Zhou Wenwen, Wang Xiaoyu, Yin Duanduan, Xue Lei, Ma Zhongfeng, Wang Zhenzhen, Zhang Qianyi, Zhao Zishu, Wang Haixia, Sun Yan, Yang Yanhong
Department of Oncology, Qinhuangdao First People's Hospital, Qinhuangdao, Hebei 066000, P.R. China.
Foundation and Clinic of Malignant Tumor, Postgraduate College, Chengde Medical University, Chengde, Hebei 067000, P.R. China.
Exp Ther Med. 2019 Aug;18(2):1350-1356. doi: 10.3892/etm.2019.7701. Epub 2019 Jun 21.
Non-small cell lung cancer (NSCLC) is the most common type of lung cancer accounting for ~80% of lung cancer cases. According to novel research, numerous microRNAs (miRs) have been suggested to function as important regulators of cancer. In addition, the expression of miR-140-5p is decreased in patients with NSCLC. Therefore, it is important to further elucidate the role of miR-140-5p in NSCLC. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used in order to investigate the expression of miR-140-5p in NSCLC tissues and matched normal tissues and to determine miR-140-5p levels following transfection with mimics into A549 lung cancer cells. Targetscan software was used to predict the oncogene target of miR-140-5p. This analysis revealed that YES proto-oncogene 1 (YES1) includes a target site for miR-140-5p binding. The results revealed that YES1 is a potential target gene of miR-140-5p, and this was further confirmed by the results of luciferase reporter assays, which demonstrated that miR-140-5p directly targeted the predicted binding site in the 3'-untranslated region of YES1. Cell Counting Kit-8 (CCK-8) and flow cytometry assays were performed to determine the levels of cell viability and apoptosis. Western blot assays was performed to investigate the expression levels of YES1 and proteins associated with apoptosis in A549 cells following transfection. The results revealed that miR-140-5p expression was significantly downregulated in NSCLC tissues compared with matched normal tissues. The expression of miR-140-5p was significantly increased following transfection with miR-140-5p mimics. The results of CCK-8 and flow cytometry assays indicated that miR-140-5p inhibited proliferation and induced apoptosis of tumor cells. Western blot analysis and RT-qPCR revealed that YES1 and B-cell lymphoma 2 (Bcl-2) mRNA and protein expression levels were markedly decreased in A549 cells, while Bcl-2 associated X (Bax) and caspase-3 expression levels increased significantly following transfection with miR-140-5p mimics compared with the negative control group. In conclusion, miR-140-5p may induce apoptosis in A549 cells by targeting YES1 and regulating the expression of apoptosis-associated proteins Bcl-2, Bax and caspase-3.
非小细胞肺癌（NSCLC）是最常见的肺癌类型，约占肺癌病例的80%。根据最新研究，许多微小RNA（miR）被认为是癌症的重要调节因子。此外，NSCLC患者中miR-140-5p的表达降低。因此，进一步阐明miR-140-5p在NSCLC中的作用很重要。采用逆转录-定量聚合酶链反应（RT-qPCR）来研究miR-140-5p在NSCLC组织和配对正常组织中的表达，并在将模拟物转染到A549肺癌细胞后测定miR-140-5p水平。使用Targetscan软件预测miR-140-5p的癌基因靶点。该分析表明，原癌基因1（YES1）包含一个miR-140-5p结合靶点。结果显示，YES1是miR-140-5p的潜在靶基因，荧光素酶报告基因检测结果进一步证实了这一点，该检测表明miR-140-5p直接靶向YES1 3'-非翻译区的预测结合位点。进行细胞计数试剂盒-8（CCK-8）和流式细胞术检测以确定细胞活力和凋亡水平。进行蛋白质免疫印迹分析以研究转染后A549细胞中YES1的表达水平以及与凋亡相关的蛋白质。结果显示，与配对正常组织相比，NSCLC组织中miR-140-5p表达显著下调。转染miR-140-5p模拟物后，miR-140-5p表达显著增加。CCK-8和流式细胞术检测结果表明，miR-140-5p抑制肿瘤细胞增殖并诱导其凋亡。蛋白质免疫印迹分析和RT-qPCR显示，与阴性对照组相比，转染miR-140-5p模拟物后，A549细胞中YES1和B细胞淋巴瘤2（Bcl-2）的mRNA和蛋白质表达水平显著降低，而Bcl-2相关X蛋白（Bax）和半胱天冬酶-3的表达水平显著增加。总之，miR-140-5p可能通过靶向YES1并调节凋亡相关蛋白Bcl-2、Bax和半胱天冬酶-3的表达来诱导A549细胞凋亡。
