Screening Of Serum MicroRNA Expression Profiles In Patients With Type 2 Diabetes Mellitus And Microvascular Complications And Its Clinical Value

Objectives:To determine the altered serum microRNA (miRNA) expression profile of type 2 diabetes patients with or without microvascular complications, and further evaluate its diagnostic,discriminative and monitoring value for these diseases.Methods:87 patients with T2DM and 87 patients with T2DMC who were hospitalized in the Department of Endocrine of Nanjing Military Region General Hospital during the period from September 2013 to May 2014 were enrolled in the present study. All the patients were diagnosed as newly onset T2DM or have diagnosed as T2DM but had not received any treatment within one month. In the meanwhile,87 ages and genders matched healthy subjects who were seeking for health examination at the physical examination center of the hospital were also recruited as normal controls. Firstly, high-throughput and high sensitivity TaqMan Low Density Array (TLDA) were applied to examine the genome-wide expression profile of 768 known miRNA in three pooled serum formed from 25 patients with T2DM,25 patients with T2DMC and 25 healthy control, respectively.Then a number of dysregulated serum miRNA in T2DM and T2DMC from TLDA combined with some reported miRNA from literature searching were selected and subsequently confirmed in individual samples in the same cohorts of TLDA (referred as training set) using an accurate quantitative real-time polymerase chain reaction technology (qRT-PCR); Next, the altered serum miRNAs from training set were further validated in another three groups samples including 62 patients with T2DM,62 patients with T2DMC, and 62 healthy controls (referred validation set). In the meanwhile, the enzyme-linked immunosorbent assay (ELISA) was applied to measure the sera levels of the oxidized low-density lipoprotein (ox-LDL), oxidized lipoprotein (a)[ox-Lp(a)], β2-glycoprotein oxidized low density lipoprotein compound(β2-GPI-ox-LDL) and β2-glycoprotein lipoprotein (a) compound[β2-GPI-Lp(a)] in the samples of all the patients and controls. Spearman rank correlation analyses were conducted to analyze the association between the altered serum miRNA the aforementioned lipoprotein and clinical indexes. Logistic regression analyses were applied to examine whether the altered miRNAs were independent risk factors for T2DM and T2DMC.Results:The TLDA results showed that the serum miRNA expression profiles of the T2DM and T2DMC patients were obviuosly different from the healthy control. Following analysis identified 53 upregulated miRNAs and 117 downregulated miRNA in the patients with T2DM and T2DMC when compared with controls.Based on the primary screening of TLDA and literature searching,12 miRNAs were then selected and confirmed in the individual samples in the same cohorts that used in TLDA screening (training set) by using qRT-PCR. The qRT-PCR data showed 8 miRNAs including miR-571, miR-661, miR-1303, miR-152, miR-1323, miR-892b, miR-770-5p and miR-886-3p were significantly elevated in the two patients’group, and furthermore, the alterations were more significant in T2DMC patients (the change> 2-fold, P< 0.05). The changing tendency of the 8 miRNAs in the validation set were consistence with that in the training set (the change>2-fold, P< 0.05). Following ROC curve analysis showed that the area under the ROC curve (AUC) of 8 serum miRNAs were ranged from 0.774 to 0.877 for diagnosing T2DM, ranged from 0.823 to 0.946 for diagnosing T2DMC, and ranged from 0.611 to 0.675 for discriminating T2DM and T2DMC, respectively. Based on the optimal cutoff, the sensitivity and specialty of 8 serum miRNAs for the diagnosis of T2DM ranged from 68.2% to 88.1% and from 63.0% to 84.5%, respectively; those for the diagnosis of T2DMC ranged from 64.8% to 84.7% and from 80.6% to 98.6%, respectively; In addition, three miRNAs were found to have the ability for the differential diagnosis of T2DM and T2DMC, and the AUC, sensitivity, and specificity for these miRNAs are: miR-886-3p (AUC= 0.675, sensitivity= 0.651, specificity= 0.628), miR-892b (AUC = 0.639, sensitivity= 0.662, specificity= 0.622) and miR-571 (AUC= 0.611, sensitivity= 0.603, and specificity= 0.608), respectively. ELISA measurement showed the levels of ox-LDL, ox-Lp(a),β2-GPI-ox-LDL and β2-GPI-Lp(a) of the T2DM and T2DMC patients were also significantly higher than those in the healthy controls (P< 0.05).Spearmanrank rank correlation analysis found that the 8 miRNA were significantly associated with fasting blood-glucose (FPG), ox-LDL and ox-Lp(a) (P<0.05). Univariate logistic regression analysis revealed that the 8 serum miRNA as well as oxidized lipoprotein and its compounds were the independent risk factors for predicting the occurrence of the patients with T2DM and T2DMC.Additional multivariate logistic regression further confirmed that miR-770, miR-661 combined serum triglycerides are still the independent risk factors after adjusted by age and gender.Conclusion:8 upregulated miRNAs including miR-571,miR-661,miR-1303, miR-152,miR-1323, miR-892b, miR-770-5p, and miR-886-3p were identified in the serums of T2DM and T2DMC patients and may have the potential to be novel auxiliary diagnosis and differential diagnosis biomarkers for T2DM and T2DMC.