Hepatitis B Virus Surface Antigen Promotes Stemness of Hepatocellular Carcinoma through Regulating MicroRNA-203a

Background and Aims:Patients with persistent positive hepatitis B surface antigen(HBsAg),even with a low HBVDNA load,have a higher risk of hepatocellular carcinoma(HCC)than those without HBV infection.Given that tumor stemness has a critical role in the occurrence and maintenance of neoplasms,this study aimed to explore whether HBsAg affects biological function and stemness of HCC by regulating microRNA,and to explore underlying mechanisms.Methods:We screened out miR-203a,the most significant down-regulated microRNA in the microarray analysis of HBsAg-positive samples and focused on that miRNA in the ensuing study.In vitro and in vivo functional experiments were performed to assess its regulatory function.The effect of miR-203a on stemness and the possible correlation with BMI1 were analyzed in this study.Results:MiR-203a was significantly down-regulated in HBsAg-positive HCC with the sharpest decrease shown in microarray analysis.The negative correlation between miR-203a and HBsAg expression was confirmed by quantitative real-time PCR after stimulation or overexpression/knockdown of HBsAg in cells.We demonstrated the function of miR-203a in inhibiting HCC cell proliferation,migration,clonogenic capacity,and tumor development in vivo.Furthermore,the overexpression of miR-203a remarkably increases the sensitivity of tumor cells to 5-FU treatment and decreases the proportion of HCC cells with stem markers.In concordance with our study,the survival analysis of both The Cancer Genome Atlas database and samples in our center indicated a worse prognosis in patients with low level of miR-203a.We also found that BMI1,a gene maintains the self-renewal capacity of stem cells,showed a significant negative correlation with miR-203a in HCC specimen(p<0.001).Similarly,opposite BMI1 changes after overexpression/knockdown of miR203a were also confirmed in vitro.Dual luciferase reporting assay suggested that miR-203a may regulate BMI1 expression by direct binding.Conclusions:HBsAg may promote the development of HCC and tumor stemness by inhibiting miR-203a,resulting in poor prognosis.miR-203a may serve as a crucial treatment target in HBsAg-positive HCC.More explicit mechanistic studies and animal experiments need to be conducted as a next step.