The Study On The Role And Mechanism Of BARD1 In Malignant Progression Of Hepatocellular Carcinoma

Objective: Hepatocellular carcinoma(HCC)is the most important primary liver cancer and is a common type of cancer in the world.In the previous genome-wide microarray analysis,we found that gene BARD1 was significantly up-regulated in HCC tissues.The purpose of this study was to investigate the potential clinical,pathological and prognostic value of BARD1 in HCC as well as its roles and mechanisms in HCC.Methods :1.The expression level of BARD1 m RNA was detected in 209 paired liver cancer tissue samples and corresponding adjacent normal liver tissues(ANLT)samples by quantitative real-time polymerase chain reaction(q RT-PCR).And the expression level of BARD1 protein in 70 matched HCC tissue samples and corresponding ANLT samples was detected by using Immunohistochemistry(IHC).2.Clinical data of 209 patients with liver cancer were collected.Kaplan-Meier analysis was used to evaluate the progression-free survival(PFS)and overall survival(OS)of patients with hepatocellular carcinoma,and the relationship between the expression of BARD1 and the prognosis of patients with liver cancer was further evaluated by univariate and multivariate analysis.3.The hepatoma cell lines SMMC7721 and Huh7 were transiently transfected with three pairs of BARD1 small interfering RNA(si RNA)and one pair of negative control sequences designed and synthesized by Shanghai Gene Pharma Technology Co.,Ltd.After 48 hours of transfection,total cellular RNA was extracted and the silencing effect of small RNA was detected by q RT-PCR.Small RNAs with the best silencing effect were used for subsequent cytological functional assays.4.The knockdown BARD1 cell model constructed by the best silencing effect si RNA-1 was used in a plate clone assay to test the effect of down-regulated of BARD1 on the proliferation of hepatoma cells.5.Using si RNA-1 to silence BARD1 for transwell invasion and migration experiments and analyze the effect of down-regulated BARD1 on malignant biological behaviors such as invasion and migration in HCC cells.6.To analyze the changes of Akt,m TOR and MMP-9 levels and Akt,m TOR phosphorylation in down-regulated BARD1 of hepatocellular carcinoma cells by Western blotting assay and explore the mechanism of BARD1 in the progress of HCC.Results:1.The expression of BARD1 m RNA and protein in HCC tissues was significantly higher than that in adjacent normal liver tissues.2.The positive expression of BARD1 in HCC patients was positively correlated with tumor-node-metastasis stage(TNM),Barcelona-Clinic Liver Cancer stage(BCLC),hepatitis B surface antigen(HBs Ag),tumor size,serum alpha-fetoprotein(AFP)level and serum aspartate aminotransferase level.Univariate analysis and multivariate analysis showed that BARD1 was an independent predictor of progression-free survival and overall survival in patients with HCC.3.In vitro cell biology experiments showed that knockdown of BARD1 expression can significantly inhibit the proliferation,invasion and migration of HCC cells.4.Silencing BARD1 can downregulate the expression of Akt,m TOR and MMP-9,and inhibit the phosphorylation of Akt(Ser473)and m TOR(Ser2248).Conclusions: BARD1 is significantly up-regulated in HCC tissues and promotes the malignant behavior of HCC cells.Meanwhile,the up-regulation of BARD1 is significantly associated with poor prognosis of HCC patients.In addition,up-regulated BARD1 can promote the progression of HCC by targeting Akt signaling pathway.Therefore,BARD1 may be a new diagnostic and prognostic biomarker for HCC.