| Objective colorectal cancer is a common neoplasm in our country, colorectal cancer mortality in the third in Western countries, only behind lung cancer and breast cancer; colorectal cancer mortality cancer in the fifth in China, and the incidence rate showed a rise year by year. The main treatment of colon carcinoma in the prophase and metaphage is surgery, patients with adanced and recurrent, its main treatments is for chemotherapy. However,multi-drug resistance (MDR) has become the biggest problem in the treatment of various malignant tumors, including colorectal cancer.lt is the main reason in the failure of chemotherapy of several cancer.In this study, our purpose is to compare the differences of short hairpin RNA (shRNA) and Tetrandrine(Tet) in the expression of MDR1mRNA,the expression of P-glycoprotein (P-gp), the effect on cell cycle and apoptosis rate, and the sensitivity to chemotherapeutic drugs,we investigate the mechanism that shRNA and Tet reverse MDR1 gene-mediated multidrug resistance in human colon carcinoma LoVo/5-Fu cells,so as to provide for a good guideline of the application of clinical medicine.Methods A eukaryotic expression plasmid of shRNA targeting on MDR1 was constructed and transiently trans fect ed into human colon carcinoma LoVo/5-Fu cells,which was as the group intervened with shRNA,then the LoVo/5-Fu cells were separately intervened with Tet and Serum-free medium as the group intervened withTet and control group.Rug sensitivity,Expression of MDR1 mRNA and P-gp was evaluated by MTT,real-time quantitative PCR (Real-time PCR) and Western blot respectively. The Cell cycle, apoptosis of cells and expression of P-glycoprotein (P-gp) were all evaluated by flow cytometry assay. Use the SPSS 10.0 statistical software to analyze experimental data.Results1. The IC50 of 5-Fu and the relative reverse rate of sensitivity of LoVo/5-Fu cells to 5-FuIn the groups intervened with shRNA,Tet and the control group, the IC50 of 5-Fu were 2.38±0.45mol/ml,4.41±0.72mol/ml,7.27±0.34mol/ml respectively.There were significant difference between group intervened with shRNA and control group (P<0.05),the relative reverse rate of sensitivity of LoVo/5-Fu cells to 5-Fu was 72.7%;There were significant difference between group intervened with Tet and control group (P<0.05),the relative reverse rate of sensitivity of LoVo/5-Fu cells to 5-Fu was 42.5%.2.The cell cycle of LoVo/5-Fu cellsAfter intervened with shRNA and Tet,the cells in the G1 cycle were both increased,in the S cycle were both reduced at the same time. ShRNA and Tet can both greatly increased the cells in the G1 cycle and reduced the cells in the S cycle,which by inhabiting the expression of MDR1 mRNA.There were significant difference between group intervened with shRNA and group intervened with Tet (P<0.05)3.The apoptotic rate of LoVo/5-Fu cellsIn the groups intervened with shRNA,Tet and the control group, the apoptotic rate were (5.88±0.44)%,(3.24±0.26)%,(1.32±0.47)% respectively.There were significant difference between group intervened with shRNA and control group (P<0.05);There were significant difference between group intervened with Tet and control group (P<0.05);There were significant difference between group intervened with shRNA and group intervened with Tet (P<0.05) 4.The expression rate of P-gp of LoVo/5-Fu cellsIn the groups intervened with shRNA,Tet and the control group, the expression rate of P-gp were (61.6±4.85)%,76.6±3.62)%,(96.9±2.25)% respectively. There were significant difference between group intervened with shRNA and control group (P<0.05);There were significant difference between group intervened with Tet and control group (P<0.05);There were significant difference between group intervened with shRNA and group intervened with Tet (P<0.05)5.The expression of MDR1 mRNA of LoVo/5-Fu cellsIn the groups intervened with shRNA,Tet and the control group, the expression of MDR1 mRNA were 232.98±81.04,569.99±85.15,1462.00±161.17 respectively.There were significant difference between group intervened withshRNA and control group (P<0.05);There were significant difference between group intervened with Tet and control group (P<0.05);There were significant difference between group intervened with shRNA and group intervened with Tet (P<0.05)6.Western blot to detected the expression of P-gp of LoVo/5-Fu cellsAfter intervened with shRNA and Tet,the expression of P-gp were both reduced.In the group intervened with shRNA,the expression of P-gp was more reduced than the group intervened with Tet.Conclusion1.ShRNA and Tet could both reverse MDR1 gene-mediated multidrug resistance in human colon carcinoma LoVo/5-Fu cells.2.The mechanism that shRNA and tetrandrine reverse MDR1 gene-mediated multidrug resistance in human colon carcinoma LoVo/5-Fu cells may relate to the down-regulation of MDR mRNA and P-gp protein.3.MDR1 shRNA and Tet could both reverse MDR1 gene-mediated multidrug resistance in human colon carcinoma LoVo/5-Fu cells, but the effect of MDR1 shRNA was better than Tet. |
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