Background and objective: Cisplatin is the first-line drug for the chemotherapy of non-small cell lung cancer (NSCLC), but the acquired chemoresistance restricted the effect of its treatment. The aim of this study is to validate the miRNAs related to the Cisplatin resistance in lung cancer and elucidate the molecular mechanisms.
Methods: We performed miRNA microarray and RT-PCR to obtain the aberrant differential expressed miRNAs between A549 and its paired Cisplatin-resistant cell line A549/DDP cells, and then we investigated the biological functions of miR-192, which is the aberrant differential expressed miRNA. After transfection of the miR-192 into A549 cells, we measured the half inhibition concentration (IC50), cell apoptosis of the trasfectant cells, and then we used biological softwares and dual-luciferase report assay to explore the target gene of the miR-192, which was further validated by RT-PCR and Western blot.
Results: MiR-192 was highly over-expressed in A549/DDP cells , whose quantity was 37.59±0.35 fold higher than that in A549 cells. Overexpression of miR-192 in A549 cells significantly conferred resistance to Cisplatin and inhibited apoptosis. By contrast, down-expression of miR-192 in A549/DDP cells remarkably restrained the Cisplatin resistance and induced apoptosis. MiR-192 binded to Bim 3'-UTR and negatively regulated Bim expression at the post-transcriptional level in lung adenocarcinoma cells.
Conclusions: Our data suggested that miR-192 induced Cisplatin-resistance and inhibited cell apoptosis in lung cancer via negative targeting Bim expression.
背景与目的 顺铂是非小细胞肺癌化疗的一线药物，但获得性耐药限制了其疗效的发挥。本研究的目的是筛选鉴定与肺癌顺铂耐药相关的microRNAs，探讨其与肺癌顺铂耐药的影响及分子机制。 方法 应用miRNA芯片及RT-PCR筛选鉴定A549与A549/DDP肺癌细胞间的差异表达miRNAs，将差异表达miR-192转染A549和A549/DDP细胞株，CCK-8检测miR-192对半数抑制浓度（half inhibition concentration, IC50）的影响，流式细胞术检测细胞凋亡，生物软件预测及双荧光素酶报告基因法寻找miR-192的靶基因，RT-PCR及Western blot检测靶基因表达水平在转染前后的变化。 结果 MiR-192在A549/DDP中显著高表达，表达量是A549细胞表达量的37.59±0.35倍。在低表达miR-192的A549细胞中过表达miR-192，细胞对顺铂的IC50显著增高，顺铂引起的细胞凋亡率显著降低；反之，在高表达miR-192的A549/DDP中抑制miR-192的表达，顺铂的IC50显著降低，顺铂引起的细胞凋亡率显著增加。miR-192可靶向作用促凋亡基因Bim的3’-UTR，并在转录后水平负向调控Bim的表达。 结论 MiR-192通过靶向负调控促凋亡基因Bim表达，诱导肺腺癌细胞株A549产生顺铂耐药，并减少顺铂引起的细胞凋亡。