Hsa-circ-000094 In Peripheral Blood Can Be Used As A Biomarker For The Diagnosis Of Type 2 Diabetes

Purpose:To explore the differential expression of circular RNA（circRNA）in peripheral blood of patients with type 2 diabetes mellitus（T2DM）and find the diagnostic marker of T2DM,the mechanism of action of circular RNA and downstream miRNA was explored.Methods:Microarray analysis was used to select several differentially expressed circRNAs from three normal patients and three T2DM patients.Enlarge the sample size（normal control,n=20;irregular glucose regulation,n=20;type 2 diabetes mellitus,n=20）to determine a circRNA which the most obvious differentially expressed by fluorescence quantitative PCR（Q-PCR）,then verified with widen sample（normal control,n=50;irregular glucose regulation,n=50;type 2 diabetes mellitus,n=50）by Q-PCR.According to bioinformatics,miRNAs with downstream expression of the most differentially expressed circular RNA were predicted,and a dual luciferase reporter gene experiment was used to construct a cloning vector containing the dual luciferase gene and the target gene,which was co-transformed with the miRNA mimic.The 293T cells were stained,and the binding activity of the circular RNA to the miRNA was determined by observing the fluorescence activity.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 1439 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,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 in the three groups,the area under the maximum curve was found by ROC curve analysis,S_IGR=0.8025（95%confidence interval[0.6655-0.9395],p=0.001）;S_T2DM=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,S_IGR=0.6733（95%confidence interval[0.5757-0.7710],P<0.001）;S_T2DM=0.7231（95%confidence interval[0.6327-0.8134],P<0.001）.Based on bioinformatics predictions,we screened five miRNAs most closely related to Hsa-circ-000094（hsa-miR-98-5p,hsa-let-7f-5p,hsa-miR-370-5p,hsa）-let-7e-5p,hsa-let-7a-5p).The results of the dual luciferase reporter gene showed that the expression of Hsa-circ-000094 and hsa-miR-370-5p were decreased after transfection into 293T cells,and the fluorescence activity was decreased by about 40%（P<0.0006）.After co-transfection into hb-let-7e-5p into 293T cells,the fluorescence activity decreased by about 20%（P<0.0018）.According to the results,Hsa-circ-000094had better binding to hsa-miR-370-5p effectively.Conclusion:The high expression of hsa-circ-000094 in peripheral blood provided a certain diagnostic basis for pre-diabetes and type 2 diabetes mellitus.Hsa-circ-000094could inhibit the activity of miR-370 by sponge adsorption,and affect the synthesis of lipid metabolism,which affected the development of diabetes.