应激相关microRNA在2型糖尿病及糖尿病前期患者中的表达规律及意义研究

摘要

目的 探索应激相关microRNA(miRNAs)在2型糖尿病(T2DM)及糖尿病前期患者中的表达规律及意义.方法 从2016年5-9月于首都医科大学宣武医院体检的职业人群中,选取新发T2DM患者70例为病例组,空腹血糖受损(IFG)患者42例为糖尿病前期组,按性别、年龄(±3岁)的群体匹配方法选择血糖、糖化血红蛋白以及血脂均正常者53例为对照组.收集研究对象一般资料,并检测其应激相关激素水平.从T2DM患者与健康对照者前期芯片数据中显示的差异表达的miRNAs中,采用生物信息学方法筛选出应激相关miRNAs(let-7b、let-7i、miR-142、miR-144、miR-155和miR-29a).采用实时荧光定量PCR法检测受试者应激相关miRNAs表达水平.结果 糖尿病前期组促肾上腺皮质激素释放激素(CRH)、皮质醇(Cor)水平高于对照组(P<0.05);病例组CRH、白介素6(IL-6)、血浆肾上腺素(E)、去甲肾上腺素(NE)、Cor水平高于对照组(P<0.05);病例组Cor水平高于糖尿病前期组(P<0.05).糖尿病前期组、病例组let-7b、miR-144、miR-29a表达水平高于对照组,miR-142表达水平低于对照组(P<0.05);病例组let-7b表达水平高于糖尿病前期组(P<0.05).所有研究对象CRH水平与let-7b、miR-144表达水平呈正相关,与miR-142表达水平呈负相关(P<0.05);所有研究对象IL-6、E、NE水平与let-7b表达水平呈正相关,与miR-142表达水平呈负相关(P<0.05);所有研究对象Cor水平与let-7b、miR-144、miR-29a表达水平呈正相关,与miR-142表达水平呈负相关(P<0.05).病例组CRH、E、NE水平与let-7b表达水平呈正相关(P<0.05);病例组IL-6水平与miR-142表达水平呈负相关(P<0.05);病例组Cor水平与miR-144、miR-29a表达水平呈正相关(P<0.05).多因素Logistic回归分析结果显示,let-7b表达水平升高能增加T2DM患病风险,miR-142表达水平升高能降低T2DM患病风险,miR-144、miR-29a表达水平升高能增加IFG患病风险(P<0.05).let-7b、miR-142、miR-144联合诊断T2DM的ROC曲线下面积(AUC)为0.880,95%CI(0.820,0.941),临界值为0.626,灵敏度为78.6%,特异度为84.9%,交叉验证平均准确率为0.853.SBP、DBP、BMI、WHR、TC、TG、HDL-C、LDL-C、let-7b、miR-142、miR-144联合诊断T2DM的AUC为0.937,95%CI(0.897,0.977),临界值为0.680,灵敏度为82.9%,特异度为94.3%,交叉验证平均准确率为0.902.结论 与健康者相比,糖尿病前期及T2DM患者血浆let-7b、miR-144表达水平上调,而血浆miR-142表达水平则下调;另外,let-7b、miR-142和miR-144可能通过调节应激反应参与糖尿病前期乃至T2DM的发生.%Objective To explore the expression pattern and significance of stress-related microRNA (miRNAs) in patients with type 2 diabetes (T2DM) and pre-diabetes.Methods From the occupational populations undergoing physical examination at Xuanwu Hospital,Capital Medical University during May to September 2016,we recruited 70 with new-onset T2DM (case group),42 with impaired fasting glucose (IFG) (pre-diabetes group),and also enrolled sex-matched and age-matched (within ±3 years range) 53 controls with normal fasting plasma glucose,glycosylated hemoglobin and blood lipids based on group-matching design.We collected their baseline characteristics and detected the level of stress-related hormones.Among the T2DM patients' significantly different expressed plasma stress-related miRNAs identified by miRNAs microarray analysis,we screened 7 plasma stress-related miRNAs (let-7b,let-7i,miR-142,miR-144,miR-155 and miR-29a) by bioinformatics analysis.Furthermore,we used qRT-PCR to detect the expression levels of these 7 miRNAs in plasma.Results Compared with the controls,both pre-diabetic and T2DM patients showed higher average levels of corticotropin-releasing hormone (CRH) and cortisol (Cor) (P<0.05),moreover,T2DM patients also presented higher average levels of interleukin-6 (IL-6),epinephrine (E),and norepinephrine (NE) (P<0.05).The average Cor level was higher in the T2DM cases than that of the pre-diabetic cases (P<0.05).Compared with the controls,the average plasma let-7b,miR-144 and miR-29a levels were significantly higher but average miR-142 level was lower in pre-diabetic and T2DM patients (P<0.05).The average plasma let-7b level was higher in T2DM patients than that in pre-diabetic patients (P<0.05). Among all the participants,plasma CRH was positively correlated with let-7b and miR-144 (P<0.05);plasma IL-6,E and NE were positively correlated with let-7b (P<0.05);plasma Cor was positively correlated with let-7b,miR-144 and miR-29 (P<0.05).Moreover,all of stress-related hormones in our study were negatively correlated with miR-142.In case group,plasm CRH,E and NE showed significant positive correlation with let-7b (P<0.05);IL-6 was negatively correlated with miR-142 (P<0.05);and Cor was positively correlated with miR-144 and miR-29a (P<0.05).Multivariate Logistic regression analysis revealed that up-regulated let-7b conferred an increased risk of future T2DM while elevated miR-142 was contributive to reducing the risk of T2DM (P<0.05);up-regulated miR-144 and miR-29a were risk factors for IFG (P<0.05).The miRNAs panel of let-7b,miR-142 and miR-144 distinguished T2DM patients from the healthy controls showed an area under the curve (AUC) of 0.880 [95%CI (0.820,0.941)],the cut-off point was 0.626 with corresponding sensitivity and specificity of 78.6% and 84.9%,respectively.The cross-validation showed that the average accuracy was 0.853.The federal panel of SBP,DBP,BMI, WHR,TC,TG,HDL-C,LDL-C,let-7b,miR-142 and miR-144 presented an AUC of 0.937 [95%CI (0.897,0.977)], the cut-off point was 0.680 with sensitivity and specificity of 82.9% and 94.3%,respectively.The cross-validation showed that the average accuracy was 0.902.Conclusion Compared with the controls,plasma let-7b and miR-144 levels were up-regulated but miR-142 level was down-regulated in pre-diabetic and T2DM patients,suggesting that let-7b,miR-142 and miR-144 may involve in the pathogenesis of pre-diabetes as well as T2DM by adjusting the stress reaction.