应用蛋白质组学探索2型糖尿病增殖期视网膜病变潜在生物标志物

To explore the potential biomarkers of proliferative diabetic retinopathy by plasma proteomics

目的利用同位素标记相对和绝对定量(isobaric tag for relative and absolute quantification,iTRAQ)蛋白质组学技术,分析糖尿病增殖期视网膜病变(proliferative diabetic retinopathy,PDR)患者与糖尿病无视网膜病变患者(diabetes patients without retina diseases,NDR)之间蛋白表达的差异,寻找PDR的候选血清标志物。方法收集2016年至2017年首都医科大学附属北京同仁医院内分泌科住院的PDR患者21例,以及性别年龄匹配的NDR患者21例。患者血清样本混匀后提取蛋白,采用iTRAQ标记,并进行液相质谱-串联质谱检测(liquid chromatograph-mass spectrometer and mass spectrometer,LC-MS/MS)分析,筛选出差异蛋白行基因本体论(Gene Ontology,GO)数据库功能富集及京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genome,KEGG)通路显著性富集分析。对相对定量结果进行独立样本t检验,通过差异倍数(fold change,FC)和P值判定蛋白表达量变化。结果以差异倍数>1.2倍(上/下调),P值≤0.05为标准,筛选出差异蛋白29个,其中PDR组上调蛋白8个,下调蛋白21个。通过对差异蛋白的GO功能显著性富集分析,结果显示,差异基因的功能可分为生物过程、细胞组分和分子功能三个板块。差异表达基因功能涉及生物调节、细胞组成、细胞代谢过程、细胞结合和催化活性等方面。KEGG通路显著性分析显示,上调最为明显的血管紧张素转换酶(angiotensin converting enzyme,ACE)参与肾素-血管紧张素系统(renin angiotensin system,RAS)、肾素分泌等通路,上调蛋白——胰岛素样生长因子I(insulin like growth factor I,IGF-1)参与HIF-1、FoxO、mTOR、PI3K-Akt和AMPK等多条与代谢相关的信号通路。下调显著的蛋白——肌球蛋白6(myosin-6)参与心肌细胞收缩和信号传导通路。结论采用iTRAQ蛋白质组学分析显示,PDR患者与NDR患者存在多种蛋白表达差异,ACE、IGF-1和Myosin-6有望作为糖尿病视网膜病变候选血清诊断标志物,未来可能为PDR的诊断和治疗提供新的靶点。

Objective To identify potential biomarkers of proliferative diabetic retinopathy(PDR)through the difference of protein expression between PDR patients and diabetes patients without retina diseases(NDR)by using isobaric tag for relative and absolute quantification(iTRAQ)proteomics.Methods A total of 42 patients admitted to the Department of Endocrinology,Beijing Tongren Hospital from 2016 to 2017 were recruited,including 21 PDR patients and 21 gender-and age-matched NDR patients.After proteins extraction from patients'serum samples,iTRAQ labeling with liquid chromatograph-mass spectrometer and mass spectrometer(LC-MS/MS)analysis was performed to screen the differential proteins.Gene Ontology(GO)enrichment and Kyoto Encyclopedia of Genes and Genome(KEGG)pathway were analyzed.Comparisons were conducted using independent sample t-tests,to determine the changes in protein expression levels through fold change(FC)and P-values.Results Totally 29 differentially expressed proteins were identified based on a FC>1.2(up/down)and a P value≤0.05.Among them,8 proteins were upregulated and 21 proteins were downregulated in the PDR group.GO enrichment analysis showed that the functions of differentially expressed genes were classified into three categories:biological processes,cellular components,and molecular functions.The differential gene expressions were involved in biological regulation,cell composition,cellular metabolic processes,and cell binding or catalytic activity.KEGG pathway analysis showed that the most significantly upregulated angiotensin converting enzyme(ACE)is involved in the renin angiotensin system(RAS)and renin secretion pathways.The upregulated protein insulin like growth factor I(IGF-1)is involved in metabolic related signaling pathways,including HIF-1,FoxO,mTOR,PI3K-Akt,and AMPK.The significantly downregulated protein myosin-6 is involved in myocardial cell contraction and signaling pathways.Conclusion The iTRAQ proteomics analysis suggested,many differential expressed proteins between PDR and NDR groups.ACE,IGF-1 and myosin-6 were potential serum diagnostic markers and maybe new therapeutic targets for diabetic retinopathy.