| Objective To investigate the expression of miR-542-3p in hepatocarcinoma(HCC)tissues and cell lines,and to explore the mechanism of miR-542-3p on the growth and cell proliferation of HCC.Thus may provide a new potential therapeutic target for liver cancer.Materials and Method 1.Surgical specimens of patients with HCC were collected to determine the expression of miR-542-3p in HCC tissues and adjacent normal liver tissues,and to analyze the effect of different expression levels of miR-542-3p on patients' overall survival.2.The expression levels of miR-542-3p in different HCC cell lines(Huh7,HepG2,Hep3 B and MHCC-LM9)were detected,and the miR-542-3p was overexpressed and downgraded respectively to verify its effect on the growth of HCC cells in vitro and vivo.In vitro,CCK-8,flow cytometry and luciferase reporter gene assay were used to detect cell proliferation,cell cycle and apoptosis,and to verify the interaction between miR-542-3p and survivin,respectively.Animal experiments were performed using nude mice to explore the effect of miR-542-3p on tumor growth in vivo.3.Bioinformatics analysis was used to find potential targets of miR-542-3p and to verify the targeting relationship between survivin and miR-542-3p by qRT-PCR,WB assay and luciferase reporter gene assay.The correlation of expression levels in liver cancer tissues was examined and the effect of miR-542-3p/survivin on HCC cell proliferation and tumor growth was verified.4.Through the overexpression of the target gene survivin,qRT-PCR,CCK-8,tumor formation in nude mice and other experiments to detect the biological role of survivin in the growth of HCC,and further verify the miR-542-3p/survivin axis on the effect of HCC cells growth.Results 1.The expression of miR-542-3p was different in HCC tissues and adjacent normal liver tissues.The expression level of miR-542-3p in HCC tissues was significantly lower than that in adjacent normal tissues(P<0.01).The expression of miR-542-3p was significantly different in HCC cell lines and normal liver cell lines.The expression level of miR-542-3p in HCC cell lines was significantly lower than that in normal liver cell lines(P<0.01).The overall survival rate of patients with high expression of miR-542-3p and low expression of miR-542-3p was different.The overall survival rate of patients with high expression of miR-542-3p was significantly higher than that of low expression level(P=0.007).2.After overexpression of miR-542-3p,the proliferation,cell growth cycle and anti-apoptosis ability of HepG2 and Huh7 cells were inhibited(P<0.05).The results of animal experiments also showed that the tumor growth ability of HepG2 and Huh7 cell lines was also significantly inhibited when the miR-542-3p was overexpressed(P<0.05).3.The results of bioinformatics analysis and further luciferase reporter assay showed that surivin is a potential target of miR-542-3p.When overexpresses miR-542-3p,the luciferase activity of luciferase vector,which contains the 3'-untranslated region of survivin and the expression of survivin protein were significantly decreased(P<0.05).After decreasing the expression of miR-542-3p,luciferase activity and the expression of survivin protein were significantly increased(P<0.05).4.Overexpression of the target gene survivin reverses the proliferation of HCC cells caused by miR-542-3p overexpression and the inhibition of tumor growth after subcutaneous tumor formation in nude mice.MiR-542-3p overexpression + Survivin overexpression group significantly increased cell proliferation ability compared with miR-542-3p,only,overexpression group(P<0.05).Animal experiments also showed that the tumor growth ability of miR-542-3p overexpression + survivin overexpression group was significantly higher than that of solely miR-542-3p overexpression group(P<0.05).5.The relationship between expression of survivin mRNA and miR-542-3p in human HCC tissues was further analyzed by qRT-PCR.It was found that there was a negative correlation between survivin mRNA and miR-542-3p expression(Pearson correlation coefficient =-0.4594;P <0.05).Conclusion The expression of miR-542-3p is down-regulated in HCC cell lines and tissues.Overexpression of miR-542-3p can inhibit the growth and proliferation of HCC cells in vitro and vivo;MiR-542-3p exerts tumor suppressive effect by directly targeting survivin.The interaction between miR-542-3p and survivin may have an important impact on the development of liver cancer;MiR-542-3p / survivin axis may be a potential diagnostic marker and therapeutic target for HCC. |
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