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

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

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,P<0.05)。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,P<0.05)。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。

Objective To investigate the expression principle and mechanism of microRNA-149（miR-149） in cisplatin-resistant A549 cells （A549/DDP）. Methods Bioinformatics methods were used to analyze microRNA expression changesin cisplatin-resistant A549 cells. A549 cells were cultured in vitro and induced to cisplatin-resistant A549/DDP cellline. Real-time fluorescence quantification PCR （qRT-PCR） was used to detect the differences in the expression ofmiR-149 in both cell lines. CCK-8 assay and flow cytometry were used to detect the effect of miR-149 overexpressionalone and the simultaneous overexpression of miR-149 and ABCC1 on cell cisplatin sensitivity. qRT-PCR and Westernblot 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 andWestern 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/DDPcells after cisplatin treatment but increase its apoptosis rate. The difference was statistically significant compared to thenegative transfected control cells（P〈0.05）. Compared with A549 cells, ABCC1 had high expression of mRNA and proteinin A549/DDP cells, and the difference was statistically significant（t=8.44 and 5.14, P〈0.05）. Overexpression of miR-149could decrease the mRNA and protein levels of ABCC1 in A549/DDP cells. The difference was statistically significantcompared to the negative transfected control cells（t=-3.14 and -4.54, P〈0.05）. Dual luciferase reporter assay showedthat 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 decreasedapoptosis after cisplatin treatment, and the differences were statistically significant （P〈0.05）. Conclusion Insufficientexpression of miR-149 may cause resistance of cisplatin to A549 cells. This mechanism is related to the inhibition ofABCC1 by miR-149.