冠心病EAT中miR-455b-3p调控脂肪细胞分化及炎症因子

miR-455b-3p regulates adipocyte differentiation and inflammatory factors in coronary heart disease EAT

目的筛选在冠状动脉粥样硬化性心脏病(CAD)患者和非冠心病(non-CAD)患者心外膜脂肪组织(EAT)中差异性表达的microRNAs(DE-miRNAs),探讨其中miR-455b-3p对脂肪细胞因子APN、Kruppel样因子4(KLF4)、白细胞介素6(IL-6)和单核细胞因子趋化蛋白1(MCP-1)分泌及对脂肪细胞分化的影响。方法收集冠心病(CAD)34例和非冠心病(non-CAD)16例EAT和血液标本,分别为实验组(CAD组)和对照组(Non-CAD组),采用miRNA芯片技术筛选出EAT中DE-miRNAs,采用实时荧光定量PCR(qRT-PCR)检测标本中miR-455b-3p的表达水平。分别将miR-455b-3p的mimics、miR-455b-3p的inhibitors和NC转染入前体3T3-L1细胞及已诱导成熟的3T3-L1细胞,分为上调组、下调组和NC组。采用qRT-PCR法检测转染24 h后细胞中APN、KLF4、IL-6、MCP-1 mRNA及脂肪细胞分化标志物过氧化物酶体增殖物激活受体-γ(PPARγ)、CCAAT/增强子结合蛋白α(C/EBPα)、脂肪酸结合蛋白4(FABP4)mRNA的相对表达水平。结果与实验组比较,对照组EAT标本中共筛选出29个DE-miRNAs(P<0.05;差异倍数>2,差异有统计学意义),其中下调10个,上调19个。miR-455b-3p是与ADIPOQ 3’-UTR有互补结合位点或密切相关的1条DE-miRNA。qRT-PCR验证结果显示,与对照组比较,miR-455b-3p在实验组中的表达均升高。与NC组比较,转染miR-455b-3p的mimics后,上调组成熟3T3-L1脂肪细胞中APN、KLF4 mRNA表达减少,IL-6、MCP-1 mRNA表达增多;脂肪细胞分化标志物PPARγ、C/EBPα、FABP4 mRNA表达升高;转染miR-455b-3p inhibitor后,下调组APN、KLF4 mRNA表达水平升高,IL-6、MCP-1 mRNA表达减少。结论在CAD组患者EAT中存在差异性表达的miRNAs,其中miR-455b-3p高表达,miR-455b-3p影响脂肪细胞因子APN、KLF4、IL-6以及MCP-1的表达,并且促进脂肪细胞分化成熟,从而影响冠心病的发生发展。

Objective To investigate the differential expression of microRNAs(DE-miRNAs)in patients with coronary atherosclerotic heart disease(CAD)and non-CAD,in which miR-455b-3p chemotaxis of adipocytokines adiponectin(APN),Kruppel-like factor 4(KLF4),interleukin-6(IL-6)and monocyte cytokines protein 1(MCP-1)secretion and its effects on adipocyte differentiation.Methods Epicardial adipose tissue(EAT)and blood samples were collected from CAD(34 cases)and non-CAD(16 cases)patients.DE-miRNAs in EAT were screened by miRNA microarray technique.Real-time quantitative PCR(qRT-PCR)was used to detect miR-455b-3p in specimens.The expression level of miR-455b-3p mimics,miR-455b-3p inhibitors and NC were transfected into precursor.The expression of APN,KLF4,IL-6,MCP-1 mRNA and adipocyte differentiation markers peroxisome proliferator-activated receptor-gamma(PPARγ)and CCAAT/enhancer binding proteinα(C/EBPα)and fatty acid binding protein 4(FABP4)mRNA were detected by qRT-PCR after 24 h of transfection.Results Compared with the non-CAD group,29 DE-miRNAs were screened out in the EAT specimens(P<0.05,difference multiples>2),of which 10 were down-regulated and 19 were up-regulated.miR-455b-3p was a DE-miRNA that had a complementary binding site or was closely related to the ADIPOQ 3'-UTR.The qRT-PCR results showed that the expression of miR-455b-3p was elevated in CAD patients compared with the non-CAD group.Compared with the miR-NC group,after transfection of miRs of miR-455b-3p,the expression of APN and KLF4 mRNA was decreased and the expression of IL-6 and MCP-1 mRNA was increased in mature 3T3-L1 adipocytes;After transfected miR-455b-3p inhibitor,APN and KLF4 mRNA expression levels increased,IL-6 and MCP-1 mRNA expression decreased;adipocyte differentiation markers PPARγ,C/EBPα,FABP4 mRNA expression increased.Conclusion There are differentially expressed miRNAs in EAT of CAD patients,in which miR-455b-3p is highly expressed.miR-455b-3p affects the expression of adipocytokines APN,KLF4,IL-6 and MCP-1,and promotes adipocyte differentiation and mature,thereby affecting the occurrence and development of coronary heart disease.