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Construction of dual function plasmid co-expressing ENDO-VEGI151 and survivin-siRNA and its anti-tumor activity
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Abstract:
Objective : To construct a dual function expressional plasmid pCDNA3.1-ENDO-VEGI151/survivin-shRNA (pEV/si-survivin), and study its effect on the proliferation and apoptosis of breast cancer MDA-MB-231 cells and vascular endothelial cells (HUEVCs), so as to evaluate its feasibility for gene therapy of cancer. Methods: The efficient siRNA sequences targeting survivin was screened in MDA-MB-231 cells; the pEV/si-survivin expression vector was constructed and transfected into MDA-MB-231 and HUEVC cells, and the expression levels of ENDO-VEGI151 and survivin were detected by the real-time PCR and Western blotting analysis; MTT assay was used to detect the proliferation inhibition in the cells of the two groups after transfection; and flow cytometry was used to detect the changes of cell cycles and apoptosis. Results: The dual function recombinant plasmid pEV/si-survivin was successfully constructed and it was correctly expressed in both MDA-MB-231 and HUEVC cells. The plasmid significantly inhibited the expression of survivin and the cell proliferation (inhibition rate being \[39.36±4.16\]% at 48 h and \[48.43±3.49)% at 72 h); it also significantly promoted cell apoptosis (\[18.33±1.48\]% vs \[4.80±1.01\]%, P<001) and induced cell cycles arrest (P<0.05) in MDA-MB-231 cells. The plasmid also significantly inhibited cell proliferation (inhibition rate being \[38.16±3.37\]%) at 48 h and \[53.75±453\]% at 72 h), promoted apoptosis, and arrested the cell cycles (P<0.05) in HUEVC cells. Conclusion: The dual function expressional plasmid pEV/si-survivin possess both angiogenesis inhibition and apoptosis promotion functions, and is expected to exert synergistic effect in vivo to improve the therapeutic outcome for patients with cancer.
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Project Supported:
Project supported by the National Natural Science Foundation of China (No.51003078), and the Science Research Foundation of Health Bureau of Shanghai (No.2008133)
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