tissues was significantly lower, P<0.05, _χ_2 test. PHF2 expression was correlated with TNM stage, P<0.05, _χ_2 test. (c) Kaplan-Meier estimates of the probability of 3-year overall survival according to low and high PHF2 expression of 60 patients with hepatocellular cancer. _P_=0.0437, log-rank test. (d) The correlation between the protein expression of miR-221 and PHF2 was measured by Pearson's correlation coefficient analysis.

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