摘要
目的 探讨miR-185-5p靶向负调控跨膜9超家族蛋白1(TM9SF1)对肺腺癌细胞增殖、迁移和自噬能力的影响。方法 在基因表达综合数据库(GEO)中下载数据集GSE51853,分析miR-185-5p在肺腺癌组织中的表达情况。采用在线数据库miRTargetLink、miRTarbase和DIANA-microT-CD预测miR-185-5p靶向结合的蛋白,使用HADb网站下载自噬相关蛋白,将4个数据库所得结果取交集,通过StarBase数据库分析交集结果VEGFA和TM9SF1的生存曲线。分别将TM9SF13'UTR(WT)或TM9SF1 3'UTR(MUT)报告质粒与miR-185-5p对照质粒(CON)或miR-185-5p过表达质粒(over-miR-185-5p)共转染HEK-293T细胞,采用双荧光素酶报告基因实验检测miR-185-5p与TM9SF1的结合位点及荧光素酶活性。采用Western blotting检测转染miR-185-5p过表达质粒后A549细胞中TM9SF1蛋白的表达情况。将A549细胞分为3组:(1)CON+NC组,转染miR-185-5p对照质粒和TM9SF1对照质粒;(2)over-miR-185-5p+NC组,转染miR-185-5p过表达质粒和TM9SF1对照质粒;(3)over-miR-185-5p+over-TM9SF1组,转染miR-185-5p过表达质粒和TM9SF1过表达质粒。采用EdU细胞增殖实验、划痕实验和Transwell迁移实验验证miR-185-5p靶向结合TM9SF1对肺腺癌细胞增殖和迁移能力的影响。分别采用stubRFPsensGFP-LC3自噬流检测慢病毒和JC-1实验检测肺腺癌细胞自噬流和线粒体膜电位(MMP)的改变。结果 GSE51853数据集中,miR-185-5p在肺腺癌组织中的表达水平明显低于正常肺组织(P<0.01);qRT-PCR检测结果显示,miR-185-5p在肺腺癌细胞NCI-H1299、A549中的表达水平低于正常肺上皮细胞BEAS-2B(P<0.01)。在线数据库miRTargetLink、miRTarbase、DIANA-microT-CD和HADb预测结果显示,miR-185-5p可靶向调控自噬相关蛋白TM9SF1。双荧光素酶报告基因实验和Western blotting检测结果分别显示,miR-185-5p可直接与TM9SF1 mRNA的3'UTR区结合,过表达miR-185-5p后靶蛋白TM9SF1的表达水平降低(P<0.05)。EdU细胞增殖实验、划痕实验和Transwell迁移实验结果显示,过表达miR-185-5p可抑制肺腺癌细胞的增殖和迁移能力,过表达TM9SF1能够削弱miR-185-5p对肺腺癌细胞增殖、迁移能力的抑制作用(P<0.05)。stub RFP-sens GFP-LC3自噬流检测慢病毒结果显示,过表达mi R-185-5p后A549细胞中自噬流加快,而同时过表达mi R-185-5p和TM9SF1后A549细胞中自噬流减慢;JC-1实验结果显示,过表达miR-185-5p后A549细胞中MMP水平降低,同时过表达miR-185-5p和TM9SF1后A549细胞中MMP水平升高。结论 miR-185-5p可能通过靶向负调控TM9SF1的表达影响肺腺癌细胞的增殖、迁移和自噬能力。
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.TM9SF13'UTR wild-type(WT)or TM9SF13'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.
作者
王晓娜
龚秀莹
赵苗苗
刘清华
李勇
王坤
尹崇高
李洪利
Wang Xiao-Na;Gong Xiu-Ying;Zhao Miao-Miao;Liu Qing-Hua;Li Yong;Wang Kun;Yin Chong-Gao;Li Hong-Li(Department of Clinical Pathology,Shandong Second Medical University,Weifang,Shandong 261053,China;Human Anatomy Teaching and Research Section,Shandong Second Medical University,Weifang,Shandong 261053,China;Medical Research Center,Shandong Second Medical University,Weifang,Shandong 261053,China;Colloge of Nursing,Shandong Second Medical University,Weifang,Shandong 261053,China)
出处
《解放军医学杂志》
北大核心
2025年第5期566-574,共9页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金(82373043)
山东省自然科学基金(ZR2022MH311)
山东省中医药科技项目(Q-2023012)
潍坊市科学技术发展计划项目(2022YX094,2023YX037)。
关键词
肺腺癌
miR-185-5p
TM9SF1
增殖
迁移
自噬
lung adenocarcinoma
miR-185-5p
transmembrane 9 superfamily protein 1
proliferation
migration
autophagy
