RNA干扰逆转肾癌细胞A498对长春新碱化疗耐药的研究

 目的　研究RNA干扰（RNAi）对肾癌细胞多药耐药基因（mdr-1）表达的抑制作用，并分析干扰前后肾癌细胞对长春新碱（VCR）药物敏感性的变化。方法　根据mdr-1基因设计3条小干扰RNA（siRNA）序列，在质粒的介导下转染肾癌A498细胞，RT-PCR法分析转染前后mdr-1 mRNA表达水平，筛选出干扰效率最高的siRNA序列；进而利用慢病毒包装siRNA重组质粒，感染A498细胞，RT-PCR法筛选沉默效果最好的细胞株进行克隆，Western blotting法检测mdr-1蛋白表达水平，MTT法对比干扰前后VCR对细胞半抑制浓度（IC50）的变化；相关数据统计通过SPSS10.0软件进行。结果　siRNA-1序列使mdr-1 mRNA表达水平下降67 ％，其稳转细胞株mdr-1蛋白表达量明显下降（P <0.01），并使VCR对细胞的IC50降低约83.25 %（P <0.01）。结论　RNAi技术可有效抑制肾癌A498细胞mdr-1基因的表达，并可显著增加其对VCR的敏感性，从而使肾癌细胞化疗耐药逆转成为可能。

Reversal of vincristine resistance in renal carcinoma cell A498 by RNA interference fragment

Objective To investigate the effect of RNA interference(RNAi)fragment on the expression of muhidrug resistancel(mdr-1)gene and the reversal of vincristine resistance in human renal carcinoma cell line.Methods Three small interfering RNA(siRNA)sequence targeted mdr-1 gene were synthesized and transfected into renal carcinoma A498 cell.The expression level of mdr-1 mRNA was assayed to identify the most effective siRNA fragment by using RT-PCR method.After the A498 cell Was infected by mdr-1-RNAi lentivirus fluid,the optimal gene silencing mdr-1-RNAi A498 cell Was identified by RT-PCR method.After the cell was cloned,the mdr-1 protein expression level was verified by Western blotting method.The IC50 of vincristine Was compared between the primary A498 cell line and the mdr-1-RNAi A498 cell line by MTT method.The statistical analysis Was performed through the SPSS10.0 statistical software.Results The siRNA-1sequence was more effective with a significant reduction of 67%in the suppression of mdr-1 mRNA expression.The expression level of mdr-1 protein and the IC50 of vincristine were significantly decreased in mdr-1-RNAi A498 cell line(P<0.01).Conclusion The RNAi could effectively inhibit the expression of mdr-1 gene and promote the sensibility to vineristine of human renal carcinoma A498 cell line,which may reverse the chemotherapy resistance of renal carcinoma.