HBV Encoded HBV-miR-2 Promotes The Malignant Phenotype Through Downregulating SMAD4 In HCC Cells

Objective: Hepatocellular carcinoma(HCC) is one of the most common tumors, with the higher incidence and mortality rates. Researches have shown that the occurrence of HCC is affected by many risk factors. HCC induced by Hepatitis B virus(HBV) infection is the most prevalent subtype of liver cancer, accounted for 80%, and it is characterized by the higher malignant degree. Accordingly, the mechanisms of the development of HCC need to be furtherexplored.Previous studies showed that cellular micro RNAs(miRNAs) were involved in the development of HCC by accelerating the malignancy. However, whether HBV can encode miRNAs that contribute to HCC is not entirely clear. In order to explore the molecular mechanism of the development of HCC induced by HBV persistent infection, we applied solexa small RNA sequencingto analyze the miRNAswith differential expression in HBV positive and negative HCC tissues, and obtained one HBV genome encoded candidate miRNA(named as HBV-miR-2) with the length of 22 nts that matched to HBV genome and homology with HBV transcripts. In this study, we mainly determined to confirm the existence of HBV encoded HBV-miR-2 and evaluate its functional role and the underlying mechanism in the HCC development.Methods: Firstly, in order to verify the existence of HBV-miR-2, HBV-miR-2 expression levels were detected in HBV positive liver cancer tissues and serum samples through RT-q PCR; Northern blot was performed to further confirm the expression of HBV-miR-2 in HBV positive Hep G2.2.15 cells. Functional experiments including MTT, cloning formation, transwell migration and invasion assays to analyze the effects of HBV-miR-2 on HCC malignant phenotype. To further explore the underlying mechanism,bioformatics were used to predict its cellular targets to elucidate the target gene which may mediate the effect of HBV-miR-2 on cell malignant behavior. Combined with the functional annotation analysis, we choose SMAD4 as a candidate target gene of HBV-miR-2 for further validation.EGFP fluorescence reporter assays were emploied to confirm the direct binding between SMAD4 and HBV-miR-2, and RT-q PCR and western blot analysis were applied to analyze the regulation relationship between HBV-miR-2 and SMAD4. In addition, we also detected the effect of target gene SMAD4 on HCC malignant phenotype. Finally, rescue experiments were used to confirm that the effects of HBV-miR-2 on cellular malignant behaviors were directly due to the suppression of SMAD4.Results: HBV-miR-2 encoded by HBV was identified and verified in HBV positive HCC tissues and serum.Northern blotting assays were used to further confirm the existence of HBV-miR-2 in HBV positive Hep G2.2.15 cells but not in HBV negative Hep G2 cells.Functionalexperiments showed that HBV-miR-2 promoted liver cancer cell proliferation, migration and invasion ability.SMAD4 was validated as a direct target of HBV-miR-2 and negatively regulated by it. In addition, we found that SMAD4 inhibited liver cancer cell proliferation, migration and invasion abilities.Moreover, ectopic expression of SMAD4 could partially counteract the malignancy phenotype induced by HBV-miR-2 in HCC cells, indicating that SMAD4 partially counteract the oncogeneic activities induced by HBV-miR-2.Conclusions: In this study, weidentified and confirmed the exsitence of HBV-miR-2 encoded by HBV. HBV-miR-2 promoted liver cancer cell malignant phenotype, including proliferation, migration and invasion. SMAD4 as a direct functional target gene of HBV-miR-2inhibited the malignant phenotype of tumor cells.