摘要
目的 分析原发性胆汁性肝硬化（PBC）患者外周血单个核细胞（PBMC）中长链非编码RNA（lncRNA）表达谱,为PBC的发病机制、临床诊断和治疗提供新的方向.方法 收集PBC及健康对照组外周抗凝血各30份,分离PBMC,从中各选取4份进行lncRNA表达谱芯片测定,并用逆转录PCR（RT-PCR）验证芯片结果.对芯片结果进行生物信息学分析,如 Cis/Trans 靶基因分析、Gene ontology（GO）分析、Pathway分析和共表达网络预测,并设计小干扰RNA（siRNA）干扰lncRNA的表达,进行靶点验证.结果 相对于健康对照组,PBC患者PBMC中共发现749个异常表达的lncRNA, 230个异常表达的信使RNA（mRNA）.PBC组核受体（NR4A3）基因表达水平下调78%,而在NR4A3基因下游20000 bp左右的lncRNA linc-pbc的表达上调2.56倍.转染针对linc-pbc的siRNA后, PBMC中linc-pbc的水平下降,而NR4A3的mRNA表达水平及蛋白质水平均上调,叉头状转录因子（FOXP3）的表达也上调.结论 linc-pbc或许通过招募多梳蛋白抑制性复合物（PRC2）,使NR4A3基因启动子区甲基化,下调NR4A3基因的表达水平,使其调控靶基因转录的活性降低,导致下游的靶基因FOXP3的表达下降,进而引起外周血及肝组织局部具有免疫抑制功能的调节性T细胞（Treg）细胞数量减少,打破机体免疫耐受平衡,促进PBC的发生和发展.
Objective Analyzing the lncRNA expression profile in peripheral blood mononuclear cells（PBMC）of primary biliary cirrhosis（PBC）patients to provide new ideas for the pathogenesis,clinical diagnosis and treatment of PBC.Methods Collected peripheral blood from 30 PBC patients and 30 healthy volunteers, then separated PBMC.Four cases from each group were selected for long-noncoding RNA （lncRNA）expression microarray detection.Reverse transcription-PCR technology in a larger sample size was used to verify the microarray results.Bioinformatic analysis such as Cis-/Trans-target genes Gene ontology（GO）and pathway analysis, co-expression networks were conducted in order to provide a theoretical basis in the pathogenesis of PBC.Transfecting small interfering RNAs（siRNAs）for linc-pbc to see changes in the expression of nuclear receptor 4A group 3（NR4A3）and forkhead boxP3（FOXP3）and cell apoptosis in transfected PBMC.Results Compared to the healthy group, 749 lncRNA and 230 coding messenger RNA（mRNA）genes were abnormally expressed.Interestingly,NR4A3 gene was down-regulated by 78%.While linc-pbc, which was about 20 000 bp downstream of NR4A3 gene, increased 2.56-fold. Then design siRNA for linc-pbc.After transfection, mRNA and protein levels of NR4A3 and FOXP3 were up-regulated.Conclusions By recruiting PRC2 complex, linc-pbc may increased the methylation level of NR4A3 gene promoter region,thus decreasing the expression of NR4A3 in PBC patients and reduced NR4A3 futher downregulated the expression of FOXP 3 and reduced the amounts of immunosuppressive Treg cells in peripheral blood and liver tissue, breaking the equilibrium state of immune tolerance, and promoted the occurrence and development of the disease.
作者
杨敏
张钧
仲人前
Yang Min , Zhang Jun, Zhong Renqian(Clinical Laboratory, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, Chin)
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2018年第5期374-379,共6页
Chinese Journal of Laboratory Medicine
作者简介
通信作者：仲人前，电子信箱：rqzhong@hotmail．com
