Effects Of Adenovirus Mediated Human VEGF Small Interfering RNA On Proferation And Apoptosis Of Osteosarcoma Cell Line MG63

[Objective] To investigate the influence of the VEGF expression and the biologicalcharacters of MG63 cell as well as the inhibition of tumor growth byAd-VEGF-siRNA, the small interfering RNA target VEGF mRNA and mutant smallinterfering RNA were designed and synthesized, and the adenovirial vectorconstructed. Then human osteosarcoma cell line MG63 were infected withAd-VEGF-siRNA to evaluate the effects of Ad-VEGF-siRNA on the proliferation andapoptosis of osteosarcoma by observation of tumor formation, tumor growth speed andtumor end volume. The synergistic mechanisms and clinical significance leadingfavorable results of Ad-VEGF-siRNA were discussed. [Methods] 1. Humanosteosarcoma cell line MG63 cells were infected with recombinant adenovirus carryingVEGF small interfering RNA.The expression levels of Bcl-2,PCNA were assayed byimmuno-cytochemistry at 24h and 48h after treatments. The cell proliferation inhibitionratio was measured by MTT assay. For the cell cycle and cell apoptosis analysis, MG63cells were examined by flow cytometry and hoechst staining respectively. 2. Anxenograft subcutaneous osteosarcoma of nude mice was successfully induced by humanosteosarcoma cell line MG63. After intratumor injection of Ad-VEGF-siRNA, theexpression of PCNA and Bcl-2 were assayed by immunohistochemistry. Further more,apoptosis of MG63 cells were detected by Tunel and transmission eletron microscope.[Results] We constrcted the recombinant adenovirus that carrying small interferingRNA against VEGF. Compared with the control groups, colorimtric MTT demonstratedthat the growth of MG63 cells were obviously inhibited by Ad-VEGF-siRNA, theeffection were positive related with MOI and infecting time. Flow cytometry detectedthat more MG63 cells dealed with Ad-VEGF-siRNA subjected to apoptosis than othergroups (P＜0.01), the apoptosis index were positive relative with MOI and infecting time.Immuno-cytochemistry demonstrated that the expression of PCNA and Bcl-2 weredown-regulated after 24 hours treated by Ad-VEGF-siRNA compared with othergroups(P＜0.01), however,after 48 hours the inhibition were increased. Hoechst stainingrevealed that MG63 cells undergoed apoptosis were easy to see after treated byAd-VEGF-siRNA, the apoptosis index were positive relative with MOI and infectingtime. An xenograft subcutaneous osteosarcoma of nude mice was successfully inducedby human osteosarcoma cell line MG63. Compared with control, the tumorigenesisof Ad-VEGF-siRNA group was greatly reduced, as demonstrated by later tumorformation, both the tumor growth speed and tumor end volume (the volume was0.91±0.05cm~3, 1.53±0.17cm~3, and 1.56±0.12cm~3, respectively). Tunel and transmissioneletron microscope revealed the sum of apoptosis MG63 cells were increased aftertherapy, however, immunohistochemistry demonstrated the expression of PCNA andBcl-2 were decreased obviously (P＜0.01). [Conclusion]The results of this study indicated that the VEGF expression,tumor growth and angiogenesis can be effectivelyinhibited by adenovirus infected widely and being obtained high titer virus to produceabundant small interfering RNA in desired tissue to cleave target VEGF mRNA highspecifically, meanwhile, tumor cells undergoed apoptosis increasing. Antiangiogenictherapy by Ad-VEGF-siRNA may provides a new effective method for gene therapy ofsolid tumors.