MicroRNA-139-3p Acts As A Tumor Suppressor By Targeting Regulation Of CDKL3 In Hepatocellular Carcinoma

Research purpose In view of the widely dysregulated expression of miR-139-3p in a variety of cancers,to investigate the biological function of miR-139-3p in hepatocellular carcinoma,to find the downstream target genes of miR-139-3p,and to clarify the molecular mechanism of miR-139-3p promoting the progression of hepatocellular carcinoma cells,so as to improve the treatment and prognosis of patients with hepatocellular carcinoma.The search for new therapeutic targets raises the possibility.MethodsBioinformatics tools were used to analyze the clinical significance of miR-139-3p with tumor prognosis,stage,and pathological type.RT-q PCR was used to verify the difference in the expression of miR-139-3p between normal liver cell lines and hepatocellular carcinoma cell lines.After transfection of miR-139-3p mimics/inhibitor in hepatocellular carcinoma cells,CCK-8 cell proliferation assay,plate colony formation and Ed U labeling were used to verify the effect of miR-139-3p on the proliferation of hepatocellular carcinoma cells.Wound healing assay and Transwell cell migration and invasion assay were used to evaluate the effect of miR-139-3p on the migration and invasion of hepatocellular carcinoma cells.Flow cytometry and apoptosis were used to verify the effect of miR-139-3p on the division cycle and apoptosis of hepatocellular carcinoma cells.Bioinformatics tools were used to analyze the expression level of CDKL3 in HCC tissue samples and prognosis overall survival time.Western blot and RT-q PCR were used to verify the high expression level of CDKL3 in HCC cells.The CDKL3 overexpression and silencing lentiviral vectors were constructed,and the transfection efficiency was verified by infecting Hep G2 and SKHep-1 human hepatocellular carcinoma cell lines.Subsequently,CCK8 cell proliferation assay and Ed U labeling were performed to detect the effect of overexpression/silencing of CDKL3 on the proliferation of hepatocellular carcinoma cells mediated by miR-139-3p mimics/inhibitor.Wound healing assay and Transwell migration assay were used to detect the effect of overexpression/knockdown of CDKL3 on the migration ability of hepatocellular carcinoma cells mediated by miR-139-3p mimics/inhibitor.Flow cytometry and apoptosis were used to verify the effect of overexpression/silencing CDKL3 on the cycle progression and apoptosis rate of hepatocellular carcinoma cells mediated by miR-139-3p mimics/inhibitor.The xenograft model in nude mice was constructed to verify the regulatory effect of miR-139-3p on the progression of hepatocellular carcinoma in vivo.At the same time,the lung metastasis model of hepatocellular carcinoma in nude mice was established to verify the effect of miR-139-3p on lung metastasis of hepatocellular carcinoma in vivo.Results1.Compared with the normal control group,the expression of miR-139-3p was downregulated in hepatocellular carcinoma tissues,and its expression level was positively correlated with the overall survival rate of hepatocellular carcinoma patients.2.miR-139-3p inhibits the proliferation,migration and invasion of hepatocellular carcinoma cells,hinders cell cycle progression,and induces apoptosis.3.Compared with the normal control group,the expression of CDKL3 in HCC tissue samples and HCC cell lines is up-regulated,and its expression level is negatively correlated with the survival time of HCC patients.It may become one of the indicators to evaluate the prognosis of HCC patients.4.CDKL3 is a direct downstream target of miR-139-3p,and miR-139-3p can negatively regulate CDKL3 expression by directly binding to its 3’UTR.5.miR-139-3p targeted and negatively regulated CDKL3 to inhibit the progression of the malignant phenotype of hepatocellular carcinoma cells,that is,overexpression of CDKL3 reversed the inhibitory effect of miR-139-3p mimics on the malignant phenotype of hepatocellular carcinoma cells.Silencing CDKL3 rescued the malignant biological behavior of miR-139-3p inhibitor in HCC cells.6.The results of in vivo experiments showed that compared with the control group,the tumor formation ability of hepatocellular carcinoma cells overexpressing miR-139-3p was significantly reduced in nude mice,the tumor volume was smaller,the growth rate was slower,the level of apoptosis was increased,and the ability of metastasis and invasion in vivo was decreased,which were opposite after silencing miR-139-3p.ConclusionmiR-139-3p can inhibit the growth,metastasis and invasion of hepatocellular carcinoma in vitro and in vivo,suggesting that it may be an effector regulating the growth of hepatocellular carcinoma.CDKL3 is a direct downstream target of miR-139-3p,and miR-139-3p targets and negatively regulates CDKL3 expression to affect the development of hepatocellular carcinoma phenotype.