Liu Zhiwu, Yao Liqiong, Tan Bangyun, Li Li, Chen Baojin
Department of Medical Laboratory Center, The First Hospital of Lanzhou University, Lanzhou, Gansu 730000, P.R. China.
Oncol Lett. 2016 Dec;12(6):5349-5355. doi: 10.3892/ol.2016.5365. Epub 2016 Nov 8.
The present study aimed to investigate the association and underlying mechanisms between microRNA-200b level and the inhibitory effect of gefitinib on non-small cell lung cancer. In total, 100 patients (43 males and 57 females; median age, 63 years) with advanced non-small cell lung cancer (NSCLC) were selected. All patients were administered with gefitinib orally (250 mg/day) and the effect of gefitinib was evaluated according to the Response Evaluation Criteria in Solid Tumors guidelines. Tumor tissue and plasma samples were collected prior to and subsequent to therapy. The microRNA-200b levels in tissues and plasma were determined by quantitative polymerase chain reaction (PCR). A549 cells were cultured and transfected with microRNA-200b mimic. Using Cell Counting Kit-8 assay, the proliferation inhibition detected was induced by 0.1 µM gefitinib in transfected or non-transfected A549 cells. Cell apoptosis and cell cycle progression were analyzed by flow cytometry and the migration of cells was observed by Transwell assay. In addition, mRNA and protein levels of insulin-like growth factor 1 receptor (IGF-1R), protein kinase B (AKT) and extracellular signal-related kinase (ERK), together with the phosphorylation of AKT and ERK in A549 cells, were determined by quantitative PCR and western blot analysis, respectively. The microRNA-200b levels in gefitinib-insensitive patients were decreased compared with gefitinib-sensitive patients. Transfection with microRNA-200b mimic increased the gefitinib induced proliferation inhibition, apoptosis and cell cycle arrest in A549 cells. Also, transfection with microRNA-200b mimic increased the migration inhibitory effect of gefitinib on A549 cells. Decreased IGF-1R expression together with reduced phosphorylation of AKT and ERK were observed following transfection of A549 cells with the microRNA 200b mimic. In conclusion, detection of microRNA-200b may predict the inhibitory effect of gefitinib on NSCLC. Upregulation of microRNA-200b led to the elevated sensitivity of glioma cells to gefitinib, and this effect may be explained as microRNA-200b being able to inhibit the expression of IGF-1R, thereby reducing the activation of downstream phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase signaling pathways.
本研究旨在探讨微小RNA-200b水平与吉非替尼对非小细胞肺癌抑制作用之间的关联及潜在机制。总共选取了100例晚期非小细胞肺癌（NSCLC）患者（43例男性和57例女性；中位年龄63岁）。所有患者口服吉非替尼（250毫克/天），并根据实体瘤疗效评价标准指南评估吉非替尼的疗效。在治疗前和治疗后采集肿瘤组织和血浆样本。通过定量聚合酶链反应（PCR）测定组织和血浆中的微小RNA-200b水平。培养A549细胞并用微小RNA-200b模拟物转染。使用细胞计数试剂盒-8法，检测0.1微摩尔吉非替尼在转染或未转染的A549细胞中诱导的增殖抑制。通过流式细胞术分析细胞凋亡和细胞周期进程，并通过Transwell试验观察细胞迁移。此外，分别通过定量PCR和蛋白质印迹分析测定A549细胞中胰岛素样生长因子1受体（IGF-1R）、蛋白激酶B（AKT）和细胞外信号调节激酶（ERK）的mRNA和蛋白水平，以及AKT和ERK的磷酸化水平。与吉非替尼敏感患者相比，吉非替尼不敏感患者的微小RNA-200b水平降低。用微小RNA-200b模拟物转染可增强吉非替尼诱导的A549细胞增殖抑制、凋亡和细胞周期阻滞。此外，用微小RNA-200b模拟物转染可增强吉非替尼对A549细胞的迁移抑制作用。在用微小RNA 200b模拟物转染A549细胞后，观察到IGF-1R表达降低以及AKT和ERK磷酸化减少。总之，检测微小RNA-200b可能预测吉非替尼对NSCLC的抑制作用。微小RNA-200b的上调导致胶质瘤细胞对吉非替尼的敏感性升高，这种作用可能解释为微小RNA-200b能够抑制IGF-1R的表达，从而减少下游磷脂酰肌醇3-激酶/AKT和丝裂原活化蛋白激酶信号通路的激活。
