血浆微RNA在骨关节炎患者中的表达谱特征及生物信息学分析

Analysis of plasma microRNA expression profiles and bioinformatics in osteoarthritis patients

目的 探讨OA患者血浆微RNA(miRNA)表达谱特征,并对差异表达的miRNA进行生物信息学分析,寻找与OA相关的血浆生物标记物.方法 收集20例OA患者及15名健康体检者静脉血,通过Agilent miRNA芯片表达谱检测OA血浆miRNA的表达谱.对差异表达的miRNA进行靶基因预测,聚类分析(GO)功能分析及KEEG通路聚类分析等生物信息学分析.最后选取独立验证样本采用方差分析对差异表达miRNA进行实时荧光定量-PCR验证,并评估效能.组间差异采用t检验分析.结果 ①与健康对照组相比,OA组筛选获得74个差异表达血浆miRNA,其中45个表达上调,29个表达下调.②预测差异性表达miRNA潜在靶基因2 731个,参与到462个KEGG通路条目中,主要富集于环磷酸腺苷(cAMP)信号通路、丝裂原活化蛋白激酶(MAPK)信号通路、破骨细胞分化等通路,主要功能集中在细胞间黏附作用、胶原合成作用、细胞内信号转导等生物学功能上.③RT-PCR显示OA患者血浆miR-20a-5p、miR-320c表达水平明显升高(t=-6.142,P<0.05;t=-3.854,P<0.05),而miR-940表达明显下调(t=2.767,P<0.05),变化趋势与芯片结果一致.受试者工作特征曲线(ROC)显示这3个miRNA的曲线下面积(AUC)分别为0.864,0.851和0.818.结论 OA患者有着独特的血浆miRNA表达谱,差异性表达的miRNA可能是OA诊断的生物标志物和潜在治疗靶点.

Objective To explore the characteristics of plasma microRNA(miRNA)profiles and bioinformatics in patients with osteoarthritis(OA)in order to search for diseases related biomarkers.Methods Blood samples from 20 cases of OA patients and 15 cases of normal control(NC)were collected to extracted total RNA in plasma.The plasma miRNA expression profile was tested by using Agilent Human miRNA array.Target gene analysis and clustering analysis were performed on differentially expressed microRNAs.Three differentially expressed miRNAs(miR-134-5p,miR-320c and miR-940)were detected by real-time quantitative polymerase chain reaction(RT-qPCR)for further confirmation of microarray data.The differences were tested using t test analysis.Results①MiRNA microarray showed that compared with NC,there were 74 differential expression genes in plasma of patients in the OA group(FC≥2,P≤0.01),among which 45 were up-regulated and 29 were down-regulated.②A total of 2731 potential target genes were predicted in three database,and involved in 462 Kyoto Encyclopedia of Genes and Genomes(KEEG)pathways.Target gene ontology(GO)functional clustering found that the main functions of miRNAs were intercellular adhesion,collagen synthesis,intracellular signal transduction,etc.The main KEGG pathways of miRNAs include mitogen-activated protein kinase(MAPK)signaling pathway,cyclic adenosine monophosphate(cAMP)signaling pathway,osteoclast differentiation signaling pathway,etc.③The expression level of miR-20a-5p and miR-320c in OA group were significantly higher than that in controls(t=-6.142,P<0.05;t=-3.854,P<0.05),while miR-940 was significantly lower than that of controls(t=2.767,P<0.05).The trend was consistent with the microarray data.The receiver operating characteristic curve(ROC)curve analyses showed that they were useful biomarkers for differentiating patients with OA from controls.Conclusion The study shows that plasma in OA patients has a specific miRNAs expression,and miRNAs play an important role in the pathogenesis of OA.