Hcv Ns5a And Trans Activate Genes Ns5atp9 Research On The Effects Of Liver Cell Apoptosis And The Mechanism

Part IConstruction of eukaryotic expression vector of HCV(Ib) NS5A andanalysis of apoptosis-related genes via microarrayObjective To construct a recombinant expression plasmid carrying HCV NS5A(Ib) mediated bypcDNA3.1/myc-His(-) and explore its effect on apoptosis-related genes.Methods HCV NS5AcDNA was obtained from HCV RNA by reverse transcriptase PCR ,then NS5A was connected topGEM-T Easy vector。Both pGEM-T-HCV NS5A and pcDNA3.1/myc-His(-) were cut bydouble-enzyme cleavage method using Xho I and Hind III to make sticky ends .Then,weconnected pcDNA3.1/myc-His(-) with NS5A. The pcDNA3.1/myc-His(-)-HCV NS5A plasmidwas transfected to HepG2,then NS5A protein expression was detected by Wesern blot. RestultsThe recombinant eukaryotic expression vector was verified by double-enzyme cleavage methodand DNA sequencing analysis, NS5A protein expression was also confirmed by Wesern blot. Aset of 11 genes related apoptosis(6 up-regulated, 5 down-regulated) whose expression wasmodified by at least twofold was selected from oligonucleotide microarrays.Conclusion Wesucceeded in constructing NS5A recombinant eukaryotic expression vector. NS5A may promoteapoptosis of HepG2 through many pathway,including death receptor pathway, mitochondrialapoptosis pathway,DNA repairment.Part IINS5ATP9 expression inhibits the apoptosis of HepG2 induced byserum starvationObjective To investigate the effect of NS5ATP9 on the apoptosis of HepG2. Methods NS5ATP9exptession plasmid (pEGFP-N1-NS5ATP9) and miR RNAi expression vector forNS5ATP9(pcDNA6.2-GW/EmGFP-miR-NS5ATP9) were transiently transfected into HepG2cells by jet PRIME. Cell apoptosis was induced by starvation.The expression of NS5ATP9 inHepG2 was detected by semiquantitative RT-PCR.Cell apoptosis was evaluated by AnnexinV/7AAD, mitochondrial membrane potentials was detected by JC-1 staining. Bax protein wassemiquantidied by Western blotting. Restults After overexpression of NS5ATP9, the percentageof early and total apoptosis decreased significantly ,detected by Annexin V/7AAD(P<0.05),when we knocked down the expression of NS5ATP9 by RNAi expression vector transfection, thepercentage of early ,late and total apoptosis increased significantly(P<0.05) .Compared tocontrol group,the percentage of cells with polarization membrane potential showed a risingtedency after overexpression of NS5ATP9(P=0.053),meanwhile cells with depolarizationmembrane potential decreased significantly(P<0.05),thenΔψm increased ,but notsignificantly(P=0.324);after knocking down of NS5ATP9 , the percentage of cells withpolarization membrane potential dereased significantly (P<0.05),meanwhileΔψm tent todecline(P=0.378).Overexpression of NS5ATP9 downregulated the expression of pro-apoptoticBax(P=0.551),and knocking down of NS5ATP9 induced upregulating of Bax significantly(P<0.05) .Conclusion Our investigation suggested that NS5ATP9 inhibits apoptosis viamitochondrial apoptosis pathway in HepG2.