Construction Of ShRNA Expression Vector Of XIAP And Its Effects On HeLa Cells

Objectives: To construct the small interfering RNA expression vector of XIAP gene and transfection hela cells of human Cervical carcionma to observe the influence of its apoptosis and cycle as well expression of XIAP mRNA. The research can provide not only new and effective target of RNA interference on XIAP gene,but also the new experiment evidence of the usage for expression vector in vivo mediated RNA interference acting as efficient gene silencing technology. Moreover it may present a new approach for gene therapy of Uterine cervix cancer,which aim at inhibiting XIAP gene activity, and helpful exploration of RNA interference applies to tumor research and gene therapy.Methods: Human Cervical carcionma cell lines HeLa were used for the current project. (1) To construct the pGPU6-GFP-XIAP-shRNA expression vector of XIAP. (2)Cultured HeLa cells, then transfected into cells by Lipofectamine TM²⁰⁰⁰,and set up four experimental groups: blank control group (blank),Lipofectamine TM2000 transfection group(mock),negative control group(NC),interference group(pGPU6-GFP-XIAP-shRNAtransfected into cells). (3) Expression level of XIAP mRNA was assayed by real-time fluorescence quantitative PCR technology. (4) Apoptosis and cycle of cells were assessed by flow cytometry;Results: (1) pGPU6-GFP-XIAP-shRNA digested by agarose gel electrophoresis, and verified the size and direction of objective gene segment inserting vector, then its targeting XIAP were successfully constructed.(2) The result of real time fluorescence quantitative RT-PCR indicated that expression of XIAP mRNA of HeLa cells transfects with the XIAP-shRNA dropped to 27% and 51% after 48 hours and 72 hours, existed some difference compared with the blank control (P<0.05), moreover, silencing effect gradually enhances at 48 hours to 72 hours; (3 ) The FCM results revealed that the apoptotic rate in XIAP-shRNA transfected group (31.60±1.29% and 37.43±1.94% ) obviously increased compared with in blank control group (1.58±1.34% and 2.12±1.44%) (P<0.05), NC group(4.58±1.63% and 5.15±1.82%) and mock group(4.19±1.64% and 4.87±1.50%) after 48 and 72 hours (p<0.05); (4) The FCM of PI simple staining indicated that interference group could cause the number of cells to raise at the time of G0/G1 and decrease at the time of S (p<0.05)Conclusion: (1) The designed and synthesized XIAP of shRNA interference sequence is cloned accurately to pGPU6-GFP expression vector indicated that shRNA expression vector constructed successfully.(2) Recombinant could more efficiently transfect to HeLa cells through lipofectamine TM2000,and then ensured the highly potent expression of recombinant in cells; (3) shRNA targeting to XIAP can successfully exert specific and effective role in gene silencing , it obviously reduced the expression of XIAP mRNA of HeLa cells; (4) shRNA targeting to XIAP can obstruct HeLa cells at the time of G₀/G₁ and induced it apoptosis. Indicated that XIAP gene recombinant polasmid of this experiment make expression silence effectively important, and can promote the apoptosis of HeLa cells, suppress the cell proliferation . So it can set foundation for the ovarian cancer gene treatment.