Hu Huihui, Xu Hangdi, Lu Fen, Zhang Jisong, Xu Li, Xu Shan, Jiang Hanliang, Zeng Qingxin, Chen Enguo, He Zhengfu
Department of Respiratory and Critical Care Medicine, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
Operation Room, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
Front Bioeng Biotechnol. 2020 Apr 8;8:259. doi: 10.3389/fbioe.2020.00259. eCollection 2020.
This study aimed to describe the mechanism of exosome-derived miR-486-5p underlying the cell cycle and progression in lung adenocarcinoma (LUAD).
Bioinformatics methods were applied for identifying the differentially expressed genes (DEGs) in the GEO-LUAD dataset, predicting where the potential target miRNA was expressed and exploring the corresponding downstream target mRNA. qRT-PCR was conducted to detect the levels of the target genes in cancer cells. Thereafter, a series of experiments were performed for cell activities evaluation, including CCK-8, EdU, colony formation assay and transwell. Besides, Western blot was applied to determine the protein levels of the migration and invasion-related factors (NEK2, E-cadherin, N-cadherin, Vimentin, MMP-2, and MMP-9). Dual-luciferase reporter gene assay was employed for validating the targeted relationship between the target genes. Furthermore, nude mouse transplantation tumor experiment was conducted to further validate the role of the target miRNA in tumor development, and immunohistochemistry was used for Ki67 detection and TUNEL was applied for cell apoptosis assay.
miR-486-5p was observed to be enriched in serum exosomes, and seen to be significantly down-regulated in cancer tissues as well as in cancer serum exosomes. It was proven that exosomes could release miR-486-5p, thus regulating LUAD progression and affecting cell cycle. Moreover, NEK2 was identified as a target of miR-486-5p both and . Enrichment analysis revealed that NEK2 was mainly activated in cell cycle and mitosis-related pathways. Meanwhile, NEK2 was found to present significant difference in different TNM stages. Furthermore, rescue experiments indicated that the inhibitory effect of miR-486-5p overexpression on LUAD progression could be abrogated when miR-486-5p and NEK2 were simultaneously up-regulated.
Exosome-derived miR-486-5p is responsible for cell cycle arrest as well as the inhibition of cell proliferation and metastasis in LUAD via targeting NEK2.
本研究旨在描述外泌体来源的miR-486-5p在肺腺癌（LUAD）细胞周期和进展中的作用机制。
应用生物信息学方法识别GEO-LUAD数据集中的差异表达基因（DEGs），预测潜在靶miRNA的表达位置，并探索相应的下游靶mRNA。进行qRT-PCR检测癌细胞中靶基因的水平。此后，进行了一系列细胞活性评估实验，包括CCK-8、EdU、集落形成试验和Transwell实验。此外，应用蛋白质免疫印迹法测定迁移和侵袭相关因子（NEK2、E-钙黏蛋白、N-钙黏蛋白、波形蛋白、基质金属蛋白酶-2和基质金属蛋白酶-9）的蛋白水平。采用双荧光素酶报告基因测定法验证靶基因之间的靶向关系。此外，进行裸鼠移植瘤实验以进一步验证靶miRNA在肿瘤发展中的作用，并采用免疫组织化学法检测Ki67，应用TUNEL法检测细胞凋亡。
观察到miR-486-5p在血清外泌体中富集，且在癌组织和癌血清外泌体中均显著下调。证实外泌体可释放miR-486-5p，从而调节LUAD进展并影响细胞周期。此外，NEK2被确定为miR-486-5p的靶标。富集分析显示NEK2主要在细胞周期和有丝分裂相关途径中被激活。同时，发现NEK2在不同TNM分期中存在显著差异。此外，挽救实验表明，当miR-486-5p和NEK2同时上调时，miR-486-5p过表达对LUAD进展的抑制作用可被消除。
外泌体来源的miR-486-5p通过靶向NEK2导致LUAD细胞周期停滞，并抑制细胞增殖和转移。
