Expression Of Vascular Endothelial Growth Factor And Its Receptor FLT-1,FLK-1 In Colorectal Carcinoma And Influence The Expression Of VEGF Gene Of Caco-2,HT29,HCT116 Cells By SiRNA-mediated Silencing

Objective: To investigate the expression and clinical significance of vascular endothelial growth factor(VEGF) and its receptor FLT-1,FLK-1 in colorectal carcinoma and the effect of blocking the VEGF expression with a specific small interfering RNA (SiRNA) on the characteristics of human colorectal carcinoma cell line Caco-2,HT29,HCT116.Methods: The expression of VEGF and FLT-1,FLK-1 was detected using an immunohistochemistry S-P method in 82 cases of colorectal carcinoma and 14 cases of normal colorectal tissue. To knockdown VEGF expression ,a small interfering RNA targeting against VEGF was synthesized and transfected into Caco-2,HT29,HCT116 cells with lipofectamine 2000. The expression of VEGF protein was detected by Immunocytochemical staining (S-P method) and Western blot analysis. The effect of suppressing proliferation of cells was detected by MTT assay.The effect of inducing cells apoptosis was detected by flow cytometry(FCM).Results: (1)The expression of VEGF and FLT-1,FLK-1 was significantly higher in tumor tissue than that in normal tissue (P<0.05). The expression of VEGF was positively correlated with lymph node metastasis and clinical stages(P<0.05).There was a significantly positive relation between FLT-1 and FLK-1(r=0.457,P<0.05) (2)Immunocytochemistry shows that: three types of cell lines(Caco-2,HT29,HCT116) of the normal control group, Lip-2000 control group and mismatch control group cytoplasm was strongly positive expression (dark brown yellow), VEGF-siRNA group cytoplasm showed weakly positive expression (Light brown yellow); Western blot indicated that: Caco-2, HT29, HCT116 cell lines VEGF -siRNA cells protein bands were significantly lower than the brightness of the normal control group, Lip-2000 control group and mismatch control group. (3) MTT test results: compared with negative control group, three of the VEGF-siRNA cell lines growth inhibition is significant (P<0.05). Tumor cell proliferation rates were significantly different between the same cell line with a VEGF-siRNA group at the different times (24h, 48h, 72h)(P<0.05), the effection showed time dependence. The same time at different cell lines (Caco-2, HT29, HCT116) of VEGF-siRNA inhibition rate between group differences were significant (P<0.05). (4)Flow cytometry results show that: compared with the blank control and negative control cells, Caco-2, HT29, HCT116 cell lines VEGF -siRNA group have a apparent hypodiploid peak. Compared with normal control group, the apoptosis rates of three VEGF-siRNA cell lines were significantly higher (P<0.01).Conclusions: VEGF and FLT-1,FLK-1 has close relationship with colorectal carcinogenesis.VEGF has related to prognosis of colorectal carcinoma .Blocking the VEGF expression with RNAi technology can significantly reduce VEGF protein level and suppress proliferation of human colorectal cancer Caco-2,HT29,HCT116 cells and induce apoptosis to a certain degree.The application of RNA interference technology which target VEGF may become a new effective gene therapy approach to colorectal cancer.