The Effect Of Specific Small Interfering RNA Against The Expression Of VEGF In Colorectal Carcinoma Of Mice

ObjectiveTo investigate the specific inhibition of the expression of VEGF of mice colorectal carcinoma cell line with RNAi technology and prove the possibility of application for RNAi in gene thrapy of colorectal cancer.MethodsSpecific small interfering RNA was introduced with VEGF mRNA as a target in cultured mice colon carcinoma cell line CT-26,Three siRNAs and one scrambled siRNA(used for a negative control)were designed on line.The three kinds of siRNAs were obtained by invitro transcription and transfected into cells with lipofectin.Constructing the specific interferring RNA expressing vector:We constructed eukaryotic expression plasmids(pSilencer4.1 CMV neo-VEGF1/ VEGF2/ VEGF3-siRNA) with pSilencer4.1 CMV neo as the plasmids,and then the clone was identified by restriction enzyme digestion and DNA sequencing technique.The recombinant vector was transferred into CT-26 colon carcinoma cells using lipofectamine.Observe the change of colorectal cancer cells after transfection: proliferation rates of cells were assayed by MTT;the change of VEGF mRNA in tumor cells were were assayed by RT-PCR;the change of VEGF protein in tumor cells were were assayed by Western blotting;ResultspSliencer4.1 CMV neo expression vector was successfully constructed and the sequence of constructed recombinant plasmid was correct by restriction enzyme digestion and DNA sequencing.The three targeting siRNA against VEGF mRNA could effectively inhibit the growth of colorectal caner cells transfected with lipofectin, however,the scrambled siRNA could not.The result of proliferation rates were assayed by MTT after transfection demonstrated that the growth of CT-26 cells were inhibited significantly in V1,V2,V3 groups than that of group Lipofectamine,cells and Scrambled(P＜0.01),especially in group V1 and V2.The result of the change of VEGF mRNA were assayed by RT-PCR after transfection demonstrated that the expression of VEGF mRNA declined significantly in V1,V2,V3 groups than that of group Lipofectamine,cells and Scrambled(P＜0.05).The result of the change of VEGF protein were assayed by Western blotting after transfection demonstrated that the content of protein in V1,V2,V3 groups was less than that of group Lipofectamine,cells and Scrambled..ConclusionThe three targeting siRNA against VEGF designed by biological software could effectively inhibit the growth of colorectal caner cells and the expression of VEGF gene.VEGF is the potential target in anti-angiogenesis therapy of colorectal cancer.Blocking the expression of VEGF in colorectal cancer by RNA interference maybe become a new method in gene therapy of colorectal cancer.