目的:运用生物信息学方法和实验验证研究肥胖相关蛋白(fat mass and obesity associated proteins，FTO)促进肾小球系膜细胞自噬，抑制炎症反应的机制。方法:免疫组化分析FTO在慢性肾小球肾炎患者肾组织中的表达;透射电镜观察过表达FTO对肾小球系膜细胞自噬的影响，Western blot检测自噬标志蛋白;荧光双标观察过表达FTO对炎症反应的影响，ELISA法检测细胞上清IL-6和TNF-α表达水平。运用catRAPID omics v2。0和Encori数据库的预测FTO相关的互作基因，并进行GO功能和KEGG信号通路富集分析。Western blot检测相关信号通路中的关键蛋白。构建PPI网络筛选核心蛋白。结果:免疫组化结果提示FTO在慢性肾小球肾炎患者肾脏组织表达显著降低(P＜0。01);过表达FTO可促进肾小球系膜细胞自噬，抑制炎症反应;通过Encori数据库和catRAPID omics v2。0数据库共得到737个FTO相关互作基因。KEGG分析结果提示Hippo信号通路、Wnt信号通路和Rap1信号通路被显著富集。Western blot实验表明过表达FTO可激活Hippo信号通路，抑制Wnt信号通路和Rap1信号通路。PPI调控网络筛选出16个核心基因。结论:FTO通过调控Hippo信号通路，Wnt信号通路，Rap1信号通路和AMPK信号通路等促进肾小球系膜细胞自噬和抑制炎症反应。CTNNB1、CTNND1、PPP2CA、YES1和ABL1等是FTO核心基因。
Study on the mechanism of fat mass and obesity associated proteins in promoting autophagy and inhibiting inflammatory response in glomerular mesangial cells based on bioinformatics analysis and experimental verification
OBJECTIVE To investigate the mechanism by which fat mass and obesity associated proteins(FTO)promote autophagy in glomerular mesangial cells and suppress inflammatory response,using bioinformatics methods and experimental vali-dation.METHODS Immunohistochemical analysis was employed to assess the expression of FTO in renal tissues of chronic glo-merulonephritis patients.Transmission electron microscopy was used to observe the effect of FTO overexpression on autophagy in glomerular mesangial cells,with Western blot used to detect autophagy marker proteins.Fluorescence double labeling was used to observe the effect of FTO overexpression on inflammatory response,and expression levels of IL-6 and TNF-α in cell superna-tant were measured by ELISA.FTO-related interacting genes were predicted using the catRAPID omics v2.0 and Encori data-bases,followed by GO function and KEGG signaling pathway enrichment analysis.Key proteins in these pathways were detected via Western blot.A PPI network was constructed to screen the core proteins.RESULTS Immunohistochemical results showed that the expression of FTO in normal kidney tissues was significantly higher than that in kidney tissues of patients with chronic glo-merulonephritis(P＜0.01).FTO overexpression could promote autophagy in glomerular mesangial cells and inhibit inflammatory response.A total of 737 FTO-related interacting genes were identified through Encori database and catRAPID omics v2.0 data-base.KEGG analysis showed that Hippo,Wnt,Rap1 and AMPK signaling pathways were significantly enriched.Western blot experiments showed that overexpression of FTO activated the Hippo signaling pathway and inhibited the Wnt and Rap1 pathways.16 core genes were identified through PPI regulatory network.CONCLUSIONS FTO promotes autophagy of glomerular mesan-gial cells and inhibits inflammatory response by regulating the Hippo,Wnt,Rap1 and AMPK signaling pathways.CTNNB1,CTNND1,PPP2CA,YES1 and ABL1 are the core genes of FTO.
fat mass and obesity associated proteinsglomerular mesangial cellsautophagyinflammationbioinformatics analysis
NETL
NSTL
万方数据
维普
