长链非编码RNA MIAT在2型糖尿病合并冠心病患者血清中的水平及对高糖诱导的心肌细胞损伤的影响  

Serum levels of long non-coding RNA MIAT in patients with type 2 diabetes mellitus and coronary heart disease and its effect on high glucose-induced cardiomyocyte injury

作　　者：孟宪杰[1] 刘蕊[2] 曹丽慧[3] 马宁 刘肖 MENG Xianjie;LIU Rui;CAO Lihui;MA Ning;LIU Xiao(Department of Endocrinology,Tangshan People's Hospital,Tangshan,Hebei 063000,China;Department of Emergency,Tangshan People's Hospital,Tangshan,Hebei 063000,China;Department of Cardiology,Tangshan People's Hospital,Tangshan,Hebei 063000,China)

机构地区：[1]唐山市人民医院内分泌科,河北省唐山市063000 [2]唐山市人民医院急诊科,河北省唐山市063000 [3]唐山市人民医院心内科,河北省唐山市063000

出　　处：《中国动脉硬化杂志》2023年第6期481-490,共10页Chinese Journal of Arteriosclerosis

基　　金：河北省医学科学研究课题计划(20221812)。

摘　　要：[目的]探讨长链非编码RNA心肌梗死相关转录本(lncRNA MIAT)在2型糖尿病(T2DM)合并冠心病(CHD)患者中的血清水平及其对高糖(HG)诱导的心肌细胞损伤的影响。[方法]选取2021年6月—12月于唐山市人民医院就诊的100例单纯T2DM患者作为T2DM组,100例单纯冠心病(CHD)患者作为CHD组,100例T2DM合并CHD患者作为T2DM+CHD组,另外选取100例健康人群作为对照组。采用实时荧光定量PCR(RT-qPCR)检测血液MIAT、microRNA-150-5p(miR-150-5p)的水平。体外培养人心肌细胞系AC16细胞,分为NG组(5.5 mmol/L正常葡萄糖)、HG组(30 mmol/L高葡萄糖)、HG+MIAT敲低阴性对照(HG+si-NC)组、HG+MIAT敲低(HG+si-MIAT)组、HG+si-MIAT+miR-150-5p抑制剂阴性对照(HG+si-MIAT+anti-NC)组、HG+si-MIAT+miR-150-5p抑制剂(HG+si-MIAT+anti-miR-150-5p)组,RT-qPCR检测细胞中MIAT和miR-150-5p的表达情况;MTT法检测细胞增殖活性;流式细胞术检测细胞凋亡;ELISA法检测乳酸脱氢酶(LDH)含量,Western blot检测细胞周期蛋白D2(CCND2)、B淋巴细胞瘤2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)和半胱氨酸蛋白酶3(Caspase-3)、剪切的Caspase-3(cleaved Caspase-3)的蛋白表达,双荧光素酶报告基因、RNA结合蛋白免疫沉淀(RIP)和RNA pull down分析miR-150-5p与MIAT、CCND2的靶向关系。[结果]与对照组、CHD组、T2DM组相比,T2DM+CHD组MIAT表达水平显著升高,分别增加了2.69倍、1.71倍、1.42倍(均P<0.05),miR-150-5p的表达显著降低,分别降低了68.63%、60.49%、46.67%(均P<0.05);相关性分析结果显示,T2DM+CHD患者中MIAT与miR-150-5p的表达水平呈负相关(r=-0.662,P<0.001)。与NG组相比,HG组AC16细胞中MIAT的表达增加了3.54倍,细胞凋亡率、LDH含量、Bax蛋白水平和cleaved Caspase-3/Caspase-3比值分别增加了5.22倍、2.19倍、2.90倍和3.83倍,miR-150-5p表达降低了75.00%,细胞增殖活性在24、48、72 h时分别降低了49.02%、52.38%、49.48%,CCND2和Bcl-2蛋白水平分别降低了72.62%和78.26%(均P<0.05);敲低MIAT使mAim To investigate the serum level of long non-coding RNA myocardial infarction-associated transcript(lncRNA MIAT)in patients with type 2 diabetes mellitus(T2DM)and coronary heart disease(CHD)and its effect on high glucose(HG)-induced myocardial cell injury.Methods From June 2021 to December 2021,100 patients with uncomplicated T2DM who visited Tangshan People's Hospital were regarded as the T2DM group,100 patients with uncomplicated coronary heart disease(CHD)were regarded as the CHD group,and 100 T2DM patients with CHD were selected as T2DM+CHD group,in addition,100 healthy people were regarded as the control group.Real-time quantitative PCR(RT-qPCR)was applied to detect the levels of blood MIAT and microRNA-150-5p(miR-150-5p).The human cardiomyocyte line AC16 cells were cultured in vitro and grouped into NG group(5.5 mmol/L normal glucose),HG group(30 mmol/L high glucose),HG+MIAT knockdown negative control(HG+si-NC)group,HG+MIAT knockdown(HG+si-MIAT)group,HG+si-MIAT+miR-150-5p inhibitor negative control(HG+si-MIAT+anti-NC)group,and HG+si-MIAT+miR-150-5p inhibitor(HG+si-MIAT+anti-miR-150-5p)group.RT-qPCR was performed to detect the expression of MIAT and miR-150-5p in cells;MTT assay was performed to detect cell proliferation viability;Flow cytometry was performed to detect apoptosis;ELISA method was implemented to detect lactate dehydrogenase(LDH)content;Western blot was performed to detect protein expressions of cyclin D2(CCND2),B cell-lymphoma-2 gene(Bcl-2),Bcl-2-associated X protein(Bax),and cysteine protease-3(Caspase-3),cleaved Caspase-3;Dual-luciferase reporter,RNA-binding protein immunoprecipitation(RIP)and RNA pull down were performed to analyze the targeting relationship of miR-150-5p to MIAT and CCND2.Results Compared with control group,CHD group and T2DM group,the expression level of MIAT was obviously increased in T2DM+CHD group by 2.69 times,1.71 times and 1.42 times(P<0.05),and the expression of miR-150-5p was obviously decreased by 68.63%,60.49%and 46.67%(P<0.05).Correlation analysis showed that

关 键 词：冠心病 2型糖尿病 心肌梗死相关转录本 microRNA-150-5p 心肌细胞 

分 类 号：R5[医药卫生—内科学]