Snail1对牛脂肪细胞增殖分化影响及作用机制的研究  

Study on the Effects and Mechanisms of Snail 1 on Proliferation and Differentiation of Bovine Adipocytes

作　　者：张文涛 刘晨阳 朱炳霖 柳丽 田媛[1] 姚宇航 成功[1,2] ZHANG Wentao;LIU Chenyang;ZHU Binglin;LIU Li;TIAN Yuan;YAO Yuhang;CHENG Gong(College of Animal Science and Technology,Northwest A&F University,Yangling 712100,China;National Beef Cattle Improvement Center,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]国家肉牛改良中心,杨凌712100

出　　处：《畜牧兽医学报》2023年第12期5008-5019,共12页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：陕西省两链融合重点专项(2022GD-TSLD-46-0102)。

摘　　要：旨在探究Snail 1基因对于牛脂肪细胞增殖分化的影响及其潜在的作用分子机制。本研究利用腺病毒在秦川牛脂肪细胞中过表达Snail 1基因并设置试验组和对照组各3个生物学重复,采用流式细胞术、CCK-8、EdU染色、油红O染色、甘油三酯测定、qRT-PCR和蛋白质印迹等试验方法探究Snail 1对牛脂肪细胞增殖和分化的影响;进一步通过RNA-Seq、双荧光素酶报告试验筛选其作用信号通路及靶基因。结果表明,过表达Snail 1抑制了细胞增殖(CCK-8)且减少了处于S复制期阳性细胞比例(EdU,P<0.05)。结合流式细胞术试验,结果表明Snail 1上调导致了细胞周期G1/S期阻滞。进一步的实时荧光定量PCR和蛋白质印迹结果表明,Snail 1抑制了细胞周期蛋白基因CCNB1、CCND 2的表达(P<0.05)。对诱导分化第6天牛脂肪细胞进行油红O染色和甘油三酯检测,结果表明Snail 1抑制了牛脂肪细胞的成脂分化和甘油三酯的生成。qRT-PCR分析表明,过表达Snail 1抑制了PPARγ(P=0.06)、FABP4(P<0.05)、LPL(P<0.05)基因表达,而PIK 3R3基因表达水平显著上调(P<0.01);蛋白质印迹结果表明,Snail 1表达显著抑制了FABP4蛋白表达。进一步通过RNA-Seq测序分析发现,Snail 1过表达引起的差异基因主要富集在PPAR、ECM受体互作、PI3K-AKT、MAPK、尼古丁成瘾及胰岛素分泌等信号通路。基于FIMO靶基因筛选联合RNA-Seq数据分析及荧光素酶试验等证实,转录因子Snail1可能通过靶向Wnt信号通路的拮抗剂SFRP2影响牛脂肪细胞的分化过程。Snail 1基因抑制牛脂肪细胞的增殖和分化过程,且可能通过“Snail1/SFRP2-Wnt/β-catenin-GSK3β”正反馈环参与了牛脂肪细胞分化调控过程。This paper aims study aimed to explore the effect of Snail 1 gene on the proliferation and differentia-tion of bovine adipocytes and its potential molecular mechanism.In this study,an adenovirus was used to overexpress the Snail 1 gene in Qinchuan cattle adipocytes.Experimental and control groups were established,each with 3 biological replicates.The effect of Snail 1 on the proliferation and differentiation of bovine adipocytes was investigated by flow cytometry,CCK-8,EdU staining,Oil Red O staining,triglyceride determination,qRT-PCR and Western blot.The signaling pathways and target genes were further screened by RNA-Seq and dual-luciferase reporter experiments.The results showed that overexpression of Snail 1 inhibited cell proliferation(CCK-8)and reduced the proportion of positive cells at the S-phase(EdU,P<0.05).Combined with flow cytometry tests,the results indicated that the up-regulation of Snail 1 led to G1/S phase arrest of the cell cycle.Furthermore,real-time qRT-PCR and Western blot results showed that Snail 1 inhibited the expression of CCNB 1 and CCND2(P<0.05).The results of Oil Red O staining and triglyceride detection on bovine adipocytes on day 6 of induced differentiation showed that Snail 1 inhibited the adipogenesis and triglyceride formation of bovine adipocytes.qRT-PCR analysis revealed that the overexpression of Snail 1 inhibited the gene expression of PPARγ(P=0.06),FABP4(P<0.05),and LPL(P<0.05),while the expression level of PIK 3R3 was significantly upregulated(P<0.01);The Western blot results domonstrated that expression of Snail 1 significantly inhibited FABP4 protein expression.Further analysis through RNA-Seq revealed that the differentially expressed genes induced by Snail 1 overexpression were predominantly enriched in signaling pathway such as the PPAR,ECM-receptor interaction,PI3K-AKT,MAPK,nicotine addiction,and insulin secretion.Based on FIMO target gene screening,combined RNA-Seq data analysis,and luciferase assay,it was confirmed that the transcription factor Snail1 potentially

关 键 词：牛 Snail 1基因 脂肪细胞 增殖和分化 RNA-SEQ 靶基因 

分 类 号：S823.81[农业科学—畜牧学]