RNA干扰抑制ERCC1对非小细胞肺癌化疗敏感性的影响

摘要

目的 探讨利用RNA干扰技术沉默切除修复交叉互补基因组1(ERCC1)表达对非小细胞肺癌耐药细胞株顺铂化疗敏感性的影响.方法 设计并合成3段靶向人的ERCC1基因的小分子干扰RNA(siRNA),构建携带ERCC1-shRNA的重组质粒表达载体,采用脂质体Lipofectamine 2000转染入人肺癌细胞株A549/DDP,荧光镜下观察并测定转染效率;应用逆转录聚合酶链反应(RT-PCR)检测转染前后ERCC1 mRNA的表达情况;应用四甲基偶氮唑蓝比色法(MTT)检测干扰ERCC1后A549/DDP细胞对顺铂敏感性的变化.结果 转染针对ERCC1的siRNA后,转染组A549/DDP细胞内ERCC1 mRNA表达均下降,转染后肺癌A549/DDP细胞对顺铂敏感性增加.结论 利用RNA干扰技术能够筛选出高效的特异阻断ERCC1基因表达的siRNA;ERCC1基因表达下调能够增加肺癌A549/DDP细胞对顺铂的敏感性,部分逆转耐药.%Objective To investigate changes of platinum-based chemotherapy sensitivity of silencing excision repair cross complementation 1(ERCC1) gene expression by using RNA interference in non-small-cell lung cancer ( NSCLC) drug resistance cell lines. Methods Three siRNA sequences targeting ERCC1 gene were designed and synthesized. Recombinant plasmid expression vector which carrying ERCCl-shRNA was constructed and transfected into A54 9/DDP cells with Lipofectamine 2000. Transfection efficiency was measured in the fluorescent microscope. The expression of ERCC1 Mrna was detected by reverse transcription-poly-merase chain reaction(RT-PCR). The change of cisplatin sensitivity after interference was test by MTT assay. Results After transfection of ERCCl-siRNA,the ERCC1 Mrna expressions in A549/DDP cells were all reduced. The sensitivity to cisplatin of A549/ DDP cell line was increased after transfection. The sensitivity to cisplatin of A54 9/DDP cell line was increased after transfection. Conclusion Highly effective and specific siRNA targeting ERCC1 gene can be successfully screened by RNA interference technique. Selective silencing of ERCC1 gene promotes the sensitivity to cisplatin in A54 9/DDP cell and can partly reverse the cisplatin resistance in human cisplatin resistant lung adeno-carcinoma cell lines A549/DDP in vitro.