摘要
目的:探讨冠心病(CAD)患者心外膜脂肪组织(EAT)和血浆中脂联素(APN,又称ADIPOQ相关差异表达miR-371b-5p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)分泌的影响,阐明miR-371b-5p在CAD中的作用机制。方法:收集CAD (CAD组,n=34)和非CAD (non-CAD组,n=16)患者的EAT和血液标本,采用实时荧光定量PCR (qRT-PCR)法检测2组患者EAT和血浆中miR-371b-5p的表达水平。将诱导成功的3T3-L1前体脂肪细胞分为空白对照组(不做任何处理)、miR-371b-5p过表达组(转染miR-371b-5p模拟物)和miR-371b-5p抑制剂组(转染miR-371b-5p抑制剂)。qRT-PCR法检测转染24h后各组3T3-L1前脂肪细胞中APN、KLF4、IL-6和MCP-1mRNA表达水平,并对miR-371b-5p进行生物信息学分析。结果:与non-CAD组比较,CAD组患者EAT和血浆中miR-371b-5p表达水平均升高(P<0.01)。与空白对照组比较,miR-371b-5p过表达组3T3-L1前脂肪细胞中APN和KLF4mRNA表达水平降低(P=0.043,P=0.045),IL-6和MCP-1mRNA表达水平升高(P=0.037,P=0.041);miR-371b-5p抑制剂组3T3-L1前脂肪细胞中APN和KLF4mRNA表达水平升高(P=0.025,P=0.027),IL-6和MCP-1mRNA表达水平降低(P=0.039,P=0.041)。miR-371b-5p预测靶基因富集于多个生物学功能及过程,KEGG通路主要富集于脂肪细胞因子等信号通路;蛋白互作,miR-371b-5p预测靶基因APN、瘦素(LEP)和AKT1编码蛋白位于互作网络中核心位置。结论:CAD患者EAT中过表达miR-371b-5p转染成熟的3T3-L1脂肪细胞后可使细胞的抗炎因子表达水平降低,炎性因子表达水平升高,miR-371b-5p有望成为CAD治疗的新靶点。
Objective:To investigate the effects of adiponectin(APN,also known as ADIPOQ)-related miR-371 b-5 p with differential expressions in the epicardial adipose tissue(EAT)and plasma in the patients with coronary artery disease(CAD)on the secretions of APN,Kruppel-like factor 4(KLF4),interleukin-6(IL-6)and monocyte chemokine 1(MCP-1),and to elucidate the mechanism of action of miR-371 b-5 p in CAD.Methods:The EAT and blood samples were collected from the patients with CAD(CAD group,n=34)and non-CAD(non-CAD group,n=16).Real-time quantitative PCR(qRT-PCR)was used to detect the expression levels of miR-371 b-5 p in the EAT and plasma of the patients in two groups.The successfully induced 3 T3-L1 preadipocytes were divided into(without any treatment),miR-371 b-5 p over-expression group(transfected with miR-371 b-5 p mimics)and miR-371 b-5 p inhibitor group(transfected with miR-371 b-5 p inhibitor).The relative expression levels of APN,KLF4,IL-6 and MCP-1 mRNA in the 3 T3-L1 preadipocytes were detected by qRT-PCR method.The miR-371 b-5 p was analyzed by bioinformatics.Results:The expression levels of miR-371 b-5 p in the EAT and plasma of the patients in CAD group were higher than those in non-CAD group(P<0.01).Compared with blank control group,the expression levels of APN and KLF4 mRNA in 3 T3-L1 preadipocytes in miR-371 b-5 p over-expression group were decreased(P=0.043,P=0.045),and the expression levels of IL-6 and MCP-1 mRNA in 3 T3-L1 preadipocytes in miR-371 b-5 p over-expression group were increased(P=0.037,P=0.041).Compared with blank control group,the expression levels of APN and KLF4 in 3 T3-L1 preadipocytes in miR-371 b-5 p inhibitor group were increased(P=0.025,P=0.027),and the expression levels of IL-6 and MCP-1 mRNA in 3 T3-L1 preadipocytes in miR-371 b-5 p inhibitor group were decreased(P=0.039,P=0.041).The target genes predicted by miR-371 b-5 p were enriched in multiple biological functions and processes.The KEGG pathway was mainly enriched in the signaling pathways such as adipocytokines.The protein interaction results showed that the target genes APN,leptin(LEP)and AKT1 encoded proteins predicted by miR-371 b-5 p were located at the core of the interaction network.Conclusion:The expression levels of anti-inflammatory factors in the cells with over-expression of miR-371 b-5 p in EAT of the patients with CAD are decreased,the expression levels of inflammatory factors are increased,and miR-371 b-5 p is expected to become a new target for CAD treatment.
引文
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