外周血Hsa-circ-000094作为2型糖尿病诊断标志物的初步研究

Hsa-circ-000094 in Peripheral blood can be used as a biomarker for the diagnosis of type 2 diabetes

目的探索2型糖尿病(T2DM)患者外周血中环状RNA(circRNA)的差异性表达,寻找T2DM的诊断标志物。方法采用微阵列分析从3个正常人和3个T2DM患者外周血中严格筛选出数个差异表达circRNAs。扩大样本量[对照组(NC)20人,糖调节异常(IGR)20人,2型糖尿病(T2DM)20人],利用荧光定量PCR(Q-PCR)测定,进一步筛选差异性最明显的一个circRNA,对确定的circRNA再进行扩大样本验证(50个NC、50个IGR和50个T2DM样本)。结果微阵列分析共发现2 953个差异表达的circRNAs,其中表达上调的为1 439个,表达下调的为1 514个。从表达上调的1 439个环状RNA中严格筛选出9个差异表达的circRNAs(hsa-circ-103838、hsa-circ-103965、hsa-circ-104227、hsa-circ-002117、hsa-circ-000094、hsa-circ-101226、hsa-circ-101720、hsa-circ-400029、hsa-circ-100633)作为候选诊断标志物。扩大样本后(n=60)Q-PCR结果显示9个circRNAs中hsa-circ-000094(别名:hsa-circ-0000247)在3组人群中差异表达最明显,通过ROC曲线分析发现其具有最大曲线下面积(AUC),SIGR=0.802 5[95%置信区间(0.665 5-0.939 5),P=0.001];ST2DM=0.77[95%置信区间(0.624-0.916),P=0.003]。为了进一步验证Hsa-circ-000094的临床诊断能力,进行再扩大样本(n=150)实验,结果显示Hsa-circ-000094在3组人群中的表达具有差异性,差异性及ROC曲线分析具有统计学意义,SIGR=0.673 3[95%置信区间(0.575 7-0.771 0),P<0.01];ST2DM=0.723 1[95%置信区间(0.632 7-0.813 4),P<0.01]。结论外周血hsa-circ-000094的高表达对IGR和T2DM提供了一定的诊断依据。

Objective The purpose of this study was to investigate the differential expression of circRNAs in human blood, as a diagnostic marker for pre-diabetes and type 2 diabetes mellitus(T2DM). Methods Microarray analysis was used to select several differentially expressed circRNAs from three normal patients and three T2DM patients. Enlarge the sample size(normal controls, n=20;subjects with impaired glucose regulation, n=20;and type 2 diabetes mellitus, n=20) to determine a circRNA which the most evident differentially expressed by fluorescence quantitative PCR(Q-PCR). Then they were verified with expanded samples(normal controls, n=50;impaired glucose regulations, n=50;type 2 diabetes mellitus, n=50) by Q-PCR. Results A total of 2 953 differentially expressed circRNAs were found in microarray analysis, of which 1 439 were up-regulated and 1 514 were down-regulated. Nine differentially expressed circRNAs were selected from the 1 439 circRNAs that were up-regulated(hsa-circ-103838, hsa-circ-103965, hsa-circ-104227, hsa-circ-002117, hsa-circ-000094, hsa-circ-101226, hsa-circ-101720, hsa-circ-400029, and hsa-circ-100633). The Q-PCR results of the expanded sample(n=60) showed that the difference expression of hsa-circ-000094(Alias: has-circ-0000247) in the nine circRNAs was the most obvious one among the 3 groups, the area under the maximum curve was found by ROC curve analysis, SIGR=0.802 5[95% confidence interval (0.665 5-0.939 5), P=0.001];ST2DM=0.77[95% confidence interval (0.624-0.916), P=0.003]. In order to verify the clinical diagnostic ability of hsa-circ-000094, the experiment was conducted to further expand the sample(n=150). The results showed that the expression of hsa-circ-000094 in the three groups was different, the difference and ROC curve analysis were statistically significant, SIGR=0.673 3[95% confidence interval (0.575 7-0.771 0), P<0.01];ST2DM=0.723 1[95% confidence interval (0.632 7-0.813 4), P<0.01]. Conclusion The higher expression of hsa-circ-000094 in peripheral blood provides a certain diagnostic basis for pre-diabetes as well as type 2 diabetes mellitus.