miR-181a和miR-181d-5p对猪前体脂肪细胞成脂分化调控的研究

doi:10.16431/j.cnki.1671-7236.2022.06.020

摘要/Abstract

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），但不影响靶基因突变型双荧光素酶报告载体中的荧光素酶活性。诱导分化结果表明，与未分化脂肪细胞（0 d）相比，miR-181a表达量在分化的第4天显著升高（P<0.05），到第6天达到极显著差异（P<0.01）；miR-181d-5p及GPD2和CREB1基因表达量在分化的第4、6天均极显著升高（P<0.01）。miR-181a和miR-181d-5p的细胞转染试验均得到相似结果，与NC组相比，mimics组miR-181a与miR-181d-5p的表达量均极显著上升（P<0.01），inhibitor组miR-181a与miR-181d-5p表达量均极显著下降（P<0.01）；mimics和siRNA组的GPD2与CREB1基因表达量均极显著下降（P<0.01），inhibitor组GPD2与CREB1基因表达量均极显著上升（P<0.01）；mimics和siRNA组PPARγ和C/EBPα基因的相对表达量均显著或极显著上升（P<0.05；P<0.01），inhibitor组PPARγ和C/EBPα基因的表达量均显著下降（P<0.05）；油红O染色结果表明，与NC组相比，mimics和siRNA组细胞脂滴数目增加，inhibitor组细胞脂滴数目减少。【结论】 miR-181a和miR-181d-5p可以分别靶向结合GPD2和CREB1基因3'-UTR区域序列，降低GPD2和CREB1基因的表达，促进猪前体脂肪细胞成脂分化。

关键词: 荣昌猪; miR-181a; miR-181d-5p; 脂肪细胞

Abstract: 【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 prediction showed that the target genes of miR-181a and miR-181d-5p were mitochondrial glycerol 3-phosphate dehydrogenase (GPD2) and cyclic adenylate response element (CREB1), respectively.GO and KEGG functional enrichment analysis revealed that the target genes of miR-181a and miR-181d-5p could inhibit triglyceride production and regulate adipocyte differentiation, and miR-181a and miR-181d-5p.The free binding energy of both miR-181a and miR-181d-5p with their target genes was less than -87.8 kJ/mol.The results of dual luciferase reporter gene assay showed that mimics transfected with miR-181a and miR-181d-5p could extremely significantly inhibit luciferase activity in the target gene 3'-UTR wild-type dual luciferase reporter vector (P<0.01), but did not affect the target gene mutant type.However, it did not affect the luciferase activity in the target gene mutant dual luciferase reporter vector.The induction of differentiation showed that miR-181a was significantly higher at day 4 (P<0.05) and reached a extremely significant difference at day 6 (P<0.01) compared with undifferentiated adipocytes (0 d).miR-181a and miR-181d-5p, as well as GPD2 and CREB1 genes, were extremely significantly higher at days 4 and 6 of differentiation (P<0.01).miR-181d-5p cell transfection assays both yielded similar results.miR-181a and miR-181d-5p expressions were both extremely significantly increased in mimics group (P<0.01) and miR-181a and miR-181d-5p expressions were both extremely significantly decreased in inhibitor group (P<0.01) compared with the NC group.The expressions of GPD2 and CREB1 genes were extremely significantly decreased in both mimics and siRNA groups (P<0.01), and the expressions of GPD2 and CREB1 genes were extremely significantly increased in inhibitor group (P<0.01).The relative expressions of PPARγ and C/EBPα genes were significantly or extremely significantly increased in both mimics and siRNA groups (P<0.05 or P<0.01), and the relative amounts of PPARγ and C/EBPα genes were significantly decreased in inhibitor group (P<0.05).The results of oil red O staining showed that the number of cellular lipid droplets increased in mimics and siRNA groups and decreased in inhibitor group compared with NC group.【Conclusion】 miR-181a and miR-181d-5p could target and bind GPD2 and CREB1 genes 3'-UTR region sequences, respectively, reduce the expression of GPD2 and CREB1 genes, and promote lipogenic differentiation of porcine precursor adipocytes.

Key words: Rongchang pigs; miR-181a; miR-181d-5p; adipose cell

中图分类号:

S857.4

王梽名, 王宇豪, 顾以韧, 龙科任, 李明洲, 金龙. miR-181a和miR-181d-5p对猪前体脂肪细胞成脂分化调控的研究[J]. 中国畜牧兽医, 2022, 49(6): 2195-2207.

WANG Zhiming, WANG Yuhao, GU Yiren, LONG Keren, LI Mingzhou, JIN Long. Study on Regulation of miR-181a and miR-181d-5p in Porcine Preadipocyte Differentiation[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2195-2207.

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