Effects of survivin shRNA-APC double gene co-expression stably transfected cell lines on the angiogenesis of HT-29 colon cancer cells subcutaneous xenografts in nude mice

Abstract

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 co-expression 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. Key words: Colon cancer; Survivin shRNA-APC double gene; VEGF; COX-2; MVD