Shi Sien, He Zefeng, Cai Jianchun, Qiu Jiangfeng
Department of Tumor Surgery, First Affiliated Hospital of Xiamen University, Xiamen, China.
Zhongguo Fei Ai Za Zhi. 2011 Apr;14(4):305-10. doi: 10.3779/j.issn.1009-3419.2011.04.01.
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.
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.
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.
Cisplatin resistance of lung cancer cells is associated with overexpression of GRP75 gene, which could regulate the expressions of p53 and bcl-2.
GRP75是热休克蛋白家族成员之一，在一些耐药癌细胞中过表达，是一种主要位于线粒体膜的分子伴侣。本研究旨在通过RNA干扰技术下调GRP75表达，探讨GRP75在癌细胞耐药机制中的作用。
通过逐渐增加顺铂浓度并结合高剂量冲击，从人肺腺癌细胞系A549亲本细胞建立顺铂耐药细胞A549/CDDP。通过慢病毒将GRP75的短发夹RNA转染到A549和A549/CDDP细胞中。应用蛋白质免疫印迹法和甲基噻唑基四氮唑（MTT）法检测沉默GRP75表达对细胞对顺铂敏感性的影响。
六组的感染率均超过90%。感染后，A549和A549/CDDP中GRP75的表达水平均下调（P<0.05）；A549/CDDP中p53的表达水平上调（P<0.05），A549/CDDP中bcl-2的表达水平下调（P<0.05）。感染前后A549/CDDP的耐药指数分别为21.52和4.14。
肺癌细胞对顺铂的耐药性与GRP75基因的过表达有关，GRP75可调节p53和bcl-2的表达。
