RESUMEN
Objective To investigate the effects of survivin shRNA-APC double gene co-expression stably transfected cell lines on the VEGF、COX-2 expressions and angiogenesis of subcutaneous exnotransplanted tumor tissues cell of HT-29 colon cancer in nude mice.Methods Forty nude mice were randomly divided into five groups,the negative control group,empty vector group,Survivin shRNA group,APC group,double-gene group.The stably transfected cell lines and HT-29 colon cancer cells were cultured,PBS suspension resulted in cell density of 2× 107/ml,injected with respective stably transfected cell lines to establish an SXT model.All the mice were sacrificed after six weeks in order to separate the subcutaneous tumor,the expressions of the VEGF,COX-2mRNA and protein were detected by Real time PCR and immunohistochemistry,CD34 antibody was used to mark the vascular endothelial cells,and the MVD values were detected by immunohistochemistry.Results Tumors were formed in the nude mice of each group.The expressions of VEGF,COX-2 mRNA in Survivin shRNA group ((50.84±3.64)％,(50.11±3.91)％),APC group((74.28±6.87)％,(72.39±6.55)％) and Survivin shRNA-APC double-gene group ((21.78±4.00) ％,(20.74±5.12) ％) were significantly lower than those in the empty vector groups((100.00±0.00) ％,(100.00±0.00) ％) or negative control group ((98.22±0.38) ％,(97.61 + 0.77)),the differences were statistically significant (P ＜ 0.05);the expressions of VEGF,COX-2 mRNA in Survivin shRNA-APC double-gene group were significantly lower than those in APC groups,Survivin shRNA group,the differences were statistically significant (P＜0.05).The expressions of VEGF,COX-2 protein in Survivin shRNA group (5.15 ± 1.02,5.26 ± 0.91),APC group (4.96 ± 1.12,4.93 ± 1.18),and Survivin shRNA-APC double-gene groups (1.81 ± 0.84,1.80± 0.81)were significantli lower than those in the negative control group (8.95± 0.55,8.77± 0.60) and empty vector group (9.17± 0.49,9.01 ± 0.80),the differences were statistically significant(P＜0.05),the expressions of VEGF,COX-2 protein in the Survivin shRNA-APC double-gene group were significantly lower than those than in APC group,Survivin shRNA group(P＜0.05);the expressions of MVD in APC group (12.14± 3.45),Survivin shRNA group (11.39 ± 2.94) and Survivin shRNA-APC double-gene group (3.96 ± 2.20) were lower than those in the negative control group (25.09 ± 5.59) and empty vector group (27.87 ± 7.36),the differences were statistically significant (P ＜ 0.05),the MVD in the Survivin shRNA-APC double-gene group was even lower than that in APC group,Survivin shRNA group,the differences were statistically significant (P ＜ 0.05).Conclusion Survivin shRNA-APC double gene coexpression stably transfected cell lines can significantly reduce the expression of the VEGF,COX-2 mRNA and protein and then inhibit the angiogenesis of transplanted tumor tissue,and its inhibitory effect is more effective than that og Survivin shRNA and APC single gene stable strain.
RESUMEN
Objective Little is known about the effect of RNAi on mitochondrial apoptotic pathways. This study aims to explore the effects of the Survivin shRNA-APC double-gene on colon cancer mitochondrial apoptosis pathway-related factors survivin,cytochrome C (Cytc),second mitochondria-derived activator of caspases (Smac),and cysteine aspartic acid specific protease 9 (Caspase-9) as well as on the apoptosis of colon cancer transplanted tumor (CCTT) cells. Methods Thirty nude mice were randomly divided into five groups of equal number,Survivin shRNA-APC double-gene,survivin shRNA,APC,empty vector and blank transfection. The CCTT model was established in the nude mice by subcutaneous injection of the colon cancer cell strains stably transfected with the Survivin shRNA-APC double-gene,survivin shRNA,APC,an empty vector and HT-29,respectively,into the mid-posterior part of the left armpit of the nude mice. The rate of tumor growth inhibition was calculated by measuring the volume and weight of the CCTTs in the nude mice. The mRNA and protein expressions of survivin,Cytc,Smac and Caspase-9 in the tumor tissue were detected by real time PCR and immunohistochemistry,respectively,and the apoptosis rate of the CCTT cells was detected by TUNEL. Results The model of CCTT was successfully established in the nude mice. Com-pared with the empty vector and blank transfection groups,the mice in the double-gene,survivin shRNA and APC groups showed sig-nificantly decreased average volume and weight of the tumor tissue (P<0.05) but increased inhibition rate of its volume and weight (P<0.05). In comparison with the survivin shRNA and APC groups,the double-gene group exhibited remarkably decreased average volume and weight of the tumor tissue (P<0.05) but increased inhibition rate of its volume and weight (P<0.05). The mRNA and pro-tein expressions of survivin were significantly lower while those of Cytc,Smac and Caspase-9 markedly higher in the double-gene,sur-vivin shRNA and APC groups than in the empty vector and blank transfection groups (P<0.05),the former even lower (P<0.05) and the latter even higher in the double-gene than in the survivin shRNA and APC groups (P<0.05). The apoptosis rate of the CCTT cells was significantly increased in the double-gene ([56.78±3.04]%),survivin shRNA ([33.61±2.02]%) and APC groups ([30.16± 1.72]%) as compared with the empty vector ([10.05±0.42]%) and blank transfection groups ([9.87±0.30])% (P<0.05),even higher in the double-gene group than in the survivin shRNA and APC groups (P<0.05). Conclusion The Survivin shRNA-APC double-gene may induce apoptosis of colon cancer transplanted tumor cells by down-regulating the expression of the apoptosis inhibitor survivin,upregulating the expressions of Cytc,Smac and Caspase-9,and suppressing the growth of the colon transplanted tumor,with more significant abilities than a single gene in regulating apoptosis-related factors,inducing cell apoptosis and inhibiting the growth of the transplanted tumor.