解放军医学杂志2025，Vol.50Issue(5)：566-574,9.DOI:10.11855/j.issn.0577-7402.1741.2025.0313
miR-185-5p靶向负调控TM9SF1对肺腺癌细胞增殖、迁移和自噬能力的影响
Effect of miR-185-5p targeted negative regulation of TM9SF1 on proliferation,migration and autophagy in lung adenocarcinoma cells
摘要
Abstract
Objective To investigate the effect of miR-185-5p-mediated targeted negative regulation of transmembrane 9 superfamily member 1(TM9SF1)on proliferation,migration and autophagy in lung adenocarcinoma cells.Methods The expression of miR-185-5p in lung adenocarcinoma tissues was analyzed using dataset GSE51853 downloaded from the Gene Expression Omnibus(GEO)database.Potential target proteins of miR-185-5p were predicted using online databases(miRTargetLink,miRTarbase,and DIANA-microT-CD),and autophagy-related proteins were obtained from HADb.The intersected results from these four databases was identified,and survival curves of vascular endothelial growth factor A(VEGFA)and TM9SF1 within the overlapping candidates were analyzed using the StarBase database.TM9SF1 3'UTR wild-type(WT)or TM9SF1 3'UTR mutant(MUT)reporter plasmids were separately co-transfected with miR-185-5p control plasmid(CON)or miR-185-5p overexpression plasmid(over-miR-185-5p)into HEK-293T cells.A dual-luciferase reporter gene assay was employed to assess the binding interaction between miR-185-5p and TM9SF1 and quantify the subsequent luciferase activity.Western blotting was used to assess TM9SF1 protein expression levels in A549 cells transfected with over-miR-185-5p.A549 cells were divided into three groups:(1)CON+NC group,co-transfected with miR-185-5p control plasmid and TM9SF1 control plasmid;(2)over-miR-185-5p+NC group,co-transfected with over-miR-185-5p and TM9SF1 control plasmid;(3)over-miR-185-5p+over-TM9SF1 group,co-transfected with both miR-185-5p and TM9SF1 overexpression plasmids.EdU cell proliferation assay,wound healing assay,and Transwell migration assay were performed to validate the effects of miR-185-5p targeted binding to TM9SF1 on proliferation and migration capacities in lung adenocarcinoma.Changes in autophagic flux and mitochondrial membrane potential(MMP)of lung adenocarcinoma cells were detected using stubRFP-sensGFP-LC3 lentivirus and JC-1 assays,respectively.Results In the GSE51853 dataset,miR-185-5p expression level was significantly lower in lung adenocarcinoma tissues compared with normal lung tissues(P<0.01).qRT-PCR analysis revealed that miR-185-5p expression was downregulated in lung adenocarcinoma cell lines NCI-H1299 and A549 compared with normal lung epithelial cells BEAS-2B(P<0.01).Bioinformatics predictions using miRTargetLink,miRTarbase,DIANA-microT-CD,and HADb databases indicated that miR-185-5p could target and regulate the autophagy-related protein TM9SF1.Dual-luciferase reporter assays and Western blotting demonstrated that miR-185-5p directly bound to the 3'UTR region of TM9SF1 mRNA,and overexpression of miR-185-5p significantly reduced the expression of target protein TM9SF1(P<0.05).EdU cell proliferation,wound healing,and Transwell migration assays demonstrated that miR-185-5p overexpression inhibited proliferation and migration capacities of lung adenocarcinoma cells,whereas TM9SF1 overexpression could attenuate this inhibition effect(P<0.05).Results of stubRFP-sensGFP-LC3 for autophagic flux analysis demonstrated that overexpression of miR-185-5p enhanced autophagic flux in A549 cells,whereas co-overexpression of miR-185-5p and TM9SF1 suppressed autophagic flux.JC-1 assays showed a decreased MMP level in A549 cells after miR-185-5p overexpression,with higher MMP level observed when miR-185-5p and TM9SF1 were co-overexpressed.Conclusion miR-185-5p may suppress proliferation,migration,and autophagy capacities in lung adenocarcinoma cells by targeting TM9SF1 through negative regulation.关键词
肺腺癌/miR-185-5p/TM9SF1/增殖/迁移/自噬Key words
lung adenocarcinoma/miR-185-5p/transmembrane 9 superfamily protein 1/proliferation/migration/autophagy分类
临床医学引用本文复制引用
王晓娜,龚秀莹,赵苗苗,刘清华,李勇,王坤,尹崇高,李洪利..miR-185-5p靶向负调控TM9SF1对肺腺癌细胞增殖、迁移和自噬能力的影响[J].解放军医学杂志,2025,50(5):566-574,9.基金项目
国家自然科学基金(82373043) （82373043）
山东省自然科学基金(ZR2022MH311) （ZR2022MH311）
山东省中医药科技项目(Q-2023012) （Q-2023012）
潍坊市科学技术发展计划项目(2022YX094,2023YX037) This study was supported by the National Natural Science Foundation of China(82373043),the Natural Science Foundation of Shandong Province(ZR2022MH311),the Shandong Province Traditional Chinese Medicine Science and Technology Project(Q-2023012),and the Weifang Science and Technology Development Plan Project(2022YX094,2023YX037) （2022YX094,2023YX037）
