熊去氧胆酸应答不佳原发性胆汁性胆管炎女性患者肝组织lncRNA、mRNA差异表达分析

Analysis of differential expression of lncRNA and mRNA in liver tissue of female primary biliary cholangitis patients with poor response to ursodeoxycholic acid

目的筛选熊去氧胆酸(ursodeoxycholic acid,UDCA)应答不佳原发性胆汁性胆管炎(primary biliary cholangitis,PBC)女性患者肝组织长链非编码RNA(long noncoding ribonucleic acid,lncRNA)、mRNA差异表达基因,并进行生物信息学分析。探讨其在PBC应答不佳中的作用及潜在功能。方法收集2016年7月至2017年7月首都医科大学附属北京地坛医院6例女性PBC患者的肝组织穿刺标本,其中3例对UDCA应答好(对照组),另外3例对UDCA应答不佳(试验组)。通过RNA提取、纯化、扩增及芯片实验,以差异倍数(fold change,FC)≥1.5倍,P<0.05作为筛选条件,利用Illumina高通量测序技术检测6例PBC患者肝组织中lncRNA、mRNA的表达水平,筛选差异表达的lncRNA和mRNA。对差异表达的LncRNA、mRNA进行基因本体(gene ontology,GO)功能分析、信号转导通路富集分析(kyoto encylopaedia of genes and genomes,KEGG分析)及蛋白互作网络分析,寻找与PBC应答不佳相关的lncRNA。结果共检测到差异表达的mRNA 599个(312个上调,287个下调)、lncRNA 1167个(685个上调,482个下调)。GO与KEGG分析发现,差异表达的mRNA与甘油三酯、长链脂肪酸代谢、胆固醇排出、糖原异生及正向调节血管生成等相关。而这些mRNA与核受体过氧化物酶体增殖激活受体(peroxisome proliferator-activated receptor,PPAR)信号通路、转化生长因子β(transforming growth factor beta,TGF-β)信号通路、脂肪酸降解通路、细胞因子受体相互作用信号通路等相关。差异表达lncRNA的靶基因与磷代谢、磷酸盐代谢、能量代谢、细胞活化、免疫效应调节、抗原加工和提呈相关。这些靶基因与病毒感染通路、泛素介导的蛋白降解通路、线粒体自噬通路、细胞因子受体相关通路、Wnt信号通路相关。结论本研究是PBC应答不佳相关lncRNA谱的补充,研究发现了一定数量差异表达的lncRNA,并预测其功能靶向基因及涉及信号通路,有望为PBC应答不佳研究提供新的参考依据。

Objective To investigate the expression profile and biological functions of long non-coding RNA(lncRNA)and mRNA in liver tissue of female primary biliary cholangitis(PBC)patients with poor response to ursodeoxycholic acid(UDCA).Methods Liver biopsy tissues were collected from 6 female patients with PBC in Beijing Ditan Hospital,Capital Medical University from July 2015 to July 2016,including 3 patients with good response to UDCA(control group)and 3 patients with poor response to UDCA(experimental group).The tissues were subjected to RNA extraction,purification,amplification and microarray experiments.The expression levels of lncRNA and mRNA were detected using illumina highthroughput sequencing technology.Differentially expressed lncRNA and mRNA were screened with fold difference(FC)≥1.5-fold and P<0.05 as screening conditions.Differentially expressed lncRNA and mRNA were analyzed by gene ontology(GO)functional analysis,signal transduction pathway enrichment(KEGG)analysis and protein interaction network analysis.Results A total of 599 differentially expressed mRNA(312 were up-regulated and 287 were downregulated)and 1167 lncRNA(685 were up-regulated and 482 were downregulated)were detected.GO and KEGG analysis revealed that differentially expressed mRNA were associated with triglyceride,long-chain fatty acid metabolism,cholesterol efflux,gluconeogenesis,positive regulation of sprouting angiogenesis and so on.These mRNA are associated with nuclear receptor peroxisome proliferator-activated receptor(PPAR)signaling pathway,transforming growth factor beta(TGF-β)signaling pathway,fatty acid degradation pathway,cytokine-cytokine receptor interaction signaling pathway and so on.The target genes of differentially expressed lncRNA are associated with phosphorus metabolism,phosphate metabolism,energy metabolism,cell activation,immune effect regulation,antigen processing and presentation.These target genes are associated with viral infection pathways,mitophagy pathways,cytokine receptor-related pathways and Wnt signaling pathways.Conclusions This study is a supplement to the lncRNA profile associated with poor response in PBC.A certain number of differentially expressed lncRNA were found,and their functionally targeted genes and signaling pathways were predicted,which is expected to provide a new reference for the study of poor response in PBC.