基于基因芯片的miRNA表达差异在结核性脑膜炎与病毒性脑膜炎诊断中的价值

Diagnostic performance of differentially expressed miRNA identified by gene chip in discriminating tuberculous meningitis from viral meningitis

目的:评价基于基因芯片的miRNA表达差异在结核性脑膜炎(tuberculous meningitis,TBM)与病毒性脑膜炎(viral meningitis,VM)诊断中的价值。方法:选取2017年12月至2019年9月在首都医科大学附属北京胸科医院和首都医科大学附属北京天坛医院治疗的TBM患者28例和VM患者27例作为验证样本进行实时荧光定量逆转录PCR(qRT-PCR)验证。选取前期收集的8例TBM患者和5例VM患者作为基因芯片样本进行全基因组miRNA微阵列分析,通过预测差异性miRNA的靶基因、基因本体(GO)富集分析、京都基因与基因组百科全书(KEGG)信号通路分析、构建蛋白质-蛋白质相互作用(PPI)网络筛选出两病间前10个差异性miRNA枢纽基因。再采用qRT-PCR对患者间表达差异明显的miR-21-5p进行验证,并通过受试者工作特征曲线(ROC)下面积(AUC)分析miRNA的诊断价值。结果:通过miRNA微阵列分析初步筛选出26个差异表达的miRNA(14个表达上调,12个表达下调)和对应的靶基因3217个。经GO富集分析、KEGG信号通路分析筛选出190个相关靶基因,对其进行PPI网络分析,筛选出前10个靶基因作为核心靶基因。对基因芯片检测样本的hsa-miR-21-5p相对表达量进行qRT-PCR验证,发现TBM患者hsa-miR-21-5p的表达水平[15.890(3.423,25.581)]较VM患者[0.807(0.614,0.955)]明显上调(Z=-2.355,P=0.019),差异倍数(TBM/VM)在基因芯片和qRT-PCR中分别是4.817和4.660,两种检测方法结果基本一致;对28例TBM和27例VM患者的hsa-miR-21-5p进行qRT-PCR结果比较,发现hsa-miR-21-5p在TBM患者的相对表达量为4.825(2.433,21.362),明显高于VM患者[0.204(0.112,0.711)],差异有统计学意义(Z=-5.000,P=0.000),且ROC曲线区分TBM与VM患者的AUC为0.893(0.806~0.980),敏感度为85.7%,特异度为88.9%。结论:相较于VM患者,作为基因芯片检测中表达差异明显的miR-21-5p在TBM患者中表达明显上调,可作为鉴别TBM与VM的潜在生物标志物。

Objective:To evaluate the diagnostic performance of differentially expressed miRNA identified by gene chip in discriminating tuberculous meningitis(TBM)from viral meningitis(VM).Methods:A total of 28 TBM patients and 27 VM patients who were treated in Beijing Chest Hospital and Beijing Tiantan Hospital affiliated to Capital Medical University from Dec 2017 to Sep 2019 were recruited for validation using Real-time Quantitative PCR(qRT-PCR).Of 8 TBM patients and 5 VM patients who were recruited earlier were selected for genome-wide miRNA analysis by gene chip.The top 10 miRNA hub genes were screened by predicting the target genes of differentially expressed miRNAs,gene ontology(GO)enrichment analysis,Kyoto encyclopedia of genes and genomes(KEGG)signaling pathway analysis,and protein-protein interaction(PPI)network.qRT-PCR was used for further validation of the expression level of hsa-miR-21-5p between two groups,and the diagnostic value of miRNA was analyzed by area under receiver operating characteristic curve(AUC).Results:Totally 26 differentially expressed miRNAs(14 were up-regulated and 12 were down-regulated)were identified by miRNA microarray analysis,and further 3217 corresponding target genes were screened out by miRTarBase database.Through GO enrichment analysis and KEGG signal pathway analysis,190 related target genes were screened out.The top 10 target genes as core target genes were defined by PPI.qRT-PCR verification of the relative expression of hsa-miR-21-5p based on gene chip samples showed that the expression level of has-miR-21-5p in TBM patients was significantly up-regulated than that in VM patients(15.890(3.423,25.581)vs.0.807(0.614,0.955);Z=-2.355,P=0.019).The fold change(TBM/VM)in gene chip and qRT-PCR were 4.817 and 4.660,respectively,which was consistent between these two techniques.qRT-PCR analysis of hsa-miR-21-5p was further performed in 28 TBM patients and 27 VM patients.The results showed that the relative expression level of hsa-miR-21-5p in TBM patients was significantly higher than that in VM patients(4.825(2.433,21.362)vs.0.204(0.112,0.711);Z=-5.000,P=0.000).The AUC for distinguishing TBM patients from VM patients was 0.893(0.806-0.980),with a sensitivity of 85.7%and a specificity of 88.9%.Conclusion:As a significantly differentially expressed miRNA in gene chip analysis,has-miR-21-5p is significantly up-regulated in patients with TBM than VM patients,which can be used as a potential biomarker to differentiate TBM from VM.