Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of the Army Medical University, Army Medical University, Chongqing, China.
Neoplasma. 2020 Jan;67(1):147-157. doi: 10.4149/neo_2019_190511N420. Epub 2019 Nov 4.
miR-186-5p has been reported to be implicated in tumorigenesis and paclitaxel resistance in non-small-cell lung cancer cells (NSCLCs). However, it remains undisclosed whether miR-186-5p takes a part in chemoresistance against diaminodichloroplatinum (cisplatin, DDP) in lung cancers, including NSCLC. Expression of miR-186-5p and sine oculis homeobox 1 (SIX1) was detected using RT-qPCR and western blot. In vitro, 50% inhibitory concentration (IC50) of DDP and cell proliferation were measured by MTT assay. The rate of apoptosis and abilities of migration and invasion were evaluated with flow cytometry and Transwell assay. The target binding between miR-186-5p and SIX1 was predicted on Diana tools software and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation. In vivo experiments, xenogeneic transplantation was conducted to monitor the tumor growth. Expression of miR-186-5p was downregulated in DDP resistant NSCLC tissues and cells (A549/DDP and H1299/DDP). Functionally, miR-186-5p overexpression could inhibit cell proliferation, migration and invasion, and promoted apoptosis rate in A549/DDP and H1299/DDP cells. Mechanically, SIX1 was identified as a downstream target for miR-186-5p and was highly expressed in A549/DDP and H1299/DDP cells. Similarly, SIX1 knockdown could also suppress DDP resistant NSCLC cell proliferation, migration and invasion, and promote apoptosis rate, which was reversed by miR-186-5p downregulation. Moreover, xenograft tumors induced by A549/DDP cells exerted cisplatin resistance, and miR-186-5p overexpression could inhibit tumor growth under DDP treatment. In conclusion, upregulation of miR-186-5p suppresses cisplatin resistance in DDP resistant NSCLC cells both in vitro and in vivo presumably by targeting SIX1.
miR-186-5p 已被报道参与非小细胞肺癌（NSCLC）细胞的肿瘤发生和紫杉醇耐药。然而，miR-186-5p 是否参与肺癌（包括 NSCLC）对二氨基二氯铂（顺铂，DDP）的化疗耐药仍未公开。使用 RT-qPCR 和 Western blot 检测 miR-186-5p 和 sine oculis homeobox 1（SIX1）的表达。体外，通过 MTT 测定法测量 DDP 的 50%抑制浓度（IC50）和细胞增殖。通过流式细胞术和 Transwell 测定法评估细胞凋亡率以及迁移和侵袭能力。在 Diana tools 软件上预测 miR-186-5p 和 SIX1 之间的靶标结合，并通过双荧光素酶报告基因测定和 RNA 免疫沉淀法进行验证。体内实验中，进行异种移植以监测肿瘤生长。DDP 耐药的 NSCLC 组织和细胞（A549/DDP 和 H1299/DDP）中 miR-186-5p 的表达下调。功能上，miR-186-5p 的过表达可抑制 A549/DDP 和 H1299/DDP 细胞的增殖、迁移和侵袭，并促进细胞凋亡率。机制上，SIX1 被鉴定为 miR-186-5p 的下游靶标，并在 A549/DDP 和 H1299/DDP 细胞中高表达。同样，SIX1 的敲低也可抑制 DDP 耐药 NSCLC 细胞的增殖、迁移和侵袭，并促进细胞凋亡率，这可被 miR-186-5p 的下调所逆转。此外，由 A549/DDP 细胞诱导的异种移植肿瘤对顺铂产生耐药性，而 miR-186-5p 的过表达可抑制 DDP 治疗下的肿瘤生长。总之，miR-186-5p 的上调通过靶向 SIX1 抑制体外和体内 DDP 耐药 NSCLC 细胞的顺铂耐药。
