Metformin Induces MiR-378 To Downregulate The CDK1,Leading To Suppression Of Cell Proliferation In Hepatocellular Carcinoma

Background:Primary liver cancer is a common malignant tumor.It has high incidence rate in Asia,Africa and southern Europe.There are more than half of all cases of liver cancer in China.Hepatocellular carcinoma(HCC)is the most common form in primary liver cancer.It has the characteristics of high malignancy,occult occultation,easy metastasis and recurrence.The current treatment is mainly based on surgery.However,most patients with HCC are in advanced stage at the time of diagnosis and cannot be treated by surgical and the prognosis is poor.Metformin is one of the most widely used oral hypoglycemic agents,and an increasing epidemiological evidence suggests that metformin is associated with a reduced risk of various cancers.It has been shown to be a possible anti-tumor molecular mechanism.Recent studies have demonstrated that metformin has an anti-tumor effect on hepatocellular carcinoma(HCC).However,the molecular mechanism of this effect requires further investigation.Objective:To investigate the effects of metformin on the proliferation and apoptosis of HCC cells and explore the molecular mechanisms involved,and to discover new biomarkers and diagnosis methods of HCC.Method:1.CCK-8(cell counting kit-8)and flow cytometry were used to detect the effect of metformin on the proliferation and apoptosis of HCC cells.The expression of miR-378 was further detected by Real-time PCR,changes in the expression of CDK1 protein and mRNA levels were detected using Real-time PCR and western blot.2.Overexpression of miR-378 in vitro by miR-378 mimic transfection,the transfection efficiency was detected by Real-time PCR.The effect of overexpression of miR-378 on proliferation and cell cycle of hepatoma cells was detected by CCK-8 and flow cytometry.3.The use of bioinformation and luciferase reporter assays revealed the binding sites for CDK1 and miR-378.The effects of overexpression of miR-378 on CDK1 mRNA and CDK1 protein were detected by Real-time PCR and Western blot.4.The efficiency of overexpression of miR-378 in transfected mice was detected by Real-time PCR.The effect of overexpression of miR-378 on CDK1 mRNAs and protein in mice was detected by Real-time PCR and western blot.5.Western blot,CCK-8 and flow cytometry were used to observe whether miR-378 inhibitor can eliminate the effects of metformin on CDK1 expression,cell proliferation,cell cycle and apoptosis.Result:1.Metformin inhibits HCC cell proliferation with changes in CDK1 and miR-378 expression.2.Overexpression of miR-378 in vitro inhibits HCC cell proliferation.3.MiR-378 directly inhibits CDK1 expression by targeting its 3'-UTR.4.Tumor growth overexpressing miR-378 in vivo and inhibiting CDK1 expression.5.Metformin inhibits CDK1 expression and cell proliferation by promoting miR-378 levels in HCC cells.Conclusion: Metformin targets down-regulated CDK1 levels and inhibits cell proliferation of hepatocellular carcinoma by inducing miR-378 targeting.