三基序蛋白25过表达对肺癌细胞H1299顺铂敏感性的影响

Effects of TRIM25 overexpression on drug sensitivity of human lung cancer cells H1299 to cisplatin

摘要:

目的：初步探讨三基序蛋白25（tripartite motif containing 25，TRIM25）过表达对人肺癌细胞株H1299顺铂敏感性的影响及其分子作用机制。方法：应用基因克隆技术构建TRIM25高表达质粒载体，瞬时转染人肺癌细胞株H1299，荧光显微镜下观察转染效率，Western blot法检测TRIM25蛋白表达水平及其过表达时丙酮酸激酶M2（pyruvate kinase isozyme type M2，PKM2）蛋白水平的变化。用顺铂分别处理转染前后的H1299细胞24 h，MTT法检测细胞耐药性，Annexin V/PI双染法流式细胞仪（flow cytometry，ＦＣＭ）检测细胞凋亡。结果：同H1299空白组和转染pGCMV/neo空载体组比较，pGCMV-trim25转染组细胞中TRIM25蛋白的表达水平明显增加（P=0.003，P=0.005）；且TRIM25过表达时，细胞中PKM2蛋白的表达水平降低（P=0.001，P=0.003）。用顺铂处理后，pGCMV-trim25转染组细胞的药物敏感性显著下降（P<0.001），转染组细胞凋亡率亦明显降低（P<0.001）。结论：TRIM25在肺癌细胞H1299中高表达时，可能通过下调PKM2而降低对顺铂的敏感性。

Abstract:

Objective：To investigate the effects of tripartite motif containing 25（TRIM25） overexpression on the chemotherapeutic drug sensitivity of human lung cancer cell line H1299 to cisplatin and the molecular mechanisms. Methods：TRIM25 overexpression plas－mid vector was constructed by gene cloning techniques and was transfected into H1299 cells. Efficiency of transfecion was observed under fluorescence microscope. Western blot was used to determine the TRIM25 protein expression level and the change of pyruvate ki－nase isozyme type M2（PKM2） protein level when TRIM25 was overexpressed. After treating untransfected and transfected H1299 cells with cisplatin for 24 h，drug resistance was detected by MTT assay and apoptosis was detected by Annexin V/PI. Results：Compared with those of H1299 blank group and pGCMV/neo empty carrier group，TRIM25 protein levels of pGCMV-trim25 transfection group were significantly increased（P=0.003，P=0.005 respectively）. Moreover，when up-regulating the TRIM25，PKM2 expression was reduced（P=0.001，P=0.003 respectively）. After cisplatin treatment，drug sensitivity as well as apoptosis rate of pGCMV-trim25 trans－fection group were significantly decreased（P<0.001，P<0.001）. Conclusions：Overexpression of TRIM25 protein in lung cancer cells H1299 could decrease the drug sensitivity to cisplatin by down-regulating expressions of PKM2.