Expression Of Survivin,VEGF And Its Receptor FLT-1 In Colorectal Carcinoma And Influence Of Proliferation And Apoptosis In Caco-2, HT29, HCT116 Cells By SiRNA-mediated Silencing Survivin Gene

Objective To investigate the expression and clinical significance of Survivin,VEGF and its receptor FLT-1 in colorectal carcinoma;to detect the expression of Survivin in human colorectal cancer Caco-2,HT29,HCT116 cells; and to explore the effect of transfection of Survivin siRNA on the proliferation and apoptosis of human colorectal cancer Caco-2,HT29,HCT116 cells, and the expression of Survivin protein in three cell lines. Methods Part 1:Immunohistochemical S-P method was used to detect the expression of Survivin, VEGF and its receptor FLT-1 in 82 cases of colorectal carcinoma and 28 cases of normal mucosa. Part 2:A siRNA directed against Survivin was transfeted into human colorectal cancer Caco-2,HT29,HCT116 cells with lipofectamine 2000.The changes of Survivin protein expression were detected by Immunocytochemical and Western Blot,The effect of suppressing proliferation of cells was detected by MTT assay.The effect of inducing cells apoptosis was detected by flow cytometry. Results Part 1:The expression rate of Survivin was 67.1% (55/82) ,VEGF and its receptor FLT-1 were 69.5%(57/82) and 82.9%(68 /82)in colorectal carcinoma, and higher than that in normal mucosa(P<0.05),respectively; Survivin protein significantly related with Duke's stage (P<0.05), The expression of VEGF had obviously association with lymph node metastasis and Duke's stage in colorectal carcinoma (P<0.05),and no relationship was observed between the expression of receptor FLT-1 and clinicopathological manifestations(P >0.05). The expression of Survivin was associated with that of VEGF in colorectal carcinoma (r=0.358, P<0.05). The expression of VEGF was associated with that of FLT-1 in colorectal carcinoma (r=0.565 P<0.05). Part 2: Three types of cells (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),Survivin-siRNA group cytoplasm showed weakly positive expression (Light brown yellow); Western blot indicated that: Caco-2, HT29, HCT116 cells'Survivin-siRNA cells protein bands were significantly lower than the brightness of the normal control group, Lip-2000 control group and mismatch control group; MTT test results: compared with mismatch control group, three of the Survivin-siRNA cells and 5-Fu group cell's growth inhibition is significant (P <0.05). Tumor cell proliferation rates were significantly different between the same cell line with a Survivin-siRNA group at the different times (24h, 48h, 72h) (P <0.05). The same time at different cells (Caco-2, HT29, HCT116) of Survivin-siRNA inhibition rate between group differences were significant (P <0.05); Flow cytometry results show that: compared with the blank control and mismatch control cells, Caco-2, HT29, HCT116 cells'Survivin-siRNA group have a apparent hypodiploid peak, cell apoptosis rates were 9.72%, 19.67%, 22.16%. With the differentiation of three cell lines is getting worse, the proportion of apoptosis increased , the trend is statistically significan(tP<0.01). Conclusions Part 1: Abnormal expression of Survivin and VEGF play an important role in carcinogenesis of colorectal carcinoma. The detect of Survivin and VEGF may be useful for evaluating the progress and prognosis of colorecta1 carcinoma.Part 2: siRNA silencing Survivin gene can inhibit the proliferation of colorectal cancer cells and induce apoptosis; Survivin may become a new gene therapy target of colorectal cancer, the application of RNA interference technology is expected to become an effective gene therapy approach to colorectal cancer.