MiR-139-5p Regulates EMT And Inhibits Invasion And Metastasis Of Hepatoma Cells By Targeting TG F-β1 XX (Genneral Surgery)

Objective:To investigate the relationship between expressions of miR-139-5P and transforming growth factor β1(TGF-β1), analyze whether TGF-β1 is the target gene of miR-139-5P, and clarify the related signaling pathway in which miR-139-5P regulates the epithelial mesenchymal trasition(EMT) and the molecular mechanism through which miR-139-5P inhibits invasion and metastasis of hepatoma cells by targeting TG F-β1.Methods:Bioinformatics methods were used at first and 20 miRNAs were found to be possible potential miRNAs of TGF-β1; miR-139-5P was found to be the best binding miRNA of TGF-β1 after further calculation and analysis, and the expression level of TGF-β1 was detected by immunohistochemistry; expressions of miR-139-5P and TGF-β1were detected with qRT-PCR and Western blot, respectively, and the correlation between expressions of them was analyzed; expression of miR-139-5P was detected with qRT-PCR and expressions of TGF-β1, E-cadherin and Vimentin were detected with Western blot in high and low metastatic hepatoma cell lines, and the relationship between expression of miR-139-5P and that of the other proteins were analyzed; in recombinant cell lines―HepG2-miR-139-5p mimics, HepG2-miR-Scramble, HepG2-miR-139-5p-PM and HepG2, qRT-PCR and western blot were used to detect the expressions of miR-139-5P and TGF-β1, respectively, and whether miR-139-5P could bind to the 3` UTR site of TGF-β1 was analyzed; changes in invasive ability of recombinant hepatoma cell lines after changed expression of miR-139-5P were detected with the transwell method. Results:(1) 20 miRNAs were found to bind with TGF-β1 by bioinformatics methods; among these imRNAs, miR-139-5p was the target miRNA with the best specificity and the strongest stability for TGF-β1.(2) Positive expression rate of TGF-β1 in hepatoma tissues was 80.4%(45/56)and that in adjacent normal liver tissues was 15.0%(3/20), with significant difference in positive expression rate between them(P<0.01).(3) miR-139-5p had low expression in hepatoma tissues, while it had high expression in adjacent normal tissues, with statistical difference between them(P<0.01); TGF-β1 had high expression in hepatoma tissues, while it had low expression in adjacent normal tissues, with statistical difference between them(P<0.01). Expressions of miR-139-5p and TGF-β1 were negatively correlated.(4) mi R-139-5p had low expression in high metastatic cell line MHCC97-H and had high expression in low metastatic cell line MHCC97-L, with significant statistical difference between them(P<0.01); TGF-β1, E-cadherin and Vimentin had high expressions in high metastatic cell line MHCC97-H and had low expressions in low metastatic cell line MHCC97-L; there was significant difference among expressions of the three proteins in different metastatic cell lines(P<0.01).(5) miR-139-5p had increased expression in cell line hepG2-miR-139-5p mimics, while TGF-β1 had down-regulated expression(P<0.05); there was no significant difference in expression level of miR-139-5p among the three cell lines―HepG2-miR-Scramble, HepG2-miR-139-5p-PM and HepG2, and there was no significant difference in expression level of TGF-β1 either. mi R-139-5p could bind to the 3`UTR site of TGF-β1.(6) Compared to the other three groups, cell line with high expression of mi R-139-5p had significantly lower count of invasive cells(P<0.01); there was no significant difference in expression level of miR-139-5p amongHepG2-miR-Scramble, HepG2-miR-139-5p-PM and HepG2, and there was also no significant difference in count of invasive cells among them(P>0.05). Conclusions:(1) TGF-β1 has high expression in hepatoma tissues, relating to the occurrence of hepatoma.(2) Expressions of mi RNA-139-5p and TGF-β1are negatively correlated in hepatoma tissues and hepatoma cells, and TGF-β1 may be the target gene of mi RNA-139-5p.(3) mi RNA-139-5p can targetedly bind to the 3`UTR site of TGF-β1 and down-regulate expressions of TGF-β1, E-cadherin and Vimentin.(4) After down-regulating TGF-β1, miRNA-139-5p regulates the EMT signaling pathway and inhibits invasion and metastasis of hepatoma cells.