Effect Of SiRNA Targeting VEGF On Cell Apoptosis And The Expression Of Survivin Of K562 Cells

PART ONE CONSTRUCTION OF THE RECOMBINANT ADENOVIRUSE ENCODING VEGF siRNAObjective: To construct an recombinant adenovirus plasmid of vascular endothelial growth factor small interfering RNA, and generating high titer recombinant adenovirus.Method: The specific human VEGF siRNA was synthesized in vitro, and subcloned into the shuttle plasmid pSES-HUS, named after pSES-VEGFsi. Then cotransfecting with adenovirus skeleton plasmid pAdeasy-1 to produce the recombinant adenoviral plasimid pAd5-VEGFsi by homologous recombinantion.The pAd5-VEGFsi was identified by enzyme cutting. Finally, We transfect pAd5-VEGFsi into package cell HEK293 by Lipofectamine and generate high titer recombinant adenoviral Ad5-VEGFsi by using ping-pong infection and CSCl gradient purification.Result: (1) The recombination plasmid pSES-VEGFsi was constructed successfully by enzyme cutting and gene screening. (2) The recombined adenoviral plasimid was constructed successfully; then it was transfected into the package cell HEK293 successfully and amplified quickly.(3) The titre of the recombinant adenovirus was 4.6x l011 pfu/ml.Conclusion: The recombinant adenovirus plasmid expressing the target gene was successfully generated, and generated high titer recombinant adenovirus Ad5-VEGFsi, made a good basic for further research.PART TWO EFFECT OF ADENOVIRUS VECTOR-MEDIATED siRNA TARGETING VEGF ON APOPTOSIS AND THE EXPRESSION OF SURVIVIN OF K562 CELLSObjective: To silence the VEGF gene in K562 cells using the adenovirus vector-mediated siRNA (Ad5-VEGFsi), detect the condition of cell apoptosis and cell proliferation. And then detected the expression of surivin, approaching the effect of VEGF in leukemia and the relationship between VEGF and survivin in pathogenesis of leukemia.Methods: The K562 cells were infected with recombinant adenovirus Ad5-VEGFsi which constructed in part one. K562 cells were divided into three groups: K562/ Ad5-VEGFsi groups, K562/Ad5 groups, and K562 groups. The VEGF and survivin mRNA expression were determined by RT-PCR. The protein level of VEGF and survivin was measured by ELISA and Western blot, respectively. Cell proliferation was measured by MTT assay and the apoptosis of K562 cells were detected by flow cytometry.Results: (1) VEGF mRNA and its protein were detected in all three groups, and the survivin mRNA and its protein were also detected in those groups. (2) The levels of mRNA and protein of VEGF in K562/Ad5-VEGFsi group were(0.25±0.06) and (1120.742±14.546)pg/ml, which were decreased significantly than that in the groups of K562/Ad5 and K562 (p<0.01).(3) The levels of survivin mRNA and its protein were (0.42±0.03) and (0.26±0.01) in K562/Ad5-VEGFsi group, which were lower than that in another groups(p<0.01). the expression of survivin would downregulate accompanied with the inhibition of VEGF expression. (4) The cells apoptosis in K562/Ad5-VEGFsi group were (16.45±0.14)% and the cells growth were inhibited(p<0.01).Conclusion: (1) The K562 cells could express VEGF mRNA and secrete the protein of VEGF. (2)The recombinant adenovirus Ad5-VEGFsi could downregulate VEGF expression efectively in K562 cells. (3) The Ad5-VEGFsi could increase the cell apoptosis and inhibite the cell proliferation. (4) After inhibition of VEGF by RNAi, the expression of survivin were decreased subsequently. The expression of VEGF was closely correlated with the expression of survivin.In a word, the overexpression of VEGF made the K562 cells have the capability of counteracting cell apoptosis and malignant proliferation. And the capability of VEGF may through up-regulating the expression of survivin gene action.