miR-140-5p对前脂肪细胞3T3-L1成脂分化的机制研究

doi:10.16431/j.cnki.1671-7236.2022.07.021

摘要/Abstract

摘要： 【目的】研究miR-140-5p在前脂肪细胞（3T3-L1）成脂分化过程中的功能及其作用机制。【方法】待3T3-L1细胞汇合度达100%时诱导其成脂分化，收集分化第―1（诱导分化前1 d）、0、1、2、3、5、7天的细胞，用实时荧光定量PCR检测miR-140-5p相对表达量；将miR-140-5p mimics、NC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，实时荧光定量PCR检测成脂标志基因CAAT增强子结合蛋白β（C/EBPβ）、CAAT增强子结合蛋白δ（C/EBPδ）与过氧化物酶体增殖物激活受体γ（PPARγ）相对表达量；利用miRandn和TargetScan在线网站预测miR-140-5p的靶基因；通过对比序列差异分析P300/CBP相关因子（PCAF）的3'-UTR序列中与miR-140-5p的结合位点序列在小鼠、人等不同物种间的序列保守性。将miR-140-5p mimics、inhibitor、NC转染3T3-L1细胞并诱导成脂分化，实时荧光定量PCR检测miR-140-5p、PCAF相对表达量，Western blotting检测PCAF蛋白水平；将PEGFP-N1-PCAF、PEGFP-N1转染3T3-L1细胞，油红O染色观察脂滴形成情况，实时荧光定量PCR检测PCAF、C/EBPδ、PPARγ基因相对表达量，Western blotting检测C/EBPβ、C/EBPδ、PPARγ蛋白表达水平。将3条PCAF siRNA（siRNA1、siRNA2、siRNA3）、siRNA NC转染3T3-L1细胞，Western blotting法检测PCAF蛋白水平，筛选最佳PCAF siRNA。将最佳PCAF siRNA和siRNA NC转染3T3-L1细胞，油红O染色观察脂滴形成情况，实时荧光定量PCR检测PCAF、C/EBPβ、C/EBPδ与PPARγ基因相对表达量，Western blotting检测C/EBPβ、PPARγ蛋白表达水平。分别将miR-140-5p mimics、NC与PGL0-PCAF 3'-UTR载体和PGLO空载体共转染293T细胞，用双荧光素酶报告试验检测miR-140-5p与PCAF的靶向关系。【结果】在诱导3T3-L1细胞成脂分化过程中，与诱导分化前1 d相比，在细胞成脂分化第1、2天时miR-140-5p相对表达量极显著升高（P＜0.01），在分化第3天时显著升高（P＜0.05）。与NC组相比，miR-140-5p mimics组脂滴数量明显增加，miR-140-5p mimics组成脂标志基因C/EBPδ与PPARγ相对表达量均极显著升高（P＜0.01）。靶基因预测结果表明，miR-140-5p与PCAF存在预期结合位点；保守性分析结果表明，靶基因PCAF结合位点序列在不同物种间具有高度保守性。与NC组相比，mimics组miR-140-5p和PCAF相对表达量均极显著升高（P＜0.01），inhibitor组miR-140-5p极显著降低（P＜0.01）、PCAF显著降低（P＜0.05），PCAF蛋白表达量显著升高（P＜0.05）。与PEGFP-N1组相比，PEGFP-N1-PCAF组脂滴数量增多，PCAF、PPARγ基因相对表达量和C/EBPβ、C/EBPδ蛋白水平极显著升高（P＜0.01），PPARγ蛋白水平显著升高（P＜0.05）。PCAF siRNA1、siRNA2与siRNA3均极显著抑制PCAF蛋白的表达（P＜0.01），siRNA3的效果最显著，因此选择siRNA3进行后续试验。与NC组相比，PCAF siRNA3组3T3-L1细胞中脂滴数量较少，PCAF、C/EBPβ、C/EBPδ、PPARγ基因相对表达量和C/EBPβ蛋白水平均极显著下降（P＜0.01）。双荧光素酶报告试验结果显示，miR-140-5p与PCAF基因之间无靶标关系。【结论】内源性miR-140-5p在3T3-L1细胞分化过程中表达升高，miR-140-5p可能通过间接上调PCAF基因表达促进3T3-L1细胞成脂分化。

关键词: miR-140-5p; P300/CBP相关因子; 3T3-L1细胞; 成脂分化

Abstract: 【Objective】 The aim of this study was to investigate the function and mechanism of miR-140-5p in the differentiation of preadipocytes 3T3-L1.【Method】 When the convergence degree of 3T3-L1 cells reached 100%,the adipogenic differentiation was induced.The cells of differentiation day ―1 (the day before differentiation induction),days 0,1,2,3,5 and 7 were collected,and the expression of miR-140-5p was detected by Real-time quantitative PCR.miR-140-5p mimics and NC were transfected into 3T3-L1 cells to induce adipogenic differentiation.Oil red O staining was used to observe the formation of lipid droplets.Real-time quantitative PCR was used to detect the relative expressions of adipogenic marker genes CAAT enhancer binding protein β (C/EBPβ),CAAT enhancer binding protein δ (C/EBPδ) and peroxisome proliferator-activated receptor γ (PPARγ).The target gene of miR-140-5p was predicted by miRandn and TargetScan online websites,the sequence conservation of the binding site sequence of P300/CBP-related factor (PCAF) 3'-UTR sequence and miR-140-5p in different species such as mice and humans was analyzed by comparing the sequence differences.miR-140-5p mimics,inhibitor and NC were transfected into 3T3-L1 cells and the cells were induced to differentiate into adipocytes.The relative expression of miR-140-5p and PCAF was detected by Real-time quantitative PCR,and the protein level of PCAF was detected by Western blotting.PEGFP-N1-PCAF and PEGFP-N1 were transfected into 3T3-L1 cells.Oil red O staining was used to observe the formation of lipid droplets.Real-time quantitative PCR was used to detect the relative expression of PCAF,C/EBPδ and PPARγ genes.Western blotting was used to detect the expression levels of C/EBPβ,C/EBPδ and PPARγ proteins.Three PCAF siRNAs (siRNA1,siRNA2 and siRNA3) and siRNA NC were transfected into 3T3-L1 cells.The protein level of PCAF was detected by Western blotting to screen the best PCAF siRNA.Optimal PCAF siRNA and siRNA NC were transfected into 3T3-L1 cells,and the formation of lipid droplets was observed by oil red O staining.The relative expression of PCAF,C/EBPβ,C/EBPδ and PPARγ genes were detected by Real-time quantitative PCR,and the expression of C/EBPβ and PPARγ protein were detected by Western blotting.miR-140-5p mimics,NC,PGL0-PCAF 3'-UTR vector and PGLO empty vector were co-transfected into 293T cells,respectively.The targeting relationship between miR-140-5p and PCAF gene was detected by double luciferase report test.【Result】 In the process of inducing adipogenic differentiation of 3T3-L1 cells,compared with the day before inducing differentiation,the relative expression of miR-140-5p was extremely significantly increased on the day 1 and day 2 of adipogenic differentiation (P＜0.01) and was significantly increased on the day 3 day of differentiation (P＜0.05).Compared with NC group,the number of lipid droplets in miR-140-5p mimics group was increased significantly,and the relative expression of lipid marker genes C/EBPδ and PPARγ in miR-140-5p mimics group were extremely increased significantly (P＜0.01).The result of target gene prediction showed that miR-140-5p had the expected binding site with PCAF.The results of conservation analysis showed that the binding site sequence of target gene PCAF was highly conserved among different species.Compared with NC group,the relative expressions of miR-140-5p and PCAF gene in mimics group were extremely significantly increased (P＜0.01),while those in inhibitor group were extremely significantly decreased (P＜0.01),PCAF was significantly decreased (P＜0.05),and the expression of PCAF protein was significantly increased (P＜0.05).Compared with PEGFP-N1 group,the number of lipid droplets in PEGFP-N1-PCAF group was increased,and the relative expression levels of PCAF and PPARγ genes and the levels of C/EBPβ,C/EBPδ protein were extremely significantly increased (P＜0.01),PPARγ protein was significantly increased (P＜0.05).PCAF siRNA1,siRNA2 and siRNA3 all extremely significantly inhibited the expression of PCAF protein (P＜0.01),and siRNA3 had the most significant effect,so siRNA3 was selected for the follow-up test.Compared with NC group,the number of lipid droplets in 3T3-L1 cells in PCAF siRNA3 group was less,the expression of PCAF,C/EBPβ,C/EBPδ and PPARγ genes and the level of C/EBPβ protein were extremely decreased significantly (P＜0.01).The results of double luciferase report test showed that there was no target relationship between miR-140-5p and PCAF gene.【Conclusion】 The experiment proved that the expression of endogenous miR-140-5p was increased during the differentiation of 3T3-L1 cells.miR-140-5p might promote the adipogenic differentiation of 3T3-L1 cells by indirectly up-regulating the expression of PCAF gene.

Key words: miR140-5p; P300/CBP-related factors; 3T3-L1 cells; adipogenic differentiation

中图分类号:

Q254

原佳慧, 张鹏翔, 吉树森, 卢嘉茵, 罗小毛, 闫艺, 王海东. miR-140-5p对前脂肪细胞3T3-L1成脂分化的机制研究[J]. 中国畜牧兽医, 2022, 49(7): 2631-2644.

YUAN Jiahui, ZHANG Pengxiang, JI Shusen, LU Jiayin, LUO Xiaomao, YAN Yi, WANG Haidong. Mechanism of miR-140-5p on Adipogenic Differentiation of Preadipocytes 3T3-L1[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2631-2644.

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