基于GEO数据库的玉液汤防治糖尿病肾病lncRNA-miRNA-mRNA转录网络的整合研究

lncRNA-miRNA-mRNA Network of Yuye Decoction(玉液汤)for the Prevention and Treatment of Diabetic Nephropathy Based on GEO Database

目的:挖掘玉液汤防治糖尿病肾病(Diabetic Nephropathy,DN)的三元转录网络,预测其防治DN的生物标志物及作用机制。方法:利用TCMSP、Pharmmapper、Uniprot数据库预测玉液汤的关键活性成分及其靶蛋白,采用GEO数据库筛查正常及DN患者的差异表达的信使核糖核酸(mRNA)和微小RNA(miRNA),将玉液汤预测靶点与DN差异表达的mRNAs(DEmRNAs)取交集得到玉液汤防治DN的靶点,利用String在线平台对其绘制蛋白质相互作用(PPI)网络,并利用Metascape平台对其进行基因本体GO功能富集分析和京都基因与基因组百科全书KEGG通路富集分析;利用Cytoscape 3.6.1软件构建玉液汤防治DN的活性成分-靶点网络。将PPI网络中的玉液汤治疗DN的靶点导入Cytoscape软件进行拓扑分析,利用Cytohubba插件筛选核心靶点,使用miRDB、miRWalk和TargetScan数据库预测核心靶点相关的miRNAs并与DN差异表达的miRNAs(DEmiRNA)取交集,将交集miRNAs导入Starbase数据库预测与之相关的长链非编码核糖核酸(lncRNA),筛选与DN有关的lncRNA;利用Discovery Studio 2019软件进行核心化合物-核心靶点的分子对接验证。链脲佐菌素(STZ)联合高脂高糖饲料诱导DN大鼠模型,给予玉液汤2.6 g/kg干预,观察各组大鼠体质量变化、空腹血糖水平及肾脏组织病理形态,检测尿蛋白、血肌酐和尿素氮含量;采用荧光定量聚合酶链式反应(Real-time PCR)验证差异基因,利用Cytoscape软件构建玉液汤防治DN的lncRNA-miRNA-mRNA的竞争性内源RNA(ceRNA)网络。结果:预测获得玉液汤防治DN的ceRNA网络由17个关键的lncRNAs、10个关键的miRNAs和13个关键的mRNAs构成。经实验验证,在ceRNA网络中,与模型对照组相比,玉液汤2.6 g/kg组大鼠尿蛋白、血肌酐和尿素氮的含量显著降低(P<0.01),核富集丰富转录本1(Neat1)、X非活性特异性转录本(Xist)、肺腺癌转移性相关转录本1(Malat1)的lncRNA表达和胱天蛋白酶1(Casp1)、组织蛋白酶B(Ctsb)、组织蛋白酶D(Ctsd)的mRNA表达显著下调,miR-361-3p、miR-193a-3p和miR-139-5p的miRNA表达显著上调(P<0.01);GO和KEGG分析发现关键靶点主要通过调控细胞凋亡、NOD样受体信号通路、MAPK信号通路、IL-17信号通路、TGF-β信号通路和PI3K-AKT信号通路等参与肾脏的炎症、纤维化、氧化应激、细胞凋亡和自噬等生物学过程,分子对接结果显示活性成分叶酸、毛蕊异黄酮、槲皮素、淫羊藿苷Ⅰ与核心靶点结合良好。结论:玉液汤可能通过影响糖尿病肾病大鼠lncRNA-miRNA-mRNA转录网络保护肾脏,抑制肾脏炎症,涉及多成分、多靶点、多途径协同作用。

Objective:To excavate the ternary transcriptional network of Yuye Decoction(YYD,玉液汤)against diabetic nephropathy(DN),and to predict its biomarkers and mechanism of action in the prevention and treatment of DN.Methods:Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Pharmmapper,and Uniprot were used to predict the key active components of YYD and their target proteins,and through GEO database,differentially expressed mRNA and miRNA in normal and DN patients were screened.The predicted targets of YYD were intersected with differentially expressed mRNAs(DEmRNAs)in DN to obtain the targets of YYD against DN.String was used to map the protein-protein interaction(PPI)network,and Metascape was utilized for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.The active component-target network of YYD in the prevention and treatment of DN was constructed by Cytoscape 3.6.1.The targets of YYD in the PPI network were imported into Cytoscape for topological analysis,and the core targets were screened using the Cytohubba plug-in.The miRDB,miRWalk,and TargetScan databases were used to predict miRNAs associated with the core targets,which were intersected with the DEmiRNAs in DN.The shared miRNAs were then imported into the Starbase database to predict the related lncRNAs,and DN-related lncRNAs were screened.Discovery Studio 2019 was used to perform the molecular docking of core compounds-core targets.Streptozotocin(STZ)combined with high-fat and high-sugar feed was given to induce DN rat model.The rats were treated with YYD 2.6 g/kg to observe the changes in body mass,fasting blood glucose,and histopathological morphology of the kidneys in each group.Additionally,urinary protein,blood creatinine,and urea nitrogen were detected.Fluorescence quantitative polymerase chain reaction(Real-time PCR)was performed to validate the differential genes,and the competitive endogenous RNA(ceRNA)network of lncRNA-miRNA-mRNA of YYD against DN was established by the Cytoscape.Results:The ceRNA network was composed of 17 key lncRNAs,10 key miRNAs and 13 key mRNAs.Experiments verified that in the ceRNA network,compared with the conditions in the model control group,urinary protein,blood creatinine and urea nitrogen were significantly reduced in the YYD 2.6 g/kg group,and the lncRNA expressions of Neat1,Xist,and Malat1,as well as the mRNA expressions of Casp1,Ctsb,and Ctsd were significantly down-regulated,while the miRNA expressions of miR-361-3p,miR-193a-3p,and miR-139-5p were markedly up-regulated.GO and KEGG analyses revealed that the key targets involved in biological processes such as inflammation,fibrosis,oxidative stress,apoptosis,and autophagy in the kidney mainly through the modulation of apoptosis,NOD-like receptor signaling pathway,MAPK signaling pathway,IL-17 signalling pathway,TGF-beta signaling pathway and PI3K-Akt signaling pathway and other pathways.Molecular docking showed that the active components,folic acid,calycosin,quercetin and Icariin I were well bound to the core targets.Conclusion:YYD may protect the kidney and inhibit renal inflammation by affecting the lncRNA-miRNA-mRNA network in DN rats,which involves multi-component,multi-target and multi-pathway synergistic effects.