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

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 affects the differentiation process of bovine adipocytes by targeting the antagonist SFRP2 of the Wnt signaling pathway.The results suggested that Snail 1 gene inhibited the proliferation and differentiation process of bovine adipocytes,and may be involved in the regulation of bovine adipocyte differentiation through the positive feedback loop of"Snail1/SFRP2-Wnt/β-catenin-GSK3β".