Study Of The Relationship Between New Melanoma Inhibitory Activity-related Gene MIA₂and Hepatitis B Virus

Hepatitis B virus (HBV) infection is limited to hepatocytes of primate and is associated with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. It is reported that there are five promoter domain in HBV genome, they control the transcription of the virus and the replication of the virus. In the domain of HBV promoter, there are serial of liver specific receptor.Liver enriched nuclear receptors such as HNF1α, HNF4α and PPARα-RXRα play essential roles in the HBV life cycle and are likely to contribute to the strict liver tropism of HBV.Recently, it is reported that there is a new discovered gene specific expressed in liver tissue, belong to MIA family, named MIA2. The new melanoma inhibitory activity-related gene MIA2transcriptional enhanced by HNF-1is exclusively expressed in liver. In this study, we want to elucidate MIA2expression exchange in the course of HBV infection and the role of MIA2in regulation of HBV transcription and replication is investigated. Our study is aimed to clarify the molecular mechanism of liver disease progression in face of HBV infection and provide thesis foundation of liver inflammation and liver carcinoma. In order to study the relationship of MIA2and HBV infection, We detected the MIA2expression level in serum of HBV chronic infection patients and healthy persons. By ELISA detection, we found MIA2level in serum of CHB patients (186.5±35.46pg/ml) is obviously lower then it in healthy persons.Then we detect MIA2mRNA and protein level by RT-PCR and ELISA assay in HepG2and HepG2.2.15cell line. Our results indicated that MIA2expression level is lower in HepG2.2.15(61.94±3.77pg/ml) then it in HepG2cell line(150.51±4.28pg/ml).In order to research the relationship of HBV infection and MIA2, cDNA of MIA2was cloned into eukarya expression vector pCMV-Tag2B, and transfected HBV infectious vector PHBV4.2into HepG2cell line to produce HBV virus particle, and detect HBsAg and HBeAg expression by ELISA and detected core associate DNA in HepG2cells by real time PCR. Our results firstly demonstrated that MIA2significantly increases HBV E and S protein expression as well as viral synthesis. Then we synthesis SiRNA of MIA2and co-transfected with PHBV4.2into HepG2cells, our results indicated that HBV protein expression level and viral synthesis both blocked. The results suggested that MIA2acts as an enhancing factor in HBV replication and may be responsible for HBV liver-tropism.Furthermore, we further investigate the molecular mechanism of HBV replication. MIA2and HBV core/Enhancer Ⅱ promoter were cotransfected into HepG2cells and luciferase were assayed. Our result indicated that MIA2activate HBV core/Enhancer Ⅱ promoter activity. It is reported that there are a serial of transcriptional factor in HBV core/Enhancer Ⅱ promoter, including HNF-1, SP1, C/EBP, HNF-4and PPARa-RXRa. By a serial interfering RNA scan, we found that interfering PPARa expression decrease the activation of MIA2to HBV core/Enhancer Ⅱ promoter, the results indicated that PPARa is a key role in the process of MIA2activating HBV core/Enhancer Ⅱ promoter. Then we demonstrated that MIA2can increase PPARa binding onto HBV core/Enhancer Ⅱ promoter.By western blot and EMSA assay, we demonstrated that MIA2can increase PPARa expression both in whole cells and in nuclear.All the results indicated that PPARa plays an important role in MIA2activating HBV core/Enhancer Ⅱ promoter.