Background and objective GRP75, a member of HSPs which is overexpressed in some resistant cancer cells, is a molecular chaperone mainly located in mitochondrial membrane.The aim of this study is to investigate the role of GRP75 on the resistant mechanisms of cancer cells by downregulating GRP75 expreesion via RNAi approach.Methods Cisplatin-resistant cell A549/CDDP was established from their parental human lung adenocarcinoma cell line A549 by combining gradually increasing concentrations of cisplatin with high dosage impact.The shRNA for GRP75 was transfected into A549 and A549/CDDP cells by lentivirus.Western blot and methyl thiazolyl tetrazolium (MTT) assay were applied to detect the influence of silencing GRP75 expression on sensitivity of the cells to cisplatin.Results The infection rate of six groups were all over 90％.After infection, the level of expression of GRP75 in both A549 and A549/CDDP were down-regulated (P＜0.05); the level of expression of p53 in A549/CDDP was up-regulated (P＜0.05) and the level of expression of bcl-2 of A549/CDDP was down-regulated (P＜0.05).The resistance index of A549/CDDP before and after infection were 21.52 and 4.14 respectively.Conclusion Cisplatin resistance of lung cancer cells is associated with overexpression of GRP75 gene, which could regulate the expressions of p53 and bcl-2.%背景与目的 GRP75(glucose regulated protein 75)属于热休克蛋白(hot shock protein,HSP)家族,是一种主要位于线粒体的分子伴侣,在某些耐药的肿瘤细胞中高表达.本研究通过慢病毒介导的RNA干扰技术沉默GRP75基因的表达,观察下调GRP75的表达对肿瘤细胞耐药性的影响并探讨GRP75在肿瘤细胞耐药机制中的作用.方法 以顺铂为诱导药物,人肺腺癌细胞系A549为诱导对象,采用逐步增加剂量与大剂量冲击相结合的方法,诱导建立耐顺铂细胞株A549/CDDP.分别将包裹GRP75-shRNA和不含干扰序列的慢病毒转染A549和A549/CDDP细胞,在荧光显微镜下观察各组细胞转染率,应用MTT比色法检测干扰前后各组细胞对顺铂的敏感性,应用Western blot检侧干扰前后各组细胞GRP75,p53,bd-2的表达.结果 各组细胞感染效率均在90%以上,转染后A549/CDDP和A549细胞中GRP75的表达均明显下调(P<0.05).转染前后A549/CDDP细胞对顺铂的耐药指数分别为21.52和4.14.转染后A549/CDDP细胞内p53的表达上调(P<0.05),bcl-2表达下调(P<0.05).结论 GRP75是A549细胞对顺铂耐药机制的相关蛋白之一,其在耐药机制中的作用与其对p53和bcl-2的调控有关.
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