上调微小RNA-200a表达对非小细胞肺癌紫杉醇化疗敏感度的影响及其机制

Effect of up regulation of miRNA-200a expression on the chemosensitivity ofa paclitaxel in non-small cell lung cancer and the underlying mechanism

目的探讨微小RNA-200a(miRNA-200a)对非小细胞肺癌紫杉醇化疗敏感度的影响及其机制。方法采用荧光定量逆转录聚合酶链反应(qRT-PCR)检测非小细胞肺癌A549细胞中miRNA-200a的表达情况;采用脂质体法将miRNA-200a模拟物及miRNA-200a阴性对照转染至非小细胞肺癌A549细胞,采用qRT-PCR检测转染效果;采用噻唑蓝(MTT)法检测上调miRNA-200a表达对紫杉醇处理的非小细胞肺癌A549细胞增殖抑制情况的影响;采用蛋白质印迹法(Western blot)检测上调miRNA-200a表达及紫杉醇处理后非小细胞肺癌A549细胞中Wnt/β-catenin信号通路相关β-catenin蛋白的表达情况。结果非小细胞肺癌A549细胞中miRNA-200a的相对表达量为(0.26±0.08),明显低于正常人肺上皮16HBE细胞的(1.02±0.03)(P﹤0.01)。转染48 h后,空白对照组、mimics NC组、miRNA-200a mimics组细胞中miRNA-200a的相对表达量分别为(1.06±0.12)、(1.18±0.25)和(5.02±0.16),3组细胞中miRNA-200a的相对表达量比较,差异有统计学意义(P﹤0.01)。miRNA-200a mimics组细胞中miRNA-200a的相对表达量明显高于空白对照组(P﹤0.01)。MTT法检测结果显示,随着紫杉醇浓度的增加,A549细胞受到的增殖抑制作用明显增强,且呈一定的浓度依赖性;与紫杉醇组相比,转染miRNA-200a mimics后,随着紫杉醇浓度的增加,A549细胞受到的增殖抑制作用增强(P﹤0.05)。Western blot检测结果显示,空白对照组、紫杉醇组、miRNA-200a mimics组和miRNA-200a+紫杉醇组细胞中β-catenin蛋白的相对表达量分别(0.78±0.03)、(0.62±0.05)、(0.48±0.05)和(0.25±0.04),4组细胞中β-catenin蛋白的相对表达量比较,差异有统计学意义(P﹤0.01)。与空白对照组相比,紫杉醇组、miRNA-200a mimics组和miRNA-200a+紫杉醇组细胞中β-catenin蛋白的相对表达量均明显降低(P﹤0.01);与紫杉醇组相比,miRNA-200a+紫杉醇组细胞中β-catenin蛋白的相对表达量明显降低(P﹤0.01)。结论上调miRNA-200a表达能够增强非小细胞肺癌A549细胞对紫杉醇化疗药物的敏感度,其作用机制与阻断Wnt/β-catenin信号通路有关。

Objective To investigate the effect of microRNA-200 a（miRNA-200 a） on paclitaxel sensitivity in nonsmall cell lung cancer and its mechanism. Method The expression of miRNA-200 a in NSCLC A549 cells was detected by qRT-PCR; the miRNA-200 a mimics and miRNA-200 a negative control were transfected into A549 cells by liposome,and the transfection effects were measured by qRT-PCR. The inhibitive effect of up-regulation of the expression of miRNA-200 a on the proliferation of paclitaxel-treated NSCLC A549 cells was determined by MTT assay. The expression of Wnt/β-catenin signaling pathway related β-catenin protein in NSCLC A549 cells after up-regulation of miRNA-200 a and paclitaxel treatment were examined by Western blot. Result The relative expression of miRNA-200 a in NSCLC cell line A549 was（0.26±0.08）, and was significantly lower than that in normal human lung epithelial 16 HBE cell line at（1.02±0.03）（P0.01）. In 48 h after transfection, the relative expression levels of miRNA-200 a in blank control group, mimics NC group and miRNA-200 a mimics group were（1.06±0.12）,（1.18±0.25） and（5.02±0.16）, and the relative expression levels of miRNA-200 a in the three groups were significantly different（P0.01）. The relative expression of miRNA-200 a in miRNA-200 a mimics group was significantly higher than that in blank control group（P0.01）. The results of MTT showed that the inhibitive effect on A549 cells was markedly increased and was dose-dependent; compared with the paclitaxel group, after transfection of miRNA-200 a mimics, the inhibition observed in A549 cells was significantly elevated as the concentration of paclitaxel increased（P0.05）. Western blot results indicated that the relative expression of β-catenin protein in blank control group, paclitaxel group, miRNA-200 a mimics group and miRNA-200 a ＋ paclitaxel group were（0.78±0.03）,（0.62±0.05）,（0.48±0.05） and（0.25±0.04）, respectively, and the relative expression of β-catenin protein in the four groups differedstatistically（P0.01）. Compared with the blank control group, the relative expressions of β-catenin protein in paclitaxel group, miRNA-200 a mimics group and miRNA-200 a＋ paclitaxel group were decreased notably（P0.01）. Conclusion Up-regulation of miRNA-200 a expression can enhance the sensitivity of A549 cells to paclitaxel chemotherapeutic drugs, and its mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.