microRNA-149在顺铂耐药非小细胞肺癌细胞中的表达变化及相关机制研究

目的探讨microRNA-149(miR-149)在顺铂耐药A549细胞(A549/DDP)中的表达规律及作用机制。方法生物信息学方法分析顺铂耐药A549细胞中的microRNA表达变化。体外培养A549细胞并诱导对顺铂耐药的A549/DDP细胞系,实时荧光定量PCR(qRT-PCR)检测miR-149在两种细胞系中的表达差异。CCK-8试验及流式细胞术检测单纯miR-149过表达以及miR-149和ABCC1同时过表达对细胞顺铂敏感性的影响。qRT-PCR及Western blot检测A549及A549/DDP细胞中ABCC1的表达差异,双荧光素酶报告试验检测miR-149以ABCC1之间的靶向关系,并利用qRT-PCR及Western blot加以证实。结果 A549/DDP细胞中的miR-149表达较低,与A549细胞相比,差异具有统计学意义(t=-4.65,P0.05)。miR-149过表达可以降低顺铂处理后A549/DDP细胞的活力但增加其凋亡率,与阴性转染对照细胞相比,差异具有统计学意义(P均0.05)。与A549细胞相比ABCC1在A549/DDP细胞中存在mRNA及蛋白水平的高表达,差异具有统计学意义(t=8.44、5.14,P均0.05)。过表达miR-149可以降低A549/DDP细胞中ABCC1的mRNA及蛋白水平,与阴性转染对照细胞相比,差异具有统计学意义(t=-3.14、-4.54,P0.05)。双荧光报告酶分析显示miR-149可直接与ABCC1 mRNA的3’UTR结合。miR-149与ABCC1同时过表达的A549/DDP细胞与单纯miR-149过表达的细胞相比,顺铂处理后细胞活力增加,凋亡减少,差异具有统计学意义(P均0.05)。结论 miR-149表达不足可能引起A549细胞对顺铂的耐药,该机制与miR-149对ABCC1的抑制作用有关。

Changes of expression of microRNA-149 in cisplatin-resistant non-small cell lung cancer cells and relevant mechanism

Objective To investigate the expression principle and mechanism of microRNA-149(miR-149)in cisplatinresistant A549 cells(A549/DDP).Methods Bioinformatics methods were used to analyze microRNA expression changes in cisplatin-resistant A549 cells.A549 cells were cultured in vitro and induced to cisplatin-resistant A549/DDP cell line.Real-time fluorescence quantification PCR(qRT-PCR)was used to detect the differences in the expression of miR-149 in both cell lines.CCK-8 assay and flow cytometry were used to detect the effect of miR-149 overexpression alone and the simultaneous overexpression of miR-149 and ABCC1 on cell cisplatin sensitivity.qRT-PCR and Western blot were used to detect the difference of ABCC1 expression in A549 and A549/DDP cells.Dual luciferase reporter assay was used to detect the targeting relationship between miR-149 and ABCC1,which was confirmed by qRT-PCR and Western blot.Results The expression of miR-149 was lower in A549/DDP cells.The difference was statistically significant compared to A549 cells(t=-4.65,P<0.05).Overexpression of miR-149 could reduce the viability of A549/DDP cells after cisplatin treatment but increase its apoptosis rate.The difference was statistically significant compared to the negative transfected control cells(P<0.05).Compared with A549 cells,ABCC1 had high expression of mRNA and protein in A549/DDP cells,and the difference was statistically significant(t=8.44 and 5.14,P<0.05).Overexpression of miR-149 could decrease the mRNA and protein levels of ABCC1 in A549/DDP cells.The difference was statistically significant compared to the negative transfected control cells(t=-3.14 and-4.54,P<0.05).Dual luciferase reporter assay showed that miR-149 bound directly to the 3''UTR of ABCC1 mRNA.Compared with cells overexpressing miR-149 alone,combined overexpression of miR-149 and ABCC1 in A549/DDP cells showed increased cell viability and decreased apoptosis after cisplatin treatment,and the differences were statistically significant(P<0.05).Conclusion Insufficient expression of miR-149 may cause resistance of cisplatin to A549 cells.This mechanism is related to the inhibition of ABCC1 by miR-149.