Construction Of EGFR ShRNA Expressing Vector And The Effect On Radiosensitivity Of Ovarian Xenograft Cancer

PART ONE CONSTRUCTED AND CONFIRMED OF EGFR SHRNA EXPRESSING VECTORObjective: To construct EGFR gene sequence-specific shRNA expression vector as a tool for the following experiments.Methods: EGFR gene highly expressed in low radiosensitivty ovarian cancer SKOV3 cells , chosen EGFR gene as targeted gene. According to guidelines for shRNA design, designed EGFR gene sequence-specific shRNA expression vector (pGensil-1-E) and sequence-nonspecific shRNA expression vector (pGensil-1-N) .In vitro synthesized the two coding sequence of short hairpin DNA single-strands, annealing, and cloned into the plasmid vector pGensil-1 of pol III promoter downstream, Confirmed by restrict endonuclease digestion and DNA sequencing.Results: The pGensil-1-E and pGensil-1-N expression vector targeting EGFR gene family was constructed successfully by Restrict endonuclease digestion and DNA sequencing.Conclusion: The expression vector targeting EGFR gene family was constructed successfully in the experiment. PART TWO THE EFFECT OF EGFR GENE SILENCED BY RNAI ON THE RADIOSENSITIVITY OF OVARIAN XENOGRAFT CANCERObjective:Sequence-specific EGFR shRNA expression vector was transfected into ovarian xenograft cancer , explored the effects of EGFR gene on the radiosensitivity of ovarian cancer by RNAi technology.Methods: Tumor-animal models with ovarian cancer SKOV3 cells was established , the nude mice were randomly divided into the control group,t he radiotherapy group and the radiotherapy + pGenesil-1-E group,the ovarian cancer tissue were locally irradiated by 6MV X-ray, monitored the tumor volume of the three groups, measured the tumor quality after treated, detected With RT-PCR,Western blot,i mmunohisto- chemistry and electron microscopy.Results:The tumor volume of the three groups were obviously difference after treated (P<0.01), the inhibit-tumor rate of the radiotherapy group and the radiotherapy + pGenesil-1-E group were 28.1%,4 6.1%, respectively; the expression of EGFR gene in radiotherapy + pGenesil-1-E group was significantly reduced and the apparent tumor apoptosis,necrosis under electron microscopy.Conclusion: The animal experiments confirmed that silenced EGFR gene by RNAi can significantly improved the radiosensitivity of ovarian cancer, provided certain experiment basis for silenced EGFR gene combined with radiotherapy in ovarian cancer.