微小RNA-21转染对人肺癌细胞株顺铂耐药性的影响及机制探讨  

The effect and mechanism of microRNA-21 on cis-dichlorodiamineplatinum resistance in lung cancer cell strain

作　　者：任丽[1] 黄琛[4] 柳雅慧 于洋[4] 林丽[2] 文柳静[3] 

机构地区：[1]天津医科大学肿瘤医院检验科天津市肿瘤防治重点实验室,300060 [2]天津医科大学肿瘤医院肺部肿瘤内科,300060 [3]天津医科大学肿瘤医院药剂科,300060 [4]天津医科大学

出　　处：《中华医学杂志》2016年第18期1454-1458,共5页National Medical Journal of China

基　　金：国家自然科学基金（81402174）

摘　　要：目的探讨微小RNA-21（miR-21）在肺癌化疗耐药中的作用及机制，为非小细胞肺癌多药耐药的逆转提供实验性及理论性依据。方法通过实时荧光定量聚合酶链反应（qRT—PCR）检测非小细胞肺癌顺铂敏感细胞株A549及耐药细胞株A549／DDP（原发A549／DDP细胞）中miR-21的表达差异。合成miR-21的干扰序列，以脂质体为载体转染A549／DDP细胞，下调细胞中miR-21表达水平；检测细胞对化疗药物顺铂敏感性的变化；流式细胞术检测肿瘤细胞凋亡及细胞周期相关指标；蛋白质免疫印迹（Westernblot）法检测生存素（Survivin）、周期蛋白D1（CyclinD1）、表皮生长因子受体（EGFR）、多药耐药相关蛋白（MRP）和脂蛋白受体相关蛋白（LRP）表达水平。结果miR-21在原发A549／DDP细胞中表达水平是顺铂敏感细胞株A549的（522．3±31．6）％，差异有统计学意义（t=48．318，P〈0．01）；miR-21表达下调后，A549／DDP细胞对顺铂的敏感性明显增加，半数抑菌浓度（IC，。）降低为（22．2±1．2）μmol／L，转染空载质粒对照组的IC50为（48．6±3．2）μmol／L，差异有统计学意义（t=5．608，P〈0．01）。与转染空载质粒对照组相比，成功转染A549／DDP细胞组细胞凋亡率为（27．7±1．1）％，显著高于原发A549／DDP细胞的凋亡率（16．8±1．1）％，差异有统计学意义（t=10．183，P〈0．01）。成功转染A549／DDP细胞组细胞周期G0／G1期比例为（37．5±1．2）％，显著低于原发A549／DDP细胞的GO／G1期比例（43．4±2．3）％，差异有统计学意义（t=8．202，P〈0．01），生存素表达水平是原发A549／DDP细胞的（71．7±4．3）％，差异有统计学意义（t=4．771，P〈0．01），CyclinD1表达水平是原发A549／DDP细胞的（69．4±4．5）％，差异有统计学意义（t=5．162，P〈0．01），EGFR表达水平是原发A549／DDP细胞的（52．3±3．2）％，差异�Objective To investigate the role of miR-21 on muhidrug resistance （MDR） in non-small cell lung cancer （ NSCLC ） and to provide experimental and theoretical basis for MDR reversal. Methods Quantitative reverse-transcription polymerase chain reaction （qRT-PCR） was used to determine the mRNA level of miR-21 both in the chemo-sensitivity cell strain A549 and the chemo-resistance cell strain A549/DDP （primary A549/DDP cells）. The miR-21 interference sequence was synthesized and transfected into A549/DDP cells by liposome as the carrier. The miR-21 expression level was knocked down and the change of chemo-sensitivity of cells was detected. Effects of miR-21 on the cell apoptosis and cell cycle in miR-21-depleted A549/DDP cells were analyzed by flow cytometry to elucidate the involvement of miR-21 in MDR reversal in NSCLC. The expression of multidrug-resistant proteins Survivin, Cyclin D1, Epidermal growth factor receptor （EGFR）, Muhidrug resistance-associated proteinl （MRP1） and Lipoprotein receptor- related protein （LRP） were detected by Western blot. Results MiR-21 level in multidrug-resistant NSCLC cell line A549/DDP was （5. 223 ±0. 316） folds higher than that in A549 cell line （t =48. 318, P 〈0.01 ）. Knockdown of miR-21 in A549/DDP cells significantly reversed their sensitivity to cis-DDP, meanwhile, the value of half maximal inhibitory concentration was significantly decreased compared with control group transfected with empty vector （ （22.2 ± 1.2） and （48.6 ± 3.2） p,mol/L, t = 5. 608, P 〈 0.01 ）. Cell apoptosis rate in A549/DDP cell with knockdown of miR-21 was significantly increased compared with primary A549/DDP cell line （ （27.7 ± 1.1 ） % and （16.8 ± 1.1 ） %, t = 10. 183, P 〈0.01）. Cell cycle GO/G1 phase in A549/DDP cell with knockdown of miR-21 was significantly decreased compared with primary A549/DDP cell line （ （37.5 ± 1.2） % and （43.4 ± 2.3 ） %, t = 8. 202, P 〈 0.01 ）. Compared with primary A549/DDP cell line, the exp

关 键 词：癌 非小细胞肺 抗药性 肿瘤 微RNAS 

分 类 号：R734.2[医药卫生—肿瘤]