Résumé
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.
Résumé
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.
Résumé
Objective Post-transcription RNA interference (RNAi) is more and more widely applied in multigene therapy.This study aimed to establish an subcutaneous xenotransplanted tumor (SXT) model of human HT-29 colon carcinoma in nude mice and investigate the effects of the survivin shRNA-APC double gene co-expression lentiviral vector on the growth of SXT.Methods Thirty-five nude mice were equally divided into five groups, double-gene survivin shRNA, survivin shRNA, APC, empty vector, and blank, and injected into the left anterior axillary with respective stably transfected cell lines and human HT-29 colon carcinoma cells, all at 2×106/mL, to establish an SXT model of human HT-29 colon carcinoma.The inhibition rate of tumor growth was calculated by measuring the size and weight of the SXT, the expressions of survivin mRNA and protein in the tumor tissue detected by real time PCR and immunohistochemistry respectively, and the apoptosis of the HT-29 colon carcinoma cells determined by TUNEL.Results The mean size and weight of the SXT were significantly reduced in the double-gene survivin shRNA-APC, survivin shRNA, and APC groups as compared with the blank and empty vector groups (P<0.05), though increased in the survivin shRNA and APC groups in comparison with the double-gene group (P<0.05).The expressions of survivin mRNA and protein in the tumor tissue were remarkably lower in the double-gene survivin shRNA-APC, survivin shRNA, and APC groups than in the blank and empty vector groups (P<0.05), even lower in the double-gene group than in the survivin shRNA, and APC groups (P<0.05).The apoptosis rate of the HT-29 colon carcinoma cells was markedly up-regulated in the double-gene survivin shRNA-APC ([56.72±3.17]%), survivin shRNA ([33.64±2.03]%), and APC groups ([31.19±1.79]%) as compared with the blank ([9.89±0.31]%) and empty vector groups ([10.06±0.43]%) (P<0.05), even more significantly in the double-gene than in the survivin shRNA and APC groups (P<0.05).Conclusion The survivin shRNA-APC double gene co-expression lentiviral vector can reduce the expression level the survivin gene, promote the apoptosis of colon carcinoma cells, and suppress the growth of the subcutaneous xenotransplanted tumor.
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Objective Studies show that the abnormal ex-pressions of APC and survivin play important roles in the development and progression of colon cancer .Survivin shRNA and APC double gene co-expression lentiviral vector was constructed to observe whether it could be successfully expressed in HT-29 colon cancer cells and whether it could impact on colon cancer cell apoptosis . Methods We selected the best shRNA interference fragment from the construc-tion of three pairs of complementary shRNA fragments and connected it with the effective fragment of APC ( aa1020-1698 ) to construct a double gene co-expression lentiviral vector .HT-29 cells were divid-ed into experimental group , empty loading group and blank group .HT-29 cells were observed by fluorescence microscopy after infec-tion.Survivin and APC expression levels were observed by real time PCR and western blot .Apoptosis was detected by caspase-3 activi-ty assay. Results ①We successfully constructed three pairs of shRNA sequences and proved that they had no human gene homolo -gous to the rest.Real time PCR analysis showed that the best sequence was shRNA 3.②After the sequence alignment of constructed shRNA vectors, three pairs of shRNA sequences were completely the same with the first designed sequence .Green fluorescence was observed in HT-29 cells by fluorescence microscope .The survivin content in experiment group (31.71 ±1.49) was significantly de-creased compared with empty loading group (100 ±0) and blank group(95.12 ±2.15)(P<0.05).The expression level of APC mR-NA was significantly increased compared with empty loading group (0 ±0) and blank group(0.51 ±0.15)(P<0.05).③The relative ratio of apoptosis in experiment group (0.573 ±0.050) was significantly increased compared with empty loading group (0.390 ± 0.040) and blank group(0.407 ±0.030)(P<0.05). Conclusion We have successfully constructed survivin-shRNA-APC double gene co-expression lentiviral vector which can be successfully expressed in HT-29 colon cancer cells , providing references for subse-quent gene therapy by the use of the carrier .
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BADH-CMO double gene,CMO gene and DREB1A gene were transformed respectively to embryonic callus of Kentucky bluegrass by particle bombardment. Then the embryonic callas of kentucky bluegrass were put in meclium for subculture which is mixed with 100mg/L hygromycin and the meclium for plantlet regeneration which mixell with 50mg/L hy gromycin about one month. Thus,the hygromycin-selectecl plants were obtained and were transplantecl into tlowerpots. The results of the PCR and Southern blot analysis indicated that the DREB1A gene,CMO gene and BADH-CMO double-gene were integrated into the genomic DNA of Kentucky bluegrass.