miR-137-3p靶向MAX基因对前脂肪细胞3T3-L1成脂分化的影响

doi:10.16431/j.cnki.1671-7236.2023.05.020

摘要/Abstract

摘要： 【目的】研究miR-137-3p在前脂肪细胞(3T3-L1)分化过程中的功能及作用机制。【方法】收集诱导分化第0、2、4、6和8天的3T3-L1细胞,利用实时荧光定量PCR检测miR-137-3p相对表达量;将miR-137-3p的模拟物(mimics)、抑制物(inhibitor)和阴性对照(NC)分别转染3T3-L1细胞并诱导成脂分化,油红O染色观察脂滴形成情况,利用实时荧光定量PCR检测成脂标志基因CAAT增强子结合蛋白α(C/EBPα)、C/EBPβ、过氧化物酶体增殖物激活受体γ(PPARγ)和脂肪酸结合蛋白4(FABP4)相对表达量,用Western blotting检测C/EBPβ、PPARγ和FABP4蛋白表达水平;利用TargetScan在线网站预测miR-137-3p的靶基因,比对候选靶基因3'-UTR区与miR-137-3p结合位点在不同物种间的序列保守性。分别将miR-137-3p mimics、NC与3'-UTR-WT和3'-UTR-Mut载体共转染HEK293T细胞,利用双荧光素酶报告试验检测miR-137-3p与MYC相关因子X(MAX)基因的靶向关系。将siMAX、siNC转染3T3-L1细胞,利用实时荧光定量PCR检测C/EBPα、C/EBPβ、PPARγ和FABP4基因相对表达量,利用Western blotting检测C/EBPβ、PPARγ和FABP4蛋白表达水平。【结果】在3T3-L1细胞的成脂分化过程中,与第0天相比,第2、4、6和8天miR-137-3p相对表达量均极显著降低(P＜0.01);与 NC组相比,miR-137-3p mimics组脂滴明显减少、变小,C/EBPα、C/EBPβ、PPARγ和FABP4基因相对表达量极显著或显著降低(P＜0.01;P＜0.05),C/EBPβ、PPARγ和FABP4蛋白表达下调,miR-137-3p inhibitor组油红O观察结果、成脂分化标志基因mRNA和蛋白表达情况则与之相反。靶基因预测结果表明,MAX的3'-UTR区序列与miR-137-3p存在预测结合位点,且在不同物种间具有高度保守性。双荧光素酶报告试验显示,miR-137-3p mimics组3'-UTR-WT双荧光素酶活性极显著降低(P＜0.01);与NC组相比,mimics组MAX基因表达水平极显著降低(P＜0.01)、inhibitor组显著升高(P＜0.05)。与siNC组相比,敲低MAX基因表达,脂滴明显减少、变小,C/EBPα、C/EBPβ、PPARγ和FABP4基因mRNA表达水平极显著下调(P＜0.01),C/EBPβ、PPARγ和FABP4蛋白表达下降。【结论】内源性miR-137-3p在3T3-L1细胞的成脂分化过程中表达水平降低,miR-137-3p可能通过下调MAX基因的表达抑制3T3-L1细胞成脂分化。

关键词: miR-137-3p; 3T3-L1细胞; MAX基因; 脂肪细胞分化

Abstract: 【Objective】 The aim of this study was to investigate the function and mechanism of miR-137-3p in the differentiation of preadipocytes 3T3-L1.【Method】 3T3-L1 cells at the 0,2,4,6 and 8 days of induced differentiation were collected and the expression of miR-137-3p was detected by Real-time quantitative PCR.miR-137-3p mimics,miR-137-3p inhibitor and negative control (NC) were transfected into 3T3-L1 cells and induced lipid differentiation.Oil red O staining was performed to observe the formation of lipid droplets.The relative expressions of lipogenic marker genes CAAT enhancer binding protein α (C/EBPα),C/EBPβ,peroxisome proliferator-activated receptor γ (PPARγ) and fatty acid binding protein 4 (FABP4) were detected by Real-time quantitative PCR.The protein expression of C/EBPβ,PPARγ and FABP4 were detected by Western blotting.The target genes of miR-137-3p were predicted using TargetScan online website,and the binding sites with miR-137-3p in the 3'-UTR region of candidate target genes were compared for sequence conservation between different species.miR-137-3p mimics,NC and 3'-UTR-WT and 3'-UTR-Mut vectors were co-transfected into HEK293T cells,respectively.Dual luciferase reporting assay was used to detect the targeting relationship between miR-137-3p and MYC-related factor X (MAX) gene.3T3-L1 cells were transfected with siMAX and siNC,and the relative expression of C/EBPα,C/EBPβ,PPARγ and FABP4 genes were detected by Real-time quantitative PCR,and the expression of C/EBPβ,PPARγ and FABP4 proteins were detected by Western blotting.【Result】 In the process of lipogenic differentiation of 3T3-L1 cells,compared with day 0,the relative expression of miR-137-3p on days 2,4,6 and 8 was extremely significantly decreased (P＜0.01).Compared with NC group,the number and size of lipid droplets in miR-137-3p mimics group were significantly decreased,the relative expression of C/EBPα,C/EBPβ,PPARγ and FABP genes in miR-137-3p mimics group were extremely significantly or significantly decreased (P＜0.01 or P＜0.05),the expression of C/EBPβ,PPARγ and FABP4 proteins were down-regulated.The results of oil red O staining,mRNA and protein expression of adipogenic differentiation marker genes in miR-137-3p inhibitor group were opposite.The results of target gene prediction indicated that the 3'-UTR region sequence of MAX had a predicted binding site with miR-137-3p,which was highly conserved among different species.Compared with NC group,the expression of MAX gene in mimics group was extremely significantly decreased (P＜0.01),while that of inhibitor group was significantly increased (P＜0.05).Compared with siNC group,knockdown of MAX gene significantly reduced the number and size of lipid droplets,the mRNA expression of C/EBPα,C/EBPβ,PPARγ and FABP4 genes were extremely significantly down-regulated (P＜0.01),and the protein expressions of C/EBPβ,PPARγ and FABP4 were decreased.【Conclusion】 The expression of endogenous miR-137-3p was decreased during the differentiation of 3T3-L1 preadipocytes,and miR-137-3p might inhibit the adipogenic differentiation of 3T3-L1 preadipocytes by down-regulating the expression of MAX gene.

Key words: miR-137-3p; 3T3-L1 preadipocytes; MAX gene; adipocyte differentiation

中图分类号:

Q254

孟超群, 李成萍, 赵薇, 秦旭勇, 周国利. miR-137-3p靶向MAX基因对前脂肪细胞3T3-L1成脂分化的影响[J]. 中国畜牧兽医, 2023, 50(5): 1928-1937.

MENG Chaoqun, LI Chengping, ZHAO Wei, QIN Xuyong, ZHOU Guoli. Effect of miR-137-3p on Adipogenic Differentiation of Preadipocytes 3T3-L1 by Targeting MAX Gene[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1928-1937.

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