糖尿病视网膜病变患者血清微核糖核酸表达谱的初步研究

doi:10.3877/cma.j.issn.2095-2007.2019.04.009

目的

本研究旨在探讨血清微核糖核酸(miRNA)成为糖尿病视网膜病变(DR)生物标志物的可能性。

方法

队列研究。2014年11月至2015年6月，招募中国医科大学附属盛京医院眼科和内分泌科患者69例作为研究对象。其中，单纯糖尿病患者30例作为单纯糖尿病组，DR患者39例作为DR组。两组采用数字表法各随机选取3例患者，应用miRNA芯片技术检测血清miRNA的表达水平；使用分层聚类分析，获得DR患者的血清miRNA表达谱；利用生物信息学方法，分析特异性表达的血清miRNA的靶基因及其相关信号通路。采用实时荧光定量聚合酶链式反应(qRT-PCR)技术，检测两组患者血清中特异性miRNA的表达水平。采用受试者工作特征曲线(ROC曲线)，评价血清中特异性miRNA对DR的诊断效能。

结果

miRNA芯片技术检测表明，在3100种成熟的miRNA中，共筛选出19种差异miRNA。其中，13种表达上调；6种表达下调。结果表明，在DR患者血清中存在特异性表达的miRNA。qRT-PCR检测表明，DR组miR-19b、miR-221和miR-18b表达均上调，差异有统计学意义(U＝256.027，125.515，254.017；P<0.05)，并且与miRNA芯片筛选的结果相符。受试者工作特征曲线结果表明，miR-19b(曲线下面积＝0.78，95%CI＝0.66~0.90)、miR-221(曲线下面积＝0.89，95%CI＝0.81~0.97)和miR-18b(曲线下面积＝0.78，95%CI＝0.67~0.90)对DR均有较高的诊断效能，其中miR-221诊断效能最高。

结论

DR患者血清中存在特异性miRNA表达谱，这些差异性表达的miRNA可能作为调控因子，通过调节靶基因调控DR的发生和发展，其中miR-221最有望成为DR的生物标志物。

关键词: 糖尿病视网膜病变, 微核糖核酸, 表达谱

Objective

The aim of this study was to study the feasibility of serum microRNA(miRNA) as a novel biomarker in patients with diabetic retinopathy(DR).

Methods

This was a cohort study. From November 2014 to June 2015, 69 patients were enrolled in Ophthalmology Department and Endocrinology Department of Shengjing Hospital, China Medical University. 30 simple diabetes mellitus(DM) patients were divided into DM group, and 39 diabetic retinopathy(DR) patients were divided into DR group. Randomly selected 3 patients from each group, and used the miRNA microarray technology to detected the expression level of serum miRNA.The hierarchical cluster analysis was used to obtain the serum miRNA expression profile of DR patients. Bioinformatics methods was used to analyze the target genes and related signaling pathways of the specific expression of serum miRNA. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) technology was used to detect the expression of miRNA in serum of the two patients. To establish the receiver operating characteristic(ROC) curve, to evaluate the diagnostic efficacy of specific miRNA in the serum of DR patients.

Results

miRNA microarray showed that in 3100 kinds of mature miRNAs, a total of 19 miRNA with significance differences were screened. Of the difference miRNA, there were 13 upregulated miRNAs, while there were 6 downregulated miRNAs. The results suggestted tha there were specific miRNA expression in tthe serum of DR patients. The qRT-PCR showed that the expression of miR-19b, miR-221 and miR-18b were significantly increased in DR group, and the difference were statistically significant (U=256.027, 125.515, 254.017; P<0.05), and in consistent with miRNA microarray screening results. The ROC curves showed that miR-19b(AUC=0.78, 95%CI=0.66-0.90), miR-221(AUC=0.89, 95%CI=0.81-0.97) and miR-18b (AUC=0.78, 95%CI=0.67-0.90) all had diagnostic efficacy for DR, and miR-221 was the most effective one.

Conclusions

The serum of patients with DR exist specific miRNA expression profiles, and these specific miRNAs may as a regulator by adjusting target gene to regulate the occurrence and development of DR. Of all the specific miRNAs markets, miR-221 may themost expective one.

Key words: Diabetic retinopathy, MicroRNA, Expression profile

表1 两组糖尿病患者血糖相关指标的比较(±s)

组别	例数	空腹血糖(mmol/l)	餐后2 h血糖(mmol/l)	糖化血红蛋白(%)
DR组	39	7.8±0.6	12.7±0.8	7.8±0.9
糖尿病组	30	7.6±0.5	12.4±1.0	7.5±0.8
t值		1.127	1.263	1.631
P值		>0.05	>0.05	>0.05

表1 两组糖尿病患者血糖相关指标的比较(±s)

组别	例数	空腹血糖(mmol/l)	餐后2 h血糖(mmol/l)	糖化血红蛋白(%)
DR组	39	7.8±0.6	12.7±0.8	7.8±0.9
糖尿病组	30	7.6±0.5	12.4±1.0	7.5±0.8
t值		1.127	1.263	1.631
P值		>0.05	>0.05	>0.05

图1 微核糖核酸芯片聚类分析图中特异性表达的微核糖核酸

图2 实时荧光定量反转录-聚合酶链反应检测微核糖核酸相对表达量的比较图　图A示miR-19b的相对表达量；图B示miR-221的相对表达量；图C示miR-18b的相对表达量；图D示miR-19b、miR-221和miR-18b的相对表达量

图3 受试者工作特征曲线评价微核糖核酸对糖尿病视网膜病变的诊断效能图　图A示miR-19b的工作特征曲线；图B示miR-221的工作特征曲线；图C示miR-18b的工作特征曲线；图D示miR-19b、miR-221和miR-18b的工作特征曲线

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Preliminary study on serum microRNA expression profile in patients with diabetic retinopathy

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Preliminary study on serum microRNA expression profile in patients with diabetic retinopathy