目的:比较人肺腺癌顺铂耐药株A549/DDP细胞与人肺腺癌顺铂敏感株A549细胞的自噬水平,观察微小RNA-126(microRNA-126,miRNA-126)对肺癌顺铂耐药细胞自噬的影响,并探讨miRNA-126在肺癌顺铂耐药中的作用.方法:MTT法检测顺铂的半数抑制浓度(half maximal inhibitory concentration,IC50);RT-qPCR检测miRNA-126转染后肺癌细胞中miRNA-126的表达量;吖啶橙染色观察肺癌细胞自噬囊泡的形成情况;Western blot检测自噬标志蛋白微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)的表达变化.结果:A549/DDP细胞自噬囊泡数量和LC3水平显著高于A549细胞(P<0.01);转染miRNA-126上调了A549/DDP细胞中miRNA-126的表达量(P<0.01);miRNA-126降低A549/DDP细胞中自噬囊泡比例和LC3-Ⅱ蛋白表达水平(P<0.01),从而抑制了顺铂耐药细胞A549/DDP的自噬水平;自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)提高A549/DDP细胞对顺铂的敏感性,miRNA-126联合3-MA进一步增加A549/DDP细胞对顺铂的敏感性(P<0.01).结论:miRNA-126通过抑制自噬逆转了肺癌A549/DDP细胞顺铂耐药.
miRNA-126 reverses cisplatin resistance in lung cancer A549/DDP cells by inhibiting autophagy
AIM:Autophagy levels in lung cancer cisplatin-resistant A549/DDP cells and cisplatin-sensitive A549 cells were compared.The effects of microRNA-126(miRNA-126)on autophagy in lung cancer cisplatin-resistant cells were assessed,and the role of miRNA-126 in lung cancer cisplatin-resistant cells was discussed.METHODS:An MTT assay was used to detect the half maximal inhibitory concentration of cisplatin.The miRNA-126 expression was de-tected by RT-qPCR.Autophagolysosomes were observed under a fluorescence microscope using acridine orange staining.Protein levels of autophagy-associated molecule microtubule-associated protein 1 light chain 3(LC3)in lung cancer cells were determined by Western blot.RESULTS:Autophagy was significantly elevated in A549/DDP cells compared with A549 cells(P<0.01).The expression level of miRNA-126 was significantly elevated in A549/DDP cells after transfection with miRNA-126(P<0.01).miRNA-126 reversed cisplatin resistance of A549/DDP cells,and the level of intracellular acid dye follicular bright red fluorescence in the A549/DDP cells was significantly reduced after transfection with miRNA-126(P<0.01).The LC3-Ⅱ protein level in A549/DDP cells was significantly reduced after transfection with miRNA-126(P<0.01),and inhibition of cell autophagy by 3-methyladenine(3-MA)enhanced cisplatin sensitivity of A549/DDP cells(P<0.01).Compared with 3-MA+NC group,miRNA-126 combined with 3-MA further increased cisplatin sensitivity of A549/DDP cells(P<0.01).CONCLUSION:miRNA-126 reverses the cisplatin resistance in lung cancer A549/DDP cells by inhibiting autophagy.
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