慢性乙型病毒性肝炎肝胆湿热证和肝郁脾虚证患者尿液中microRNAs的表达研究

Expression and significance of urinary microRNAs in chronic hepatitis B patients with liver-gallbladder dampness-heat syndrome and liver depression and spleen deficiency syndrome

目的研究慢性乙型病毒性肝炎(慢乙肝)肝胆湿热证和肝郁脾虚证患者尿液中microRNAs(miRNAs)的表达差异,探索不同证候的特征性miRNAs,为慢乙肝辨证分型提供生物学标志物和客观化依据。方法收集慢乙肝肝胆湿热证和肝郁脾虚证患者(各53例)及健康者(24例)的尿液标本,采用miRNAs表达谱芯片技术检测miRNAs表达谱,分析差异表达的miRNAs;采用实时定量RT-PCR(RT-qPCR)技术检测慢乙肝肝胆湿热证和肝郁脾虚证患者尿液中部分差异表达的miRNAs相对表达量;进行miRNAs潜在靶基因的预测、GO和pathway分析。结果①miRNAs表达谱芯片检测结果显示,与肝郁脾虚证比较,慢乙肝肝胆湿热证患者有21条miRNAs呈高表达,1条miRNA呈低表达(差异倍数>1.5,P<0.05)。②RT-qPCR结果显示,与肝郁脾虚证比较,慢乙肝肝胆湿热证患者尿液中miR-483-3p和miR-4700-3p呈显著高表达(P<0.05),miR-4745-5p呈显著低表达(P<0.05);与健康者比较,慢乙肝肝胆湿热、肝郁脾虚证患者尿液中miR-483-3p呈低表达(P<0.05)、miR-4745-5p呈高表达(P<0.05),慢乙肝肝郁脾虚证患者尿液中miR-4700-3p呈低表达(P<0.05)。③miR-483-3p、miR-4700-3p和miR-4745-5p的联合ROC曲线AUC为0.810 6,对于鉴别肝胆湿热证和肝郁脾虚证患者具有一定的诊断价值。④生物信息学方法显示,证候差异表达miRNAs所调控的潜在靶基因,GO大都与生物学调控、代谢过程、多细胞有机体的发展、应激反应和细胞增殖等相关,pathway大都与翻译后蛋白质修饰、TGF-β信号通路、TGF-β家庭成员的信号、TGF-β受体复合物的信号传导等通路有关。结论慢乙肝肝胆湿热、肝郁脾虚证患者尿液中存在差异表达的miRNAs,miR-483-3p、miR-4700-3p和miR-4745-5p或许为慢乙肝肝胆湿热、肝郁脾虚证的潜在标志性分子,这些miRNAs可能分别通过调控其相应的靶基因而影响证候的发生发展。

Objective To investigate the expression difference of microRNAs(miRNAs) in urine of chronic hepatitis B(CHB) patients with liver-gallbladder dampness-heat(LGDH) syndrome and liver depression and spleen deficiency(LDSD) syndrome, to explore the characteristic miRNAs of different syndromes and to provide biological markers and objective basis for CHB syndrome differentiation and classification. Methods Urine samples were collected from 53 subjects with LGDH syndrome, 53 subjects with LDSD syndrome and 24 healthy subjects. The expression levels of urinary miRNA were detected by miRNA microarray and miRNAs with differential expressions were analyzed;The quantitative reverse transcription-polymerase chain reaction(RT-qPCR) was used to detect the relative expressions of some differentially expressed miRNAs in urine of CHB patients with LGDH syndrome and LDSD syndrome. Prediction, GO and pathway analysis of miRNAs potential target genes were carried out. Receiver-operator characteristic(ROC)curves were generated to determine the specificity and sensitivity of each individual miRNA. The target genes of miRNAs were predicted and Gene Ontology(GO) and pathways were analyzed using bioinformatics methods. Results(1) The microarray of miRNAs expression profiles showed that 21 miRNAs were upregulated and 1 miRNA was down-regulated in CHB patients with LGDH compared with LDSD(fold change > 1. 5,P<0. 05).(2) The results from RT-q PCR revealed that miR-483-3 p and miR-4700-3 p were in significantly higher expression(P<0.05) and miR-4745-5 p was in significantly lower expression(P<0.05) in the urine of CHB patients with LGDH syndrome compared with those with LDSD syndrome;miR-483-3 p was in lower expression(P<0.05) and miR-4745-5 p was in higher expression(P<0.05) in the urine of CHB subjects with LGDH syndrome LDSD syndrome and miR-4700-3 p was in lower expression(P<0.05) in the urine of CHB subjects with LDSD syndrome compared with healthy subjects.(3)ROC curve analysis exhibited the AUC(0.8106) and showed that these three miRNAs of miR-483-3 p,miR-4700-3 p and miR-4745-5 p were sensitive and specific enough to distinguish LGDH and LDSD subjects.(4)Bioinformatics methods showed that the difference of syndromes expressed potential target genes regulated by miRNAs. GO was mostly related to biological regulation,metabolic process,development of multicellular organisms,stress response and cell proliferation;Pathway was mostly related to post-translational protein modification,TGF-β signaling pathway,TGF-β family member signaling,TGF-β receptor complex signaling and other pathways. Conclusion The miRNAs in the urine of CHB patients with LGDH syndrome and LDSD syndrome are differentially expressed. miR-483-3 p,miR-4700-3 p and miR-4745-5 p may be potential markers for syndrome differentiation in CHB and these miRNAs may affect the development of syndromes by regulating their related target genes.