Novel-miR-10 Induced By Hepatitis B Virus Facilitates Malignancy In Hepatoma Cells

[Objective] Hepatocellular carcinoma(HCC) is one of the most common and aggressive human malignancies. Due to the lack of optimize strategies for the early diagnosis and therapy, the prognosis of HCC is poor. Hepatitis B virus(HBV) infection is a major risk factor for hepatocellular carcinoma. The HBV infection lead to chronic hepatitis, which may eventually develop into HCC. The process and mechanism from the HBV associated chronic hepatitis to HCC is complex and unclear. microRNAs are a class of about 22 nt non-coding RNA regulating the gene expression at the posttranscriptional level. Previous study has shown that HBV infection lead to the dysregulation of miRNAs, which may play important roles in HBV infection and HBV associated HCC. miRNAs may regulate oncogenes or tumor suppressors to regulate cell viability, proliferation, migration and invasion and thus affect the development of HCC. We suspect that the dysregulation of miRNAs resulted from HBV infection may contribute to the HBV associated HCC. Here, we aimed to explore the different expression profile of miRNAs in HBV infection HCC and their roles on the development of HCC. We presented a new machenism by which HBV promoted the development of HCC. [Methods] Solexa sequencing was used to screen the dysregulated miRNAs in HBV positive and negative HCC patients. qRT-PCR was used to verify the effect of HBV on the expression of novel-miR-10. Dual luciferase reporter assay was used to test the influence of HBV, HBx and transcription SP1 and CREB1 on the activity of the novel-miR-10 promoter. The MTT assay, colony formation assay, transwell migration and invasion assay were used to demonstrate the biological function of novel-miR-10 on the HCC cells. The FACS was adopted to test the apoptosis rate of HCC cells and western bolt assay was used to test the EMT process of HCC cells. Candidate targets of novel-miR-10 were screened by bioinformatics methods and the EGFP reporter assay, real-time PCR assay and western blot assay were used to verify it. The MTT assay, colony formation assay, transwell migration and invasion assays were used to explore the influence of TNFRSF19 and RAB43 on the malignant phenotypes of HCC cells. The rescue assays demonstrated that the effect of novel-miR-10 on HCC cells was mediated by TNFRSF19 and RAB43. [Results] Through the solexa sequencing we found some novel miRNAs and one of them novel-mi R-10 shows higher level in HBV positive HCC tissues than the HBV negative HCC tissues. Ectopic expression of HBV1.3copy and HBx protein induces the expression of novel-miR-10 and the activity of novel-miR-10 promoter. The transcription factor SP1 inhibites and CREB1 promotes the activity of novel-miR-10 promoter and CREB1 mediates the promotion of HBx on the novel-miR-10 promoter. The ectopic expression of novel-miR-10 promotes the cell viability, colony formation rate, migration and invasion activities in HCC cells. TNFRSF19 and RAB43 as two targets of novel-miR-10 and decreasing the expression of these two genes counteracts the inhibition of the aggressive malignance induced by ASO-novel-miR-10. [Conclusions] HBV promoted the expression of novel-miR-10 mediated by the promotion effect of HBx and CREB1 on the activities of novel-miR-10 promoter. Novel-miR-10 promoted the malignant phenotypes included the cell viabilities, cell proliferation, migration and invasion abilities and acted as an oncogene in HCC cells, which is mediated by targeting the TNFRSF19 and RAB43 genes. All the results present a new mechanism of HBV inducing the development of HCC.