miR-181a和miR-181d-5p对猪前体脂肪细胞成脂分化调控的研究  

Study on Regulation of miR-181a and miR-181d-5p in Porcine Preadipocyte Differentiation

作　　者：王梽名 王宇豪[1] 顾以韧[2] 龙科任 李明洲[1] 金龙[1] WANG Zhiming;WANG Yuhao;GU Yiren;LONG Keren;LI Mingzhou;JIN Long(College of Animal Science and Technology,Sichuan Agricultural University,Chengdu611130,China;Sichuan Provincial Key Laboratory of Animal Genetics and Breeding,Sichuan Academy of Animal Science,Chengdu 610066,China)

机构地区：[1]四川农业大学动物科技学院,成都611130 [2]四川省畜牧科学研究院动物遗传育种四川省重点实验室,成都610066

出　　处：《中国畜牧兽医》2022年第6期2195-2207,共13页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(U19A2036、32102512);四川省科技计划育种攻关项目(2021YFYZ0009、2021YFYZ0030);雅安市科技计划项目(21SXHZ0022);四川省科技创新人才项目(2022CXRC00x)。

摘　　要：【目的】探究miR-181a和miR-181d-5p在荣昌猪原代前体脂肪细胞分化中的作用及机制。【方法】通过比对种子序列差异分析miR-181a和miR-181d-5p在猪、人、大鼠和小鼠不同物种间的序列保守性;实时荧光定量PCR检测miR-181a和miR-181d-5p在猪肌肉和背部皮下脂肪组织中表达;利用miRandn和TargetScan在线软件预测miR-181a和miR-181d-5p的靶基因,并进行GO和KEGG富集分析;使用RNAhybrid在线软件预测miR-181a和miR-181d-5p与其靶基因的结合自由能。取7日龄雄性荣昌猪背部皮下脂肪组织分离原代前体脂肪细胞。分别设计miR-181a和miR-181d-5p的靶基因野生型和突变型载体,并与对应的miRNA共转染前体脂肪细胞,进行双荧光素酶报告基因试验验证miRNA与靶基因的靶标关系,测定miR-181a和miR-181d-5p与其靶基因的结合情况。构建miR-181a和miR-181d-5p的模拟物(miR-181a mimics、miR-181d-5p mimics)、抑制物(miR-181a inhibitor、miR-181d-5p inhibitor)、阴性对照(NC)及其靶基因的干扰siRNA(Gene-siRNA),转染细胞6 h后进行诱导分化。6 d后,分别对不同处理的细胞通过实时荧光定量PCR检测基因表达情况,油红O染色鉴定甘油三酯生成情况。【结果】保守性分析结果表明,miR-181a和miR-181d-5p在猪、人、大鼠和小鼠间具有高度序列保守性;miR-181a与miR-181d-5p在猪不同组织中定量结果显示,miR-181a与miR-181d-5p在猪脂肪组织中特异性高表达;靶基因预测结果表明,miR-181a与miR-181d-5p的靶基因分别为线粒体甘油3-磷酸脱氢酶(GPD2)和环腺苷酸反应元件(CREB1);GO和KEGG功能富集分析发现,miR-181a和miR-181d-5p的靶基因可以抑制甘油三酯生成,调节脂肪细胞分化,且miR-181a和miR-181d-5p与其靶基因的自由结合能均<-87.8 kJ/mol;双荧光素酶报告基因试验结果表明,转染miR-181a与miR-181d-5p的mimics可以极显著抑制靶基因3′-UTR野生型双荧光素酶报告载体中的荧光素酶活性(P<0.01),但不影响靶�【Objective】The aim of this study was to investigate the role and mechanism of miR-181a and miR-181d-5p in the differentiation of primary precursor adipocytes in Rongchang pigs.【Method】The sequence conservation of miR-181a and miR-181d-5p among different species of pig,human,rat and mouse was analyzed by comparing seed sequence differences.miR-181a and miR-181d-5p expression in pig muscle and subcutaneous adipose tissue was detected by Real-time quantitative PCR.miRandn and TargetScan online softwares were used to predict miR-181a and miR-181d-5p target genes with GO and KEGG enrichment analysis.The free energy of miR-181a and miR-181d-5p binding to their target genes was predicted using RNAhybrid online software.Primary precursor adipocytes were isolated from subcutaneous adipose tissue on the back of 7-day-old male Rongchang pigs.Wild-type and mutant vectors of miR-181a and miR-181d-5p target genes were designed and cotransfected with the corresponding miRNAs in the precursor adipocytes.A dual luciferase reporter gene assay was performed to verify the targeting relationship between miRNA and target genes,and the binding of miR-181a and miR-181d-5p to their target genes was measured.miR-181a and miR-181d-5p mimics(miR-181a mimics,miR-181d-5p mimics),inhibitor(miR-181a inhibitor,miR-181d-5p inhibitor),negative control(NC)and interfering siRNAs of their target genes(Gene-siRNA)were constructed,transfected the cells.The transfected cells were induced to differentiate 6 h later.After 6 d,gene expression and triglyceride production were determined by Real-time quantitative PCR,oil red O staining was used to detect triglyceride in the treated cells,respectively.【Result】The results of conservativeness analysis showed that miR-181a and miR-181d-5p were highly sequence conserved among pig,human,rat and mouse.miR-181a and miR-181d-5p were quantified in different tissues of pigs,which showed that miR-181a and miR-181d-5p were specifically highly expressed in porcine adipose tissues.The results of target gene pre

关 键 词：荣昌猪 miR-181a miR-181d-5p 脂肪细胞 

分 类 号：S857.4[农业科学—临床兽医学]