Xie Wei-Bin, Liang Li-Hui, Wu Kai-Ge, Wang Le-Xing, He Xin, Song Cheng, Wang Yun-Qi, Li Yun-Hui
Department of Integrated Traditional Chinese and Western Medicine, Hunan Cancer Hospital and The affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, China.
Department of Geriatrics, Hunan Provincial People's Hospital, Changsha, China.
Cell Physiol Biochem. 2018;46(2):654-663. doi: 10.1159/000488634. Epub 2018 Mar 28.
BACKGROUND/AIMS: Programmed death ligand1(PD-L1) plays a role in the development and progression of non-small cell lung cancer (NSCLC). This study aimed to identify miRNA(s) that are responsible for regulation of expression of PD-L1 in NSCLC, and to investigate the role of PD-L1 in regulation of the cell cycle in NSCLC.
We predicted the target miRNA of PD-L1, which was miR-140, using the online tools TargetScan and miBase. In NSCLC cells obtained from clinical specimens, in addition to A549 and NCI-H1650 cell cultures, western blots were used to detect the level of expression of proteins, while real-time PCR was used to determine the level of expression of PD-L1, miR-140, cyclin E, and β-actin. Transfection with miR-140 mimics, miR-140 inhibitors, and PD-L1 siRNA were conducted using commercial kits. To determine whether miR-140 directly binds PD-L1, a luciferase reporter gene with wild type or mutated PD-L1 was used. Cell viability was measured with the MTT assay, and PI staining was used for cell cycle analysis.
We found low expression of miR-140 and high expression of PD-L1 and cyclin E in NSCLC cells. Over-expression of miR-140 suppressed the expression of PD-L1 by directly binding its 3' UTR, and was also associated with decreased expression of cyclin E and inhibition of cellular proliferation in A549 and NCI-H1650 cells. Inhibition of PD-L1, in the absence of manipulations to miR-140, also decreased the expression of cyclin E.
We conclude that miR-140 directly suppresses PD-L1 and inhibits the miR-140/PD-L1/cyclin E pathway in NSCLC.
背景/目的：程序性死亡配体1（PD-L1）在非小细胞肺癌（NSCLC）的发生和发展中起作用。本研究旨在鉴定负责调控NSCLC中PD-L1表达的微小RNA（miRNA），并研究PD-L1在NSCLC细胞周期调控中的作用。
我们使用在线工具TargetScan和miBase预测了PD-L1的靶标miRNA，即miR-140。在从临床标本中获取的NSCLC细胞中，除了A549和NCI-H1650细胞培养物外，蛋白质印迹法用于检测蛋白质表达水平，而实时定量PCR用于测定PD-L1、miR-140、细胞周期蛋白E和β-肌动蛋白的表达水平。使用商业试剂盒进行miR-140模拟物、miR-140抑制剂和PD-L1小干扰RNA（siRNA）的转染。为了确定miR-140是否直接结合PD-L1，使用了带有野生型或突变型PD-L1的荧光素酶报告基因。采用MTT法测定细胞活力，PI染色用于细胞周期分析。
我们发现NSCLC细胞中miR-140表达低，PD-L1和细胞周期蛋白E表达高。miR-140的过表达通过直接结合其3'非翻译区（UTR）抑制PD-L1的表达，并且还与A549和NCI-H1650细胞中细胞周期蛋白E表达降低和细胞增殖抑制相关。在未对miR-140进行操作的情况下，抑制PD-L1也会降低细胞周期蛋白E的表达。
我们得出结论，miR-140直接抑制NSCLC中的PD-L1并抑制miR-140/PD-L1/细胞周期蛋白E通路。
