Construction Of Eukaryotic Expression Plasmid Expressing ShRNA Targeting EGFR Gene Family And The Effect Of Proliferation On SKOV3

PART ONE CONSTRUCTION OF EUKARYOTIC EXPRESSION PLASMID EXPRESSING SHRNA TARGETING EGFR GENE FAMILYObjective: To construct the eukaryotic expression plasmid experssing shRNA targeting EGFR gene family as a tool for following experiments in vitro and in vivo.Methods: EFGR family highly expressed in low radiosensitive ovarian cancer cells SKOV3, chosen highly expressed homologous genes in EFGR family as targeted gene. According to guidelines for shRNA design, designed experimental shRNA and negative control shRNA. In vitro synthesized 2 of the coding sequence of short hairpin DNA single-strands, annealing, and cloned into the corresponding vector pGensil-1 of pol III promoter downstream, named pGensil-1-E and pGensil-1-C。Confirmed by restrict endonuclease digestion and DNA sequencing.Results: restrict endonuclease digestion and DNA sequencing results: both are equal to the design. Eukaryotic expression plasmid experssing shRNA targeting homologous genes in EFGR family was constructed successfully.Conclusion: Using RNA interference technology, our studies successfully constructed eukaryotic targeting EGFR family gene and obtained expression plasmid experssing shRNA, pGensil-1-E and pGensil-1-C.PART TWO THE EFFECT ON INHIBITION OF RNAI-MEDIATED EGFR FAMILY TO CELL PROLIFERATION OF SKOV₃ HUMA OVARIAN CANCINOMAObjective: transfect constructed plasmid vector pGensil-1-E and pGensil-1-C to SKOV₃ cells, and to study the inhibitory effect of it by RNA interference.Methods: SKOV₃ cells were transfected by pGensil-1 with liposome 2000. Stabilized transfected SKOV₃ cells were constructed by selection with G418, inhibitory effect of HER mRNA detected by semi-quantitative RT-PCR, inhibitory effect of HER2 protein was detected by Western-blot. Observed the effect of knockdown of HER2 on proliferation ability of SKOV₃ cell by MTT assay.Results: stabilized transfected SKOV₃ cells were constructed successfully. MTT results: instantaneous transfected pGensil-1-E group cells proliferation inhibition rates were 59.7﹪,81.2﹪and 87.8﹪compared with the blank group cells at 24h,48h,72h respectively .The results of semi-quantitative RT-PCR : HER2 mRNA expression drop to 32.9﹪and 21.7﹪respectively in instantaneous and stabilized transfected SKOV₃ cells compared with the blank group cells. Western-blot results: stabilized transfected SKOV₃ cells P185 protein expression were knockdown to 21.8﹪compared with the blank group cells.Conclusion: obtained stabilized transfected SKOV₃ cells, in which both HER2 mRNA and protein expression were knockdown apparently. Which can provide theoretic directions and experimental evidences for enhancing radiotherapy sensitivity of ovarian carcinoma in our future study.