Long-Fragment RNA Inhibits Hepatitis B Virus Gene Replication And Expression In HepG2.2.15 Cell Line

Objective:To evaluate the inhibitory effects of long antisense RNA on HBV replication in HepG2.2.15 cells.Methods : The coding region of HBV S gene was cloned into pTARGET vector in sense and antisense orientations and the recombinant plasmids were transfected into HepG2.2.15 cells, and the HepG2.2.15 cells were divided into HBS2(antisense RNA) group,HBS4(sense RNA) group,interferon group, poly:c group and control group. HBsAg and HBeAg in the culture supernatant were detected by ELISA. HBV DNA,HBV cccDNA in the cells and HBV DNA in the supernatant were quantified by real time PCR.β-IFN mRNA in the cells was quantified by real time RT-PCR. The rate of apoptosis was detected by Cellular DNA Fragmentation ELISA.Results:(1)PCR and sequencing results displayed that recombinant plasmids were constructed successfully.(2)ELISA result showed that the level of HBsAg and HBeAg in the supernatant of HBS2 group, interferon group and poly:c group were lower than control group(P<0.05); the level of HBsAg in the supernatant of HBS4 group was higher than control group(P<0.05),but the level of HBeAg of HBS4 group had no difference with control group(P>0.05).(3)Real time PCR result displayed that the level of HBV DNA both in supernant and cells of HBS2 group, interferon group and poly:c group were lower than control group(P<0.05),but HBS4 group had no difference with control group(P>0.05),the level of HBV cccDNA in the cells of HBS2 group, interferon group and poly:c group were lower than control group(P<0.05);HBS4 group had no difference with control group(P>0.05).(4)Real time RT-PCR result indicated that the level ofβ-IFN mRNA in the cells of HBS2 group and poly:c group were higer than control group(P<0.05), HBS4 group had no difference with control group.(5) Cellular DNA Fragmentation ELISA result showed that the rates of apoptosis of HBS2 group, interferon group and poly:c group were higher than control group(P<0.05), HBS4 group had no difference with control group(P>0.05).Conclusion:Antisense RNA might be a useful tool to repress HBV replication,also could induce the apoptosis of HepG2.2.15 cells.