微RNA-221诱导PC-9肺癌细胞对吉非替尼的耐药机制  

Mechanism of miR-221 contributes to gefitinib resistance in PC-9 cells

作　　者：高园园 康小红[1] 王颖[1] 赵可雷[1] 王亚楠 寇卫政[1] 苗战会[1] 路平[1] Gao Yuanyuan, Kang Xiaohong, Wang Ying, Zhao Kelei, Wang Ya＇nan, Kou Weizheng, Miao Zhanhui, Lu Ping.(Department of Oncology, the First Affiliated Hospital of Xinxiang Medical University, Xinxiang 453100, China)

机构地区：[1]新乡医学院第一附属医院肿瘤科,河南新乡453100

出　　处：《中华医学杂志》2018年第42期3447-3452,共6页National Medical Journal of China

基　　金：国家自然科学基金（81503414）

摘　　要：目的探讨微RNA（miR）－221诱导PC－9肺癌细胞对吉非替尼的耐药机制。方法应用慢病毒空载对照（LV－NC）和LV－miR－221转染PC－9细胞并建立细胞稳转株PC－9/NC和PC－9/miR－221。荧光定量PCR法检测稳转细胞株中miR－221的相对表达量；细胞计数试剂盒（CCK－8法）检测吉非替尼（0～4 μmol/L）对细胞生长增殖的影响；流式细胞术检测吉非替尼对细胞凋亡的影响；荧光定量PCR法和免疫荧光法检测凋亡酶激活因子－1（APAF－1）mRNA和APAF－1蛋白相对表达量；双荧光素酶报告基因实验检测APAF－1与miR－221的关系；Western印迹检测表皮生长因子受体（EGFR）及其磷酸化（p－EGFR）和APAF－1、剪切的半胱氨酸/天冬氨酸蛋白水解酶（Cleaved－caspase－3）蛋白相对表达量。结果PC－9/NC细胞中miR－221的相对表达量显著低于PC－9/miR－221细胞[（1.00±0.082）比（40.24±0.017）]（P〈0.01）；PC－9/NC细胞半数抑制浓度（IC50）显著低于PC－9/miR－221细胞[（IC50 ＝0.1 μmol/L）比（IC50〉4 μmol/L）]（P〈0.05）；PC－9/NC细胞凋亡率显著高于PC－9/miR－221细胞[（33.42±4.28）%比（10.27±1.12）%]（P〈0.05）；PC－9/NC细胞中APAF－1mRNA的表达量显著高于PC－9/miR－221细胞中[（1.000±0.069）比（0.701±0.072）]（P〈0.05），PC－9/NC细胞中APAF－1蛋白的相对表达量明显高于PC－9/miR－221细胞。共转染荧光素酶质粒pmir－REPORT－APAF－1－wt和miR－221a模拟物后，miR－221a抑制荧光素酶活性显著高于转染miRNA阴性对照组（P〈0.01）；吉非替尼均可下调PC－9/NC和PC－9 /miR－221细胞中的p－EGFR，PC－9/miR－221细胞中APAF－1、Cleaved－caspase－3蛋白明显低于PC－9/NC细胞，吉非替尼上调PC－9/NC细胞中的APAF－1、Cleaved－caspase－3等促凋亡蛋白表达水平显著高于PC－9/miR－221细胞（P〈0.05）。结论miR－221可能通过下调APAF－1的表达诱导PC－9细胞对吉ObjectiveTo investigate the effect of microRNA-221 （miR-221） overexpression on gefitinib resistance in PC-9 cells and study its underlying mechanisms.MethodsPC-9 cells were transfected with LV-NC and LV-miR-221 to establish cell stabilizing strains （PC-9/NC and PC-9/miR-221）, then they were used to detect the relative expression of miR-221 and apoptotic protease activating factor-1 （APAF-1） mRNA by real-time fluorescence quantitative PCR （qRT-PCR）. The effects of gefitinib （0-4 μmol/L） on the growth and proliferation of cell stabilizing strains were detected by CCK-8 Assay. After gefitinib treatment, cell apoptosis was detected by Flow Cytometry Assays. The expression of epidermal growth factor receptor （EGFR）, phosphorylated epidermal growth factor receptor （p-EGFR）, APAF-1 and cleaved cysteinyl aspartate specific proteinase-3 （Cleaved-caspase-3） were detected by Western blot. Dual-Luciferase Reporter Assay was used to evaluate the relationship between APAF-1 and miR-221.ResultsThe relative expression of miR-221 in PC-9/NC cells was significantly lower than that in PC-9/miR-221 cells[（1.00±0.082） vs （40.24±0.017）]（P〈0.01）. The half maximal inhibitory concentration （IC50） in PC-9/NC cells was significantly lower than that in PC-9/miR-221 cells[（IC50=0.1 μmol/L） vs （IC50〉4 μmol/L）]（P〈0.05）. Apoptosis rate of PC-9/NC cell was significantly higher than PC-9/miR-221[（33.42±4.28）% vs （10.27±1.12）%]（P〈0.05）; APAF-1 mRNA expression was significantly higher in PC-9/NC cells than PC-9/miR-221[（1.000＋ 0.069） vs （0.701±0.072）]（P〈0.05）, and the expression of APAF-1 protein in PC-9/NC cells was significantly higher than that of PC-9/miR-221 cells. The dual luciferase reporter gene results showed that miR-221a inhibited luciferase activity significantly stronger than transfected miRNA negative control group after co-transfection of luciferase plasmids pmir-REPORT-APAF-1-wt and miR-221a mimics （P〈0.01）. p-EGFR was down-

关 键 词：肺肿瘤 微RNAS 吉非替尼 抗药性  肿瘤 

分 类 号：R734.2[医药卫生—肿瘤]