Experimental Research On Inhibition Of Survivin And VEGF Gene Expression By Double Target Of SiRNA In Osteosarcoma

Objective To study the inhibitory effect of a single strand of double-target siRNA on VEGF and Survivin gene in osteosarcoma cell lines(MG-63 and Saos-2),as well as the effects on cell migration,angiogenesis,proliferation and apoptosis.By exploring the mechanism of double target siRNA,we expand the technology of nucleic acid interference and lay a foundation for the development of multi-strand and multi-target siRNA interference technology.This study will provide theoretical basis and technical support for the research and development of drugs related to multi-target siRNA nucleic acid interference.Methods1.Two single-target siRNA(siRNA-Survivin and siRNA-VEGF)were synthesized.On this basis,a double-target siRNA(siRNA-VEGF&Survivin)targeting both VEGF and Survivin genes were designed and synthesized.2.MG-63 and Saos-2cells were transfected with siRNA-Survivin?siRNA-VEGF and siRNA-VEGF & Survivin.Detecting the transfection efficiency by fluorescence microscopy3.QRT-PCR and Western Blot were used to detect the m RNA and protein expression of VEGF and Survivin gene in MG-63 and Saos-2 cells4.The protein localization and expression of Survivin and VEGF were documented by Immunofluorescence staining and immunohistochemistry.5.The growth level was detected by MTT assay and the apoptosis was detected by Flow cytometry(FCM)in MG-63 and Saos-2 cells.6.MG-63 and Saos-2 cell migration and angiogenesis were detected by scratch test and capillary tube assay respectively.Results1.A single target siRNA and a double target siRNA sequence targeting to VEGF and Survivin were successfully constructed2.MG-63 and Saos-2 cells were transfected with all groups of siRNA successfully,and the transfection rate accounted for 80%.3.Western Blot,QRT-PCR,immunofluorescence staining and immunohistochemistry experiment result indicates that the expression levels of VEGF and Survivin in single-target siRNA group and single-strand double-target siRNA group were lower than those in control group and non-transfected group,and the difference was statistically significant(P < 0.05).There was no significant difference in the level of expression of RNA and protein between single-stranded and double-target siRNA groups(P > 0.05).4.Double-target siRNA can significantly inhibit the proliferation,migration and angiogenesis of osteosarcoma cells,and promote the apoptosis of osteosarcoma cells.The effect of double-target siRNA group is better than that of single-target siRNA group.Conclusions1.Silencing of Survivin and VEGF gene by the the single and double target of siRNA can down-regulates the expression of VEGF and Survivin in MG-63 and Saos-2 cells effectively2.It has confirmed that single-target siRNA and double-target siRNA can significantly inhibit migration and angiogenesis of osteosarcoma(MG-63 and Saos-2)in vitro experiments.3.It has showed that both single-target siRNA and a single-strand double-target siRNA can inhibit the proliferation and promote apoptosis of osteosarcoma(MG-63 and Saos-2)in vitro experiments.4.The double target of siRNA appears to be superior to inhibit cell proliferation,migration,angiogenesis and promote apoptosis compared with single target siRNA.5.There was no correlation between VEGF and Survivin genes,and a single double target siRNA had a double inhibitory effect on proliferation and apoptosis of osteosarcoma cells compared with a single target siRNA...