Th17细胞功能相关基因OX40L筛选及其与miR-146a-3p靶向关系验证、对Th17细胞迁移调控作用机制探讨  

Screening of OX40L gene related to Th17 cell function and verification of its targeted relationship with miR-146a-3p,and its regulatory mechanism on Th17 cell migration

作　　者：黄韵 李春美 李作孝 HUANG Yun;LI Chunmei;LI Zuoxiao(Department of Neurology,The Affiliated Hospital of Southwest Medical University,Luzhou 646000,China)

机构地区：[1]西南医科大学附属医院神经内科,四川泸州646000

出　　处：《山东医药》2024年第36期29-35,39,共8页Shandong Medical Journal

基　　金：泸州市人民政府—西南医科大学科技战略合作基金项目(2023LZXNYDJ049)。

摘　　要：目的筛选Th17细胞功能相关基因OX40L,验证其与微小核糖核酸146a-3p(miR-146a-3p)的靶向关系,并探讨其对Th17细胞迁移的调控作用机制。方法①通过在GEO数据集中搜索multiple sclerosis,筛选出GSE57098基因数据集,R语言获取CD4+T细胞和Th17细胞的差异基因,DAVID 6.8在线分析软件获取GO分析及KEGG分析,STRING在线分析网站获取蛋白互作关系,Cytoscape软件获取Th17细胞功能相关的Hub蛋白,选取关键蛋白OX40L,并用qPCR进行验证。②使用Starbase预测miR-146a-3p与OX40L存在结合位点,双荧光素酶报告实验验证miR-146a-3p与OX40L靶向关系。③体外培养生长良好的Th17细胞,给予miR-146a-3p模拟物和抑制剂及其对照处理,qRT-PCR检测Th17细胞miR-146a-3p mRNA,CCK8增殖实验检测各组Th17细胞增殖情况,Transwell小室侵袭实验检测各组Th17细胞迁移情况,流式细胞术检测各组Th17细胞凋亡比例;qRT-PCR法和Western blot法检测各组OX40L mRNA和蛋白,ELISA法检测各组Th17细胞TNF-α。结果①GSE57098数据集中Th17细胞和CD4+T细胞有差异基因2130个,其中表达上调1515个,表达下调615个,OX40L在表达上调的蛋白中排名靠前。OX40L在KEGG分析中参与Cytokine-cytokinereceptorinteraction途径。②miR-146a-3p与OX40LmRNA3'UTR呈靶向结合关系,miR-146a-3p可抑制OX40L基因的转录表达。③与对照组比较,miR-146a-3pmimic组Th17细胞TLR4和p-NFκBp65蛋白表达降低,分泌TNF-α能力降低,细胞迁移能力降低,P均<0.05。结论OX40L在Th17细胞基因表达谱发生明显变化,其表达上调是造成多发性硬化发病的机制之一;miR-146a-3p通过靶向调节OX40L的表达,进一步抑制TNF-α途径中NF-κB p65的翻译水平,降低Th17细胞的迁移。Objective To screen the Th17 cell function-related gene OX40L,to verify its targeted relationship with microRNA 146a-3p(miR-146a-3p),and to observe its regulatory effect on Th17 cell migration.Methods①The GSE57098 gene dataset was screened out by searching for multiple sclerosis in the GEO dataset,differential genes between CD4+T cells and Th17-like cells were obtained using R language,GO analysis and KEGG analysis were performed using DAVID 6.8 online analysis software,protein interactions were obtained from STRING online analysis website,Hub proteins related to Th17 cell function were obtained using Cytoscape software,and the key protein OX40L was selected and validated by qPCR.②The binding site between miR-146a-3p and OX40L was predicted by Starbase,and double luciferase reporter assay was used to verify the targeted relationship between miR-146a-3p and OX40L.③Th17 cells that grew well were cultured in vitro,and were treated with miR-146a-3p mimics,inhibitors,and controls.MiR-146a-3p was detected by qRT-PCR,and the proliferation of Th17 cells was detected by CCK-8.The migration of Th17 cells was detected by Transwell,the apoptosis ratio of Th17 cells was detected by flow cytometry,qRT-PCR and Western blotting were used to detect OX40L mRNA and protein,and ELISA was used to detect the TNF-α level in each group.Results①There were 2130 differentially expressed genes between Th17 cells and CD4+cells in GSE57098,of which 1515 were up-regulated and 615 were down-regulated.OX40L ranked high among up-regulated proteins,which was involved in the Cytokine Receptor Interaction pathway by KEGG analysis.②MiR-146a-3p had a targeted binding relationship with OX40L mRNA3'UTR,and miR-146a-3p could inhibit the transcriptional expression of OX40L gene.③Conclusions OX40L had significant changes in the gene expression profile of Th17 cells,and its up-regulation was one of the main mechanisms of multiple sclerosis;miR-146a-3p further inhibited the translation level of NFκB p65 in the TNF-α pathway and reduced the mig

关 键 词：多发性硬化 OX40L基因 TH17细胞 微小核糖核酸146a-3p TNF-α途径 

分 类 号：R744.51[医药卫生—神经病学与精神病学]