环状RNA hsa_circ_0031739与2型糖尿病关系的初步研究

A preliminary research on the relationship of hsa_circ_0031739 and type 2 diabetes mellitns

目的筛选高糖处理人脐静脉内皮细胞中差异表达环状RNA，分别在细胞模型中和2型糖尿患者外周血中进行验证，为探索2型糖尿病的生物学诊断标志物奠定基础。方法以脐静脉内皮细胞作为实验模型，设置对照组（n＝3）与高糖组（n＝3），分别使用正糖（5.5 mmol/L）及高糖（30.0 mmol/L）条件进行处理，运用高通量测序技术初步筛选差异表达环状RNA。通过PCR和实时定量PCR（q－PCR）技术，在内皮细胞模型中对差异表达环状RNA进行验证。随后，在2型糖尿患者外周血（n＝32）及正常人外周血（n＝28）中，进一步验证上述差异表达环状RNA。运用t－检验、相关性分析和ROC曲线，对实验结果进行分析。结果高通量测序共筛选出1 087个差异表达环状RNA，其中554个上调，533个下调。从中挑选出6个上调环状RNA在脐静脉内皮细胞模型中初步验证，发现，PCR结果及q－PCR结果与测序结果基本一致。再从中挑选出3个上调最为明显的环状RNA在外周血中进一步验证。本研究发现，环状hsa_circ_0031739在外周血样本中的差异表达也具有统计学意义（0.015±0.002 5 vs. 0.006±0.001 3，P＝0.005 9〈0.05），ROC曲线下面积为0.730，并且与血糖和糖化血红蛋白水平正相关（r＝0.317，P＝0.013 7〈0.05；r＝0.348，P＝0.006 4〈0.05）。结论人脐静脉内皮细胞作为糖尿病研究中常用的细胞模型，高糖处理后存在差异表达环状RNA谱。环状RNA hsa_circ_0031739在细胞模型和2型糖尿病外周血样本中的差异表达均具有统计学意义，与血糖及糖化血红蛋白相关，具有一定的诊断意义。因此，环状RNA hsa_circ_0031739有望成为糖尿病新型生物学诊断标志物，有助于2型糖尿病的综合诊断和疾病机理研究。

ObjectiveIn order to establish a basis for exploring diagnostic biomarkers of type 2 diabetes mellitus （T2DM）, our research screened differentially expressed circRNAs in high glucose treated human umbilical vein endothelial cells （HUVECs） and validated these circRNAs in both cell models and peripheral blood samples of T2DM patients.MethodsThe research used HUVECs as experimental model. The control group （n=3） and high glucose group （n=3） were set up and treated under normal condition （5.5 mmol/L） and high glucose condition （30.0 mmol/L）, respectively. The research utilized high-throughput sequencing technology to preliminarily screen differentially expressed circRNAs. Differentially expressed circular RNAs were validated in the endothelial cell model by PCR and real-time quantitative PCR （q-PCR） techniques. Subsequently, in the peripheral blood samples of T2DM patients （n=32） and control individuals （n=28）, the differentially expressed circRNAs were further validated. Finally, t-Test, correlation analysis and ROC curve were used to analyze the experimental results.ResultsTotal 1 087 differentially expressed circular RNAs were screened by high-throughput sequencing; and of these, 554 were up-regulated, 533 were down-regulated. Six increased circRNAs were selected and validated in HUVEC model; and their PCR and q-PCR results matched with sequencing results. Further validation was conducted in peripheral blood samples for three most up-regulated circRNAs. This study found that the differential expression of hsa_circ_0031739 in peripheral blood samples was statistically significant （0.015±0.002 5 vs. 0.006±0.001 3, P=0.005 9〈0.05）. The area under the ROC curve （AUC） was 0.730 and the expression level of hsa_circ_0031739 was positively correlated with blood glucose （GLU） and glycated hemoglobin （GHb） （r=0.31 7, P=0.013 7〈0.05; r=0.348, P=0.006 4〈0.05）.ConclusionHUVECs were commonly utilized as cell models in the study of T2DM, and differentially expressed circular RNA profiles existed afterhigh glucose treatment. The differential expression of hsa_circ_0031739 was significant in both high glucose treated HUVECs and peripheral blood samples of T2DM patients, and the expression level of hsa_circ_0031739 was correlated with GLU and GHb, which has certain diagnostic significance. Therefore, hsa_circ_0031739 may become a new diagnostic biomarker for T2DM and be helpful for the comprehensive diagnosis and mechanism research of T2DM.Key words：