miR-146b-3p通过RAF1/p38MAPK/COX-2信号通路影响糖尿病发生脑梗死的机制研究

The Effect of miR-146b-3p on Cerebral Infarction in Patients with Diabetes through RAF1/p38MAPK/COX-2 Signaling Pathway

目的探讨miR-146b-3p通过RAF1/p38MAPK/环氧化酶-2(COX-2)信号通路影响糖尿病脑梗死的发生机制。方法选取我院2015年4月—2017年12月210例糖尿病病人为研究对象,通过随访研究调查病人发生血栓情况,有15例患有脑梗死,作为伴有糖尿病的脑梗死(DMCI)组,另选取15例无脑梗死病人为对照组。采用酶联免疫吸附法(ELISA)测定两组病人白介素-6(IL-6)、COX-2含量;采用聚合酶链式反应(PCR)及免疫印迹方法检测相关基因的mRNA及蛋白表达。结果两组一般资料及相关指标比较差异无统计学意义(P>0.05);与对照组相比,DMCI组血中IL-6和COX-2表达升高,但存在一定的局限性;IL-6诱导外周血单核细胞2 h后,COX-2的mRNA和蛋白表达明显增加,COX-2表达受IL-6调控;与对照组相比,miR-146b-3p mRNA在DMCI组表达低于正常组(0.16±0.07与1.12±0.16,P<0.001);与对照组相比,DMCI组RAF1 mRNA的表达增加(P<0.01),且其蛋白表达也增加;DMCI组中COX-2、p-p38MAPK表达增加,p38MAPK蛋白表达减少,说明DMCI病人激活了p38MAPK的信号通路;当用10 ng/mL的IL-6处理外周血单核细胞(PBMC)时,可激活STAT3磷酸化,且当IL-6处理PBMC 1 h后,miR-146b-3p mRNA表达下降,当IL-6处理12 h后,发现miR-146b-3p mRNA在IL-6处理12 h时(0.20±0.12)表达低于对照组(1.02±0.15,P<0.05),还发现IL-6处理中COX-2的mRNA(4.98±0.69)和蛋白质表达高于对照组;DMCI组中miR-146a mRNA表达降低,与miR-146b的表达趋势一致。结论miR-146b-3p的表达通过介导RAF/p38MAPK/COX-2信号通路与糖尿病病人脑梗死的发展密切相关,miR-146b-3p可作为糖尿病脑梗死病人检测和干预的新靶点。

Objective To observe the effect of miR-146b-3p on cerebral infarction in patients with diabetes through RAF1/p38MAPK/COX-2 signaling pathway.Methods Two hundred and ten patients with diabetes mellitus were selected.Based on follow-up studies,15 patients with cerebral infarction were selected as the study subjects(DMCI group),and the other 15 patients were selected as the control group.The levels of interleukin-6(IL-6)and cyclooxygenase-2(COX-2)were detected by ELISA.The mRNA and protein expressions of related genes were detected by PCR and Western Blotting.Results There was no statistical difference in general data and relevant indicators between two groups.Compared with the control group,the levels of IL-6 and COX-2 of patients in the DMCI group significantly increased,but there were certain limitations.The mRNA and protein expressions of COX-2 in peripheral blood mononuclear cells induced by IL-6 increased significantly after 2 hours,and the expression of COX-2 was regulated by IL-6.miR-146b-3p expression of patients in the DMCI group was significantly lower than that in the control group(0.16±0.07 vs 1.12±0.16,P<0.001).RAF1mRNA expression of patients in the DMCI group was significantly higher than that in the control group(P<0.01),and protein expression of RAF1 increased(P<0.001).The expressions of COX-2 and p-p38MAPK in the DMCI group increased,while the expression of p38MAPK protein was decreased,indicating that DMCI patients activated the p38MAPK signaling pathway.STAT3 phosphorylation was significantly activated when peripheral blood mononuclear cells(PBMC)were treated with 10 ng/mL IL-6.The expression of miR-146b-3p decreased when IL-6 treated with PBMC for 1 h.After 12 h of IL-6 treatment,the expression of miR-146b-3p in the 12 h IL-6 group(0.20±0.12)was lower than that in the control group(1.02±0.15,P<0.05).The expression of COX-2 mRNA(4.98±0.69)and protein in the IL-6 group were higher than those in the control group.The expression of miR-146a decreased in the DMCI group,which was consistent with the expression trend of miR-146b.Conclusion The expression of miR-146b-3p closely related to the development of cerebral infarction in patients with diabetes by mediating RAF/p38MAPK/COX-2 signaling pathway,and miR-146b-3p might be used as a new target for detection and intervention in diabetic patients with cerebral infarction.