miRNA-223对非小细胞肺癌A549/DDP细胞顺铂耐药性的影响

Influence of miRNA-223 on drug-resistance of non-small cell lung cancer A549/DDP cells to cisplatin

目的探讨microRNA-223(miR-223)在非小细胞肺癌(NSCLC)A549/DDP细胞对顺铂(DDP)耐药性方面的影响及可能机制。方法采用实时荧光定量PCR(qRT-PCR)检测DDP耐药肺腺癌细胞株A549/DDP及其亲本细胞株A549中miR-223的水平,将A549/DDP细胞分为3组:对照组、空转染组(转染无关序列)和抑制组(转染miR-223 inhibitor),于转染24、48、72及96 h后分别采用qRT-PCR和CCK-8法检测转染效果及细胞增殖情况,CCK-8法评价转染后A549/DDP细胞对DDP的药物敏感性变化,采用流式细胞术检测转染48 h后的细胞凋亡和细胞周期情况,Western blotting检测转染48 h后耐药基因蛋白P-糖蛋白(P-gp)、多药耐药相关蛋白1(MRP1)及肺耐药相关蛋白(LRP)的表达。结果 A549/DDP细胞中的miR-223水平较高,为A549细胞的(7.14±0.26)倍(P<0.05);抑制组转染后的miR-223水平降低,转染96 h后的miR-223水平分别降至对照组的(67.15±2.84)%和空转染组的(65.80±3.47)%,差异均有统计学意义(P<0.05);与对照组相比,抑制组转染后的增殖抑制率、凋亡率及G0/G1期细胞比例均升高,而S和G2/M期细胞比例及3种耐药基因蛋白水平均降低,以上差异均有统计学意义(P<0.05)。DDP对抑制组细胞的半数抑制浓度(IC50)值为(15.67±1.30)μg/ml,低于对照组的(33.71±2.61)μg/ml(P<0.05)。结论 miR-223可增加A549/DDP细胞对DDP的耐药性,可能与耐药基因蛋白表达上调有关;降低miR-223水平可抑制A549/DDP细胞增殖、诱导凋亡及细胞周期阻滞,并下调耐药蛋白的表达,从而增加A549/DDP细胞对DDP的敏感性。

Objective To explore the influence of miRNA-223 （ miR-223） on drug-resistance of non-small cell lung cancer A549/DDP cells to dichorodiamine platinum （ DDP ） and its possible mechanism. Methods The real-time fluorescence quantitative PCR （ qRT-PCR） was used to measure the miR-223 level of A549/DDP cells. According to the experimental protocol, A549/DDP cells were divided into 3 groups：control group, empty vector transfection group （ cells transfected with unrelated sequence） and inhibi-tor group （ cells transfected with miR-223 inhibitor） . The qRT-PCR and CCK-8 were applied to detect the effect of transfection and pro-liferation at 24, 48, 72 and 96 h post-transfection. The changes of drug-resistance of A549/DDP cells to cisplatin were measured by CCK-8. The cell cycle and apoptosis at 48 h post-transfection were detected via flow cytometry. The changes of expression of multidrug resistance protein, such as P-glycoprotein （ P-gp） , multidrug resistance associated protein 1 （ MRP1） and lung resistance related pro-tein （LRP）, were evaluated by Western blotting. Results A higher level of miR-223 was observed in A549/DDP cells than in A549 cells with a （7.14±0.26）-fold increase. There was a decreased miR-223 level in inhibitor group after transfection, which was further decreased compared to （67.15±2.84）% of the control group and （65.80±3.47）% of the empty vector transfected group at 96 h post-transfection （ P〈0.05） . Compared with the control group, there were elevated inhibitory rates of proliferation, early and late apoptotic rates and proportion of cells in G0/G1 phase but reduced proportion of cells in S and G2/M phases and three protein levels related to re-sistance genes in inhibitor group. The inhibited concentration of 50% （IC50） for DDP was （15.67±1.30） μg/ml in inhibitor group, lower than （ 33.71 ± 2.61） μg/ml in control group. Conclusion MiR-223 can increase the drug resistance of A549/DDP cells to＆amp;nbsp;DDP, possibly by increasing the gene expression related to resistance. The reduced level of miR-223 resulted in the inhibition of the proliferation, induction of apoptosis and cell cycle arrest and the down-regulation of drug resistance protein.