Study Of HBV Replication Regulated By Arid2 And ITS Molecular Mechanisms

Objective:HBV infection is a serious disease and the leading cause of death around the world, each year about 1 million people died of HBV related diseases.SWI/SNF is a multi-subunit chromatin remodeling complex that use energy derived from adenosine triphosphate (ATP) hydrolysis to change chromatin structures for nuclear processes. Human SWI/SNF composed of two subtypes called BAF (BRG1/hBRM-associated factors) and PBAF (Polybromo-associated BAF), each of these two complexes has some unique subunits and differs in chromatin remodelling activity of mononucleosomes and nucleosomal arrays. BAF200 encoded by the Arid2 (AT-rich interactive domain 2) was initially identified in the PBAF complex. It is reported that Arid2 play an important role in the virus replication progress as a specific subunit. Recent studies have found that Arid2 can affect the human immunodeficiency virus long terminal repeat (HIV LTR) and regulate the transcriptional activity. In our study, we focuses on the regulation of HBV replication by Arid2 in HBV stable replication cell lines, and further explore the related molecular mechanisms Arid2 involved in the regulation of HBV replication.Methods:1) Western blot and Southern blot were applied to explore the correlation between Arid2 gene expression and the HBV replication level in the HBV stable replication model.2)RT-PCR, ELISA and Southern blot were used to determine the effects of knockdown or overexpression of Arid2 on HBV replication biomarkers in the HBV stable replication model(HepG2-HBV1.1 and HepG2.2.15) 3)Real-time PCR was employed to screen for the HBV replication transcription factor which may be influenced by Arid2 gene expression. Besides, We used Co-immuno precipitation (Co-IP) and chromatin immunoprecipitation (ChIP) experiments to study the molecular mechanisms Arid2 involved in the regulation of HBV replication.Results:Western blot and Southern blot revealed that HBV replication level were significantly upregulated relative to Arid2 gene expression level in the HBV stable replication model; We found that the HBV DNA copies, pgRNA and HBcAg expression were downregulated in the siArid2 group than the control group by qRT-PCR and Southern blot method(P<0.05); HBeAg in the supernant of the siArid2 group was significantly decreased than the control group by ELISA. However, HBsAg level in the supernant had no significant difference between the siArid2 group and control group(P>0.05). Realtime-PCR confirmed that the expression of transcription factor PGC-la was significantly reduced by silencing Arid2 gene expression; It was further showed that PGC-1 a bined onto HBV Enhancer, and the HBV Enhancer II mainly. Knockdown of Arid2 induced significantly decreased recruitments of PGC-la to the HBV Enhancer Ⅱ by ChIP assay; Co-IP results confirmed that Arid2 protein can not physically interact with transcription factor PGC-1α to regulate the transcriptional activity of HBV. Otherwise, it was also verified that the HBV DNA copies, pgRNA, HBcAg expression in the cytoplasm and the HbeAg in the supernant were upregulated in the Arid2 overexpression model. In addition, overexpression of Arid2 can also upregulate the expression of PGC-la and increase recruitments of PGC-la to the HBV Enhancer Ⅱ.Conclusion:These experiments indicated that HBV replication level was positively related with the Arid2 expression level. Its mechanisms was likely that Arid2 upregulated the expression level of PGC-la and facilitated the binding of PGC-la to HBV enhancer II and resulted in regulation of HBV replication...