糖尿病肾病患者血浆细胞游离DNA中羟甲基化修饰lncRNA的筛选及其ceRNA调控网络构建  

Screening of hydroxymethylated lncRNAs in cell-free DNA of plasma from patients with diabetic kidney disease and construction of ceRNA regulatory network

作　　者：储金林 马睿瑶 铁璐[2] 李琳琳[1] CHU Jinlin;MA Ruiyao;TIE Lu;LI Linlin(School of Pharmacy,Xinjiang Medical University,Urumqi 830011,China;不详)

机构地区：[1]新疆医科大学药学院,乌鲁木齐830011 [2]北京大学基础医学院药理学系

出　　处：《山东医药》2022年第26期1-5,共5页Shandong Medical Journal

基　　金：北京市科协金桥工程种子资金C类项目(ZZ16019)。

摘　　要：目的筛选糖尿病肾病(DKD)患者血浆细胞游离DNA(cfDNA)中5-羟甲基胞嘧啶(5hmC)修饰的关键ln⁃cRNA,并构建其ceRNA调控网络。方法收集14例糖尿病患者和17例DKD患者的血浆样本,利用5hmC-Seal技术对cfDNA进行全基因组5hmC富集,通过加权基因共表达网络(WGCNA)方法筛选差异关键lncRNA,然后使用Star⁃base预测lncRNA的靶向miRNA,经PCR法验证糖尿病肾病模型小鼠肾组织中可预测出靶向miRNA的相关lncRNA mRNA的表达水平,再通过TargetScan、miRDB预测miRNA的靶向mRNA,使用Metascape对mRNA进行富集分析,运用Cytoscape进行构建内源竞争RNA调控网络。结果DKD组与糖尿病组的差异羟甲基化基因共1644个,WGCNA分析得到关键差异lncRNA 15个。通过数据库预测出下游靶向miRNA的lncRNA有2个,分别为LINC00863和LINC00641,对于DKD的诊断准确性均较高(AUC≥0.8),在DKD小鼠肾组织中验证lncRNA的表达水平与预测结果一致。将预测得到的靶向miRNA的下游mRNA与DKD组织转录组差异基因验证分析得到246个靶mRNA,并成功构建DKD的lncRNA-miRNA-mRNA调节网络。结论从DKD患者血浆cfDNA中筛选得到5hmC修饰的关键差异lncRNA 15个,并对其中有下游靶向miRNA的LINC00863和LINC00641成功构建其ceRNA调控网络;LINC00863和LINC00641可能成为DKD诊断标志物和治疗靶点。Objective To screen out the key long non-coding RNAs(lncRNAs)modified by 5-hydroxymethylcyto⁃sine(5hmC)in plasma cell-free DNA(cfDNA)of patients with diabetic kidney disease(DKD)and to construct a regulato⁃ry network of competitive endogenous RNA(ceRNA).Methods Plasma samples of 14 diabetic patients and 17 DKD pa⁃tients were collected,cfDNA was enriched for 5hmC genome-wide by 5hmC-Seal technology,and differentially key ln⁃cRNAs were screened by the weighted gene co-expression network(WGCNA)method,and then Starbase database was used to predict the targeted microRNA(miRNA)of lncRNA.Polymerase chain reaction was used to verify the expression level of lncRNA mRNA in the kidney tissues of DKD model mice that could predict the target miRNA.Then,TargetScan and miRDB database were used to predict the target mRNA of miRNA,and Metascape was used for mRNA enrichment analysis.Finally,Cytoscape was used to construct the ceRNA regulatory network.Results There were 1644 differen⁃tially hydroxymethylated genes between the DKD group and the diabetes group,and 15 key differential lncRNAs were iden⁃tified by WGCNA analysis.There were two lncRNAs that targeted downstream miRNAs predicted from the database,LINC00863 and LINC00641,which had high diagnostic accuracy for DKD(AUC≥0.8).The expression level of lncRNAs in the kidney tissues of DKD mice was verified to be consistent with the predicted results.A total of 246 targeted mRNAs were obtained by analyzing the downstream mRNAs of the predicted target miRNAs and DKD tissue transcriptome differen⁃tial genes,and the lncRNA-miRNA-mRNA regulatory network of DKD was successfully constructed.Conclusions Fif⁃teen key differential lncRNAs modified by 5hmC are screened out from the plasma cfDNA of DKD patients,and their ceR⁃NA regulatory networks are successfully constructed for LINC00863 and LINC00641,which target downstream miRNAs.Meanwhile,LINC00863 and LINC00641 may become diagnostic markers and therapeutic targets for DKD.

关 键 词：糖尿病肾病 5-羟甲基胞嘧啶 长链非编码RNA 加权基因共表达网络分析 

分 类 号：R915[医药卫生—微生物与生化药学]