The In Vitro Study Of The Antiviral Immunity Mechanism Of MiR-155 On Chronic HBV Infection

ObjectiveTo explore the effect of over-expressed miR-155 on the HBV replication and expression, and evaluate the immune response of the HepG2.2.15 cells to miR-155.Methods1. The pre-miR-155 was amplified from total DNA of HepG2.2.15 cells by PCR. The target gene fragment was digested by EcoR I and BamH I, then was cloned into the pmR-mCherry plasmid. Restriction digestion and sequencing were performed to evaluate the recombinant.2. The pmiR-155 plasmid was transfected into HepG2.2.15 cells by liposome-mediated method, while the empty plasmid group (pmR-mCherry plasmid), the transfection reagent group, the blank control group and the control group (HepG2 cells) were set as controls. The expression of cherry was detected by fluorescence microscope. The intracellular expression of miR-155 was detected by RT-qPCR.3. The ultra purified recombinant plasmid pmiR-155 were transfected into HepG2.2.15 cells, while the empty plasmid group (pmR-mCherry plasmid), the transfection reagent group, the blank control group and the control group (HepG2 cells) were set as controls, and collected the cells and supernatant after transfection. The expression of SOCS-1 mRNA were detected by RT-PCR, the SOCS-1 protein were investigated by Western Blot, and the changes of IFN-γ and IL-2 were detected by ELISA.4. The ultra purified recombinant plasmid pmiR-155 were transfected into HepG2.2.15 cells, while the empty plasmid group (pmR-mCherry plasmid), the transfection reagent group and the blank control group were set as controls. The expression of HBsAg and HBeAg were detected by chemiluminescent assay, and the changes of HBV DNA were investigated by RT-qPCR.Results1. The pmiR-155 eukaryotic expression vector was successfully constructed comfirmed by the colony PCR, enzyme digestion and DNA sequencing.2. The cherry protein expressed well in the transfection cells as shown by fluorescence. The efficiency of transfecion reached above 50%. The level of miR-155 in HepG2.2.15 cells transfected with the recombinant plasmid was obviously higher than the other groups (P<0.05).3. RT-PCR showed the expression of SOCS-1 mRNA in the recombinant group were less than others (0.63±0.09) (P<0.05). The expression of SOCS-1 protein markedly decreased by western blot (0.37±0.07) (P<0.05). ELISA showed the secretion of IFN-γ and IL-2 in the recombinant group both increased than other groups (P<0.05).4. The chemi luminescent assay showed that the HBsAg and HBeAg in the recombinant group both reduced than other groups. The HBsAg and HBeAg were suppressed with (83.96±1.52)%, (79.60±8.71)% by vector pmiR-155 on the 48h of post-transfection. The HBV DNA in the recombinant group were markedly suppressed with (51.87±0.36)%, (43.67±1.51)% and (68.21±6.02)% by real-time quantitive PCR, respectively on the 24h,48h and 72h of post transfection.ConclusionOver-expression miR-155 can enhance the immune response and inhibit the replication and expression of HBV in the chronic HBV infection.