Analysis Of The Effect Of HBx Knockdown To HBV Replication And Calcium Channels In The Mitochondrial, As Well As Expression Profile Changes In HepG2.2.15 Cells

Background:HBx is encoded by the smallest of the four HBV open reading frames (ORFs). It acts as a multifunctional protein and has been found to play a key role in HBV replication. The Calcium-PyK2 (calcium-dependent proline-rich tyrosine kinase-2) pathway induced by HBx is a well defined section of HBV replication process. Intracellular calcium channels and calcium signaling may directly participate in the replication of HBV. In vivo and in vitro experiment proved that HBx could bind to MPTP (Mitochondrial permeability transition pore) on mitochondrial membrane, but whether it could affect the function of MPTP and the replication of HBV still needs further study. The mechanisms of HBV replication inhibited after knockdown of HBx remain undefined.Objectives: We attempted to inhibit the replication of HBV through the knockdown of HBx by siRNA and explore the relation between MPTP and virus replication, then obtain the expression profiles of HepG2.2.15 pre-and after knockdown of HBx, thereby providing the basis for further study of the functions of HBV and its biological mechanism.Methods:In this study HBx siRNA1694 was transfected to HepG2.2.15 cell line which contain the entire HBV genome, and the titers of HBsAg and HBeAg, and the copy numbers of HBV DNA were quantitatively detected by microparticle enzyme immunoassay (MEIA), and real-time PCR respectively. Flow cytometer was used to measure the fluorescence intensity variation of Calcein caused by different state of MPTP on mitochondrial membrane. The expression profiles of pre-and after the knockdown of HBx in HepG2.2.15 were obtained using Affymetrix microarray, and the changes in expression level of genes were verified with real-time PCR.Results:HBx siRNA1694 could downregulate the expression of HBx dramatically, and suppress the secretion of HBsAg and HBeAg and reduce the copy numbers of HBV DNA. Treatment with HBx siRNA1694 or CsA could antagonize the reduction of the intracellular Calcein fluorescence intensity caused by Ca2+ releasing agent. Expression level of 385 genes showed more than 2 folds changes after knockdown of HBx with the P value less than 0.05. Among them,288 showed downregulation and 97 showed upregulation. Eight of these genes including HSPB8 and PIK3CB were verified.Conclusions:The HBV X gene siRNA1694 could inhibit the replication of HBV effectivity via the downregulation of the expression of HBx mRNA, and the effect of inhibition is closely related with the function of MPTP. Expression profiles of HepG2.2.15 pre- and after knockdown of HBx was obtained and verified, indicating a good repeatability and reliability of our microarray data.