CAJ | 학술논문

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。
목적：탐토microRNA?223（ miR?223）재비소세포폐암（ NSCLC） A549／DDP세포대순박（ DDP ）내약성방면적영향급가능궤제。방법채용실시형광정량PCR（ qRT?PCR）검측DDP내약폐선암세포주A549／DDP급기친본세포주A549중miR?223적수평，장A549／DDP세포분위3조：대조조、공전염조（전염무관서렬）화억제조（전염miR?223 inhibitor），우전염24、48、72급96 h후분별채용qRT?PCR화CCK?8법검측전염효과급세포증식정황，CCK?8법평개전염후A549／DDP 세포대DDP적약물민감성변화，채용류식세포술검측전염48 h후적세포조망화세포주기정황，Western blotting검측전염48 h후내약기인단백P?당단백（P?gp）、다약내약상관단백1（MRP1）급폐내약상관단백（LRP）적표체。결과 A549／DDP세포중적miR?223수평교고，위A549세포적（7?14±0?26）배（P＜0?05）；억제조전염후적miR?223수평강저，전염96 h후적miR?223수평분별강지대조조적（67?15±2?84）％화공전염조적（65?80±3?47）％，차이균유통계학의의（P＜0?05）；여대조조상비，억제조전염후적증식억제솔、조망솔급G0／G1기세포비례균승고，이S화G2／M기세포비례급3충내약기인단백수평균강저，이상차이균유통계학의의（P＜0?05）。 DDP대억제조세포적반수억제농도（IC50）치위（15?67±1?30）μg／ml，저우대조조적（33?71±2?61）μg／ml（ P＜0?05）。결론 miR?223가증가A549／DDP 세포대DDP 적내약성，가능여내약기인단백표체상조유관；강저miR?223수평가억제A549／DDP세포증식、유도조망급세포주기조체，병하조내약단백적표체，종이증가A549／DDP 세포대DDP적민감성。
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 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.