Ye Zaiting, Fang Bingmu, Pan Jiongwei, Zhang Ning, Huang Jinwei, Xie Congying, Lou Tianzheng, Cao Zhuo
The Sixth Affiliated Hospital of Wenzhou Medical University/Lishui People's Hospitlal, Lishui, Zhejiang 323000, P.R. China.
The Central Hospital of Lishui City, Lishui, Zhejiang 323000, P.R. China.
Oncol Rep. 2017 Jun;37(6):3244-3252. doi: 10.3892/or.2017.5619. Epub 2017 May 3.
The present study determined the role and mechanism of miR-138 in non-small cell lung cancer (NSCLC). In total, 45 freshly resected clinical NSCLC tissues were collected. The expression of miR-138 in tissues and cell lines were determined by real-time quantitative PCR. miR-138 mimics were transfected into A549 and Calu-3 cells in vitro, and then the effects of miR-138 on lung cancer cell proliferation, cell cycle, invasion and metastasis were investigated by CCK-8 assay, Transwell and flow cytometry, respectively. The protein expression of the potential target gene Sirt1 in lung cancer cells were determined by western blot analysis. Dual-luciferase reporter assay was performed to further confirm whether Sirt1 was the target gene of miR-138. The expression of miR-138 was significantly lower in lung cancer tissues and was negatively correlated to the differentiation degree and lymph node metastasis of lung cancer. In vitro experiment results showed that miR-138 inhibited lung cancer cell proliferation, invasion and migration. It was verified that miR-138 could downregulate Sirt1 protein expression, inhibit epithelial-mesenchymal transition (EMT), decrease the activity of AMPK signaling pathway and elevate mTOR phosphorylation level. Dual-luciferase reporter assay demonstrated that miR-138 could directly regulate Sirt1. Downregulation of Sirt1 alone can also cause the same molecular and biological function changes. Western blot analysis and confocal microscopy results indicated that overexpression of miR-138 or interference of Sirt1 expression could inhibit lung cancer cell autophagy activity possibly through AMPK-mTOR signaling pathway. miR-138 plays a tumor suppressor function in lung cancer. It may inhibit the proliferation, invasion and migration of lung cancer through downregulation of Sirt1 expression and activation of cell autophagy. The downregulation of miR-138 is closely related to the development of lung cancer.
本研究确定了miR - 138在非小细胞肺癌（NSCLC）中的作用及机制。总共收集了45例新鲜切除的临床NSCLC组织。通过实时定量PCR测定组织和细胞系中miR - 138的表达。将miR - 138模拟物体外转染至A549和Calu - 3细胞，然后分别通过CCK - 8测定、Transwell实验和流式细胞术研究miR - 138对肺癌细胞增殖、细胞周期、侵袭和转移的影响。通过蛋白质免疫印迹分析确定肺癌细胞中潜在靶基因Sirt1的蛋白表达。进行双荧光素酶报告基因检测以进一步确认Sirt1是否为miR - 138的靶基因。miR - 138在肺癌组织中的表达显著降低，且与肺癌的分化程度和淋巴结转移呈负相关。体外实验结果表明，miR - 138抑制肺癌细胞增殖、侵袭和迁移。证实miR - 138可下调Sirt1蛋白表达，抑制上皮 - 间质转化（EMT），降低AMPK信号通路活性并提高mTOR磷酸化水平。双荧光素酶报告基因检测表明miR - 138可直接调控Sirt1。单独下调Sirt1也可引起相同的分子和生物学功能变化。蛋白质免疫印迹分析和共聚焦显微镜结果表明，miR - 138过表达或Sirt1表达干扰可能通过AMPK - mTOR信号通路抑制肺癌细胞自噬活性。miR - 138在肺癌中发挥肿瘤抑制功能。它可能通过下调Sirt1表达和激活细胞自噬来抑制肺癌的增殖、侵袭和迁移。miR - 138的下调与肺癌的发生发展密切相关。
