MicroRNA-126通过IRS-1/AKT/ERK调控CD4^+T细胞功能的分子机制研究  

Study on the mechanism of microRNA-126 regulating the function of CD4^+T cells through IRS-1/AKT/ERK pathways

作　　者：胡燕[1] 王小妹[1] HU Yan;WANG Xiaomei(Center for Medical Laboratory and Pathology,the First Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410000,China)

机构地区：[1]湖南中医药大学第一附属医院医学检验与病理中心

出　　处：《重庆医学》2020年第2期178-182,共5页Chongqing medicine

基　　金：湖南省教育厅项目（13C688）

摘　　要：目的利用基因芯片技术和实时荧光定量PCR技术,结合Targets scan软件和Western blot技术,初步探讨微小RNA-126(miR-126)调控CD4+T细胞功能的分子机制。方法提取野生型小鼠(对照组)和miR-126基因敲减小鼠(miR-126敲减组)脾淋巴细胞总RNA,送基因芯片测序,筛选出差异表达基因;运用Targets scan和miR-Walk软件预测miR-126可能作用的靶分子,经筛选后将胰岛素受体底物-1(IRS-1)作为miR-126的靶分子;进一步利用实时荧光定量PCR检测两组小鼠脾CD4+CD62L+T细胞中IRS-1的水平变化,Western blot技术检测两组小鼠脾淋巴细胞蛋白激酶B(AKT)和细胞外调节蛋白激酶(ERK)及其磷酸化水平。结果基因芯片分析筛选出大量差异表达的基因,与对照组比较,IRS-1的表达上调2.7005倍。Target scan和miR-Walk软件预测miR-126可与IRS-1的3′非翻译区(3′UTR)结合,提示IRS-1是miR-126作用的靶分子。PCR显示miR-126敲减组小鼠CD4+T细胞中IRS-1的表达明显上调(P<0.05),Western blot显示miR-126敲减组小鼠CD4+T细胞中AKT、ERK及其磷酸化水平明显增加(P<0.05)。结论MiR-126通过IRS-1/AKT/ERK信号通路调控CD4+T细胞的功能。Objective To explore the underlying mechanism of microRNA-126(miR-126)regulating the function of CD4+T cells by micro-Array,real-time PCR,Targets scan software and Western blot.Methods The total RNA of spleen lymphocytes in the wild type mice(the control group)and miR-126 knock-down mice(the miR-126KD group)was extracted and sequenced by NimbleGene expression profiling,and the differentially expressed genes were screened.The target genes of miR-126 were predicted by Targets scan and miR-Walk software.Then insulin receptor substrate-1(IRS-1)was selected as the target gene of miR-126,and the expression of IRS-1 in CD4+T cells of the two groups was detected by real-time PCR.At last,the relative expression of phosphorylation of AKT and ERK in CD4+T cells of the two groups was detected by Western blot.Results Gene expression profile analysis revealed a large number of differentially expressed genes.Compared with the control group,IRS-1 was up-regulated by 2.7005 times.Targets scan and miR-Walk software predicted that miR-126 could bind with the 3′-untranslated regions(3′UTR)of IRS-1,suggesting that IRS-1 was the target gene of miR-126.Real-time PCR showed that the expression of IRS-1 of CD4+T cells in the miR-126KD group was up-regulated significantly(P<0.05),the phosphorylation of AKT and ERK of CD4+T cells in the miR-126KD group significantly increased by Western blot(P<0.05).Conclusion MiR-126 can regulate the function of CD4+T cells through IRS-1/AKT/ERK pathways.

关 键 词：MIR-126 基因敲除技术 抗原 CD4 胰岛素受体底物-1 

分 类 号：R392.1[医药卫生—免疫学]