表 1(Table 1)
图 1(Fig. 1)
Snail1对牛脂肪细胞增殖分化影响及作用机制的研究
引用本文
张文涛, 刘晨阳, 朱炳霖, 柳丽, 田媛, 姚宇航, 成功. Snail1对牛脂肪细胞增殖分化影响及作用机制的研究[J]. 畜牧兽医学报, 2023, 54(12): 5008-5019.
ZHANG Wentao, LIU Chenyang, ZHU Binglin, LIU Li, TIAN Yuan, YAO Yuhang, CHENG Gong. Study on the Effects and Mechanisms of Snail1 on Proliferation and Differentiation of Bovine Adipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5008-5019.
Snail1对牛脂肪细胞增殖分化影响及作用机制的研究
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
2. 国家肉牛改良中心, 杨凌 712100
摘要：旨在探究Snail1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Snail1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Snail1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Snail1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Snail1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, Snail1抑制了细胞周期蛋白基因CCNB1、CCND2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Snail1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Snail1抑制了PPARγ(P=0.06)、FABP4(P < 0.05)、LPL (P < 0.05)基因表达, 而PIK3R3基因表达水平显著上调(P < 0.01);蛋白质印迹结果表明, Snail1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, Snail1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。Snail1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-GSK3 β"正反馈环参与了牛脂肪细胞分化调控过程。
关键词：牛 Snail1基因 脂肪细胞 增殖和分化 RNA-Seq 靶基因
Study on the Effects and Mechanisms of Snail1 on Proliferation and Differentiation of Bovine Adipocytes
1. College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China;
2. National Beef Cattle Improvement Center, Yangling 712100, China
2. National Beef Cattle Improvement Center, Yangling 712100, China
Abstract: This paper aimsstudy aimed to explore the effect of Snail1 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 Snail1 gene in Qinchuan cattle adipocytes. Experimental and control groups were established, each with 3 biological replicates. The effect of Snail1 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 Snail1 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 Snail1 led to G1/S phase arrest of the cell cycle. Furthermore, real-time qRT-PCR and Western blot results showed that Snail1 inhibited the expression of CCNB1 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 Snail1 inhibited the adipogenesis and triglyceride formation of bovine adipocytes. qRT-PCR analysis revealed that the overexpression of Snail1 inhibited the gene expression of PPARγ (P=0.06), FABP4 (P < 0.05), and LPL(P < 0.05), while the expression level of PIK3R3 was significantly upregulated (P < 0.01);The Western blot results domonstrated that expression of Snail1 significantly inhibited FABP4 protein expression. Further analysis through RNA-Seq revealed that the differentially expressed genes induced by Snail1 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 Snail1 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-GSK3 β".
Key words:
cattle Snail1 gene adipocyte proliferation and differentiation RNA-Seq target gene
肉牛肌内脂肪含量与牛肉的风味、多汁性和嫩度密切相关[1-2]。肌内脂肪含量是衡量牛肉品质等级的重要指标之一[3]。肌内脂肪的形成与脂肪细胞的增殖和分化密切相关，而脂肪细胞增殖分化的过程本质上是基因表达模式的改变，受到了一系列转录因子的协同调控[4-5]。然而，目前关于肉牛脂肪沉积调控的研究仍处于起步阶段，相关功能基因及其转录调控机制有待进一步挖掘和研究[1, 6]。
Snail1属于锌指转录因子超家族，它是中胚层形成的关键基因。Snail家族成员编码转录抑制子，通过结合下游基因启动子区E-box(CAGGTG)位点参与基因表达调控[7-8]。前人研究表明，Snail1在上皮-间充质转化、免疫调节和细胞干性维持等方面均具有广泛的生物学功能[9-12]。此后大量研究也表明，Snail1在癌细胞的侵袭、迁移和增殖过程同样发挥了重要作用[13-14]。近年来研究发现，Snail1也参与了脂肪细胞的分化调控[15]。Snail1可通过结合人脂联素(Adiponectin，ADPN)启动子区E-box元件，进而抑制Adiponectin表达[16]。同时，Snail1可通过抑制3T3-L1细胞过氧化物酶体增殖物激活受体γ(PPARγ)和CCAAT增强子结合蛋白α(CEBP/α)的表达来抑制脂肪细胞分化，染色质免疫沉淀试验等进一步证实了Snail1通过直接结合PPARγ启动子区E-box元件来抑制PPARγ的转录活性[16]。有研究表明，Wnt-GSK3 β信号通路可通过磷酸化修饰调控Snail1的蛋白水平，进而参与了细胞增殖调控[17-19]。然而，目前关于Snail1在牛脂肪细胞增殖和分化中的作用及其靶基因和信号通路仍未知，有待进一步研究。
1 材料与方法 1.1 主要材料本试验所用牛前体脂肪细胞通过酶消化法从肾周脂肪组织分离，具体方法参考先前文献[20]。脂肪组织采自西北农林科技大学国家肉牛改良中心秦川肉牛良繁场秦川牛新生牛。
1.2 主要仪器CO2细胞培养箱(Thermo Fisher)；实时荧光定量PCR仪(Biorad)；倒置荧光显微镜(Olympus)；活细胞工作站(Biotek)；化学发光成像系统(Biorad)；流式细胞仪(BD)；多功能酶标仪(TECAN)。
1.3 主要试剂胎牛血清(FBS, PAN)；细胞周期染色试剂盒(联科生物)；Cell Counting Kit-8(同仁生物)；Cell-LightTM EdU Apollo567 In Vitro Kit(广州锐博)；脂肪细胞分化诱导I液(每100 mL：IBMX 300 μL, 0.5 mmol·L-1; 胰岛素1 mL, 2 μmol·L-1; 地塞米松10 μL, 1 μmol·L-1; 罗格列酮7.8 μL, 1 μmol·L-1)；脂肪细胞分化诱导II液(每100 mL：胰岛素1 mL，2 μmol·L-1)；PrimeScriptTM RT reagent Kit with gDNA Eraser(TaKaRa)；TB Green Premix Ex TaqTM II(Tli RNaseH Plus, TaKaRa)；LipofectamineTM3000试剂盒(ThermoFisher)。
1.4 方法1.4.1 腺病毒包装及转染 Snail1过表达腺病毒(Ad-Snail1)及其空载对照(Ad-NC)由百恩维生物科技有限公司(深圳)包装完成。腺病毒滴度为1.02×1011 PFU·mL-1。腺病毒侵染试验具体参照先前文献[21]。
1.4.2 流式细胞术 培养牛前体脂肪细胞汇合度达到40%~50%时，利用Snail1过表达腺病毒按照MOI=22侵染细胞，每组生物重复3次。细胞侵染48 h后，消化并收集细胞，按照细胞周期染色试剂盒说明书(联科生物)进行处理，流式细胞仪(BD FACSAriaTM III，BD)上机对牛脂肪细胞的细胞周期进行分析。
1.4.3 CCK-8细胞增殖试验 将牛脂肪细胞接种于48孔板，每孔4 000个细胞。分别用Ad-NC、Ad-Snail1处理24、48、72 h后，按照CCK-8说明书(Cell Counting Kit-8，同仁生物)操作，于450 nm处测定吸光度(Infinite M200PRO，TECAN)。
1.4.4 EDU染色 将牛前脂肪细胞接种到12孔板培养，直到汇合度达到40%~50%进行腺病毒侵染，每组生物学重复3次。细胞侵染48 h后，按照EdU染色试剂盒(Cell-LightTM EdU Apollo567 In Vitro Kit，广州锐博)进行染色，检测细胞增殖情况。
1.4.5 牛前脂肪细胞的分化 将牛前脂肪细胞接种于6孔板中，置于37 ℃下，5% CO2培养箱中(Thermo Fisher)培养至融合度达80%时进行Ad-NC、Ad-Snail1腺病毒侵染。侵染48 h后，细胞中加入诱导I液进行诱导分化。诱导I液处理2 d后，更换诱导II液进行分化维持，之后每2 d更换一次诱导II液。
1.4.6 油红O染色 诱导分化第6天的牛脂肪细胞经PBS润洗3次，4%多聚甲醛室温固定30 min。然后用PBS冲洗3次，1%油红O工作液避光孵育30 min。之后用PBS多次冲洗细胞，最后在倒置荧光显微镜下拍照。
1.4.7 甘油三酯测定 诱导分化第6天的牛脂肪细胞加入胰酶消化，然后加入等体积10% FBS培养液终止消化，转移至离心管，1 000 r·min-1离心5 min，弃上清，加入细胞裂解液，混合后静置10 min。将180 μL的上清液转移到新的离心管中，70 ℃加热10 min，然后2 000 r·min-1离心5 min取上清，配制标准品作标准曲线。在96孔板中加入工作液190 μL和10 μL上清样品，避光37 ℃孵育10~15 min，利用酶标仪550 nm波长进行甘油三酯测定。
1.4.8 RNA逆转录及实时定量PCR分析 采用Trizol法提取牛脂肪细胞总RNA，于-80 ℃保存。按照TaKaRa逆转录试剂盒及SYBRⓇ Primix Ex TaqTMⅡ试剂盒(TaKaRa)说明书进行RNA逆转录和实时荧光定量PCR。
1.4.9 蛋白质印迹 用于增殖和分化检测的牛脂肪细胞经预冷的PBS冲洗3次后加入预冷的RIPA和蛋白酶抑制剂，冰上孵育15 min以裂解细胞。裂解的细胞12 000 r·min-1, 4 ℃，离心10 min，分离取上清液，通过BCA法对提取的蛋白进行定量。在提取的蛋白中加入5×蛋白上样缓冲液，然后100 ℃煮沸10 min，-80 ℃保存备用。Western blot试验蛋白上样量为20 μg。用于Western blot分析的抗体概要信息列于表 1。
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表 1 抗体信息 Table 1 Antibody information |
1.4.10 转录组测序 将牛脂肪细胞接种于6孔板中，用Ad-NC、Ad-Snail1侵染48 h后进行成脂诱导分化。收集分化第6天细胞并送至广州基迪奥生物技术公司进行Illumina HiSeq2000 RNA-seq测序。利用Omicshare生物信息学云平台(https://www.omicshare.com/)对测序数据进行分析，按照|Fold change|≥1.5、FDR < 0.05标准进行差异表达基因(DEGs)筛选。利用Omicshare在线工具对差异表达基因进行KEGG通路富集分析。利用String在线工具和Cytoscape软件构建目标基因集的蛋白相互作用(PPI)网络。在线工具MEME软件包中FIMO(https://meme-suite.org/meme/tools/fimo)对牛注释基因起始上游2 000 bp，下游100 bp进行Snail1潜在的E-box结合位点分析，联合转录组测序DEGs筛选Snail1蛋白潜在结合靶基因。
1.4.11 荧光素酶报告分析 pFLAG-Snail1过表达载体和SFRP2启动子区报告载体pSFRP2-luc+均由北京生工合成并克隆。将HEK293A细胞接种于48孔细胞培养板。当细胞融合度达到60%时，按照LipofectamineTM3000试剂盒(ThermoFisher)操作步骤将pFLAG-Snail1(0、0.25、0.5、1.0 μg)+ pSFRP2-luc+或pbasic-luc+质粒进行共转染，每组3个生物学重复，海肾荧光素酶质粒pRL-TK作为内参校正。转染36 h后，通过酶标仪进行双荧光素酶活性检测(Dual-Glo Luciferase Assay System，Promega)。
2 结果 2.1 Snail1腺病毒侵染及过表达效率检测将Ad-Snail1腺病毒以最佳感染复数MOI=22侵染牛前体脂肪细胞，检测其过表达效率。通过对照组pEGFP-N1质粒转染组绿色荧光蛋白检测发现，腺病毒显著提高了外源基因转染效率(图 1A)。实时荧光定量PCR和Western Blot结果表明，Ad-Snail1腺病毒能显著上调细胞中Snail1基因的表达。与对照组相比，Snail1基因mRNA平均表达量提高了255.76倍(图 1B，P < 0.01)，蛋白表达量较对照组明显提高(图 1C)。结果表明，Ad-Snail1腺病毒过表达效果良好，可进行下一步的试验。
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A. pEGFP-N1质粒，Ad-NC、Ad-Snail1转染/侵染牛前体脂肪细胞48 h荧光检测(标尺：200 μm)；B. qRT-PCR检测Snail1基因mRNA表达水平；C. Western blot检测Snail1蛋白过表达效率。*.P < 0.05；**.P < 0.01，下同 A. Fluorescent detection of pEGFP-N1 plasmid, Ad-NC, and Ad-Snail1 transfection/infection into cattle preadipocytes for 48 hours(bar: 200 μm); B. Detection of Snail1 mRNA level by qRT-PCR; C. Western blot analysis to detect the overexpression efficiency of Snail1 protein. *. P < 0.05;**. P < 0.01, the same as below 图 1 Snail1基因过表达效率检测 Fig. 1 Snail1 gene overexpression efficiency detection |
CCK-8检测结果表明，与对照组(NC)相比，Snail1过表达48、72、96 h后，细胞数量明显减少(P < 0.05), 说明Snail1抑制了牛前体脂肪细胞的增殖过程(图 2A)。EdU染色结果显示，Snail1过表达后减少了EdU阳性细胞数量(P < 0.05), 表明DNA复制受到抑制(图 2B)。流式细胞术检测结果发现，Snail1过表达显著上调了G0/G1细胞数量(P < 0.05)，下调G2/M期细胞数量(P < 0.05)，S期细胞呈现一定下降趋势。上述结果表明，Snail1过表达引起G1/S期阻滞，抑制细胞增殖(图 2C)。进一步，通过实时荧光定量PCR和Western Blot对增殖相关标志基因与蛋白检测分析发现，Snail1过表达抑制了细胞周期相关基因(CCNB1, CCND2)的mRNA(P < 0.05)和蛋白的表达(图 2D)。综上所述，这些结果表明Snail1通过下调细胞周期相关基因的表达，使得细胞G1/S期阻滞，从而抑制前脂肪细胞的增殖。
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A. CCK-8检测细胞活力；B. EdU染色检测DNA复制活性：阳性细胞EdU染色(红色)，细胞核Hoechst染色(蓝色)(标尺：100 μm)；C. 流式细胞术测定牛前体脂肪细胞细胞周期；D. Snail1过表达48 h后细胞周期相关基因表达检测 A. Cell viability analysis by CCK-8;B. The activity of DNA replication detection by EdU staining: Positive cells were stained with EdU (Red), nucleus were stained with Hoechst (blue)(bar: 100 μm); C. The percentages of bovine preadipocytes in different cell-cycle phases were determined by flow cytometry; D. Relative expression analysis of cell cycle related genes after 48 hours of Snail1 overexpression treatment 图 2 Snail1抑制牛前脂肪细胞增殖 Fig. 2 Snail1 inhibited the proliferation of cattle preadipocytes |
为了探究Snail1对牛前体脂肪细胞成脂分化的影响，对Ad-Snail1侵染并诱导分化第6天的牛脂肪细胞进行油红O染色和甘油三酯测定。结果发现，Snail1过表达抑制了牛脂肪细胞的成脂分化和甘油三酯的生成(图 3A，3B)。qRT-PCR和Western blot分析表明，Snail1过表达后，PPARγ mRNA表达量下降(P = 0.06)，但蛋白表达量无明显变化；脂肪酸结合蛋白4 (FABP4) mRNA表达量显著降低(P < 0.05)，蛋白水平呈现相同下降趋势；脂蛋白脂酶(LPL) mRNA表达量显著降低；磷酸肌醇3激酶调节亚基3 (PIK3R3) mRNA表达水平极显著升高(P < 0.01，图 3C)。上述结果表明，Snail1过表达抑制了牛前体脂肪细胞成脂分化。
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A.油红O染色结果(标尺：50 μm); B.甘油三酯测定结果; C.Snail1基因过表达且诱导分化6 d后，细胞分化相关标记基因mRNA表达水平检测；D.蛋白表达水平检测 A. Oil Red O staining results(bar: 50 μm); B. Triglyceride content analysis; C. The relative mRNA expression level detection of cell differentiation-related marker genes after overexpression of Snail1 gene and induced differentiaion for 6 days; D. Protein expression level detection 图 3 Snail1抑制牛脂肪细胞成脂分化 Fig. 3 Snail1 inhibited adipogenic differentiation of cattle adipocytes |
利用RNA-Seq对转录因子Snail1影响脂肪细胞分化的潜在作用信号通路进行分析。热图结果显示，Ad-Snail1、Ad-NC组能明显分为两组，表明测序结果良好(图 4A)。与对照组相比较，Snail1基因过表达组有791个基因表达显著上调，549个基因表达显著下调(|Fold change|≥1.5)(图 4B, 4C)。KEGG富集分析结果表明，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(图 4D)。
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A.聚类分析；B.差异表达基因(DEGs)分析; C.差异表达基因(DEGs)火山图; D. 差异表达基因(DEGs)KEGG通路富集分析 A. Cluster analysis; B. Differentially expressed gene (DEGs) analysis; C. Volcanic map of differentially expressed genes (DEGs); D. KEGG pathway enrichment analysis of DEGs 图 4 RNA-Seq分析 Fig. 4 RNA-Seq analysis |
随机挑选RNA-Seq结果中8个差异基因进行qRT-PCR检测，通过qRT-PCR与RNA-Seq差异倍数相关性分析发现，RNA-Seq/qRT-PCR差异倍数相关系数为0.88(P < 0.001)，结果表明RNA-Seq测序结果可靠(图 5A、5B)。
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A. qRT-PCR验证；B. RNA-seq/qRT-PCR差异倍数相关性分析 A. qRT-PCR verification of RNA-Seq results; B. Correlation analysis of fold changes of RNA-Seq/qRT-PCR 图 5 RNA-Seq测序数据验证 Fig. 5 Verification of RNA-Seq data |
为了获得Snail1作用关键的潜在靶基因，利用String在线工具对脂肪细胞分化相关的下调基因(|Fold change|≥2)进行蛋白互作(PPI)网络构建(图 6)。进一步利用Cytoscape mCODE对互作网络功能模块进行聚类分析，发现下调基因富集到了5个子网络集(表 2)。
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图 6 Snail1过表达后差异基因PPI网络构建 Fig. 6 PPI network construction of DEGs after Snail1 overexpression |
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表 2 mCODE筛选子网络 Table 2 mCODE filtlers subnetworks |
利用MEME包中的FIMO工具搜索牛注释基因启动子区域(上游2 000 bp，下游100 bp)，筛选潜在的结合靶基因。通过Pearson相关系数对预测的靶基因和RNA-Seq数据进行分析，结合KEGG富集信号通路筛选发现，Wnt信号通路拮抗基因SFRP2基因可能是Snail1的潜在靶基因(图 6，表 3)。RNA-Seq数据表明，Snail1过表达后SFRP2下调3.3倍，且该基因位于PPI互作网络中核心子网络5的Wnt信号通路中(图 6，表 2)。此外，SCD、ATGL等脂肪代谢相关基因也可能是Snail1的潜在靶基因(表 3)。
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表 3 Snail1靶基因预测及表达相关性分析 Table 3 Prediction of Snail1 target gene and correlation analysis of its expression |
进一步通过荧光素酶试验对Snail1潜在靶基因SFRP2进行验证。JASPAR软件分析发现，在SFRP2启动子区域发现了Snail1转录因子的5个潜在结合位点，其中包含FIMO软件预测的388~397位置结合位点(图 7A)。pFLAG-Snail1和pSFRP2-luc+共转染293 T细胞36 h后进行荧光素酶检测(pRL-TK为内参)。结果表明，SFRP2启动子活性随着Snail1表达质粒浓度的升高而被显著抑制，呈现剂量依赖效应(图 7B)。阴性对照组pBasic-luc+中荧光素酶含量无明显变化(图 7C)。以上结果表明，SFRP2可能是Snail1直接作用的靶基因。
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A. SFRP2启动子中Snail1的潜在结合位点示意图；B.荧光素酶检测Snail1过表达对pSFRP2-luc+启动子活性的影响；C.阴性对照pBasic-luc+质粒 A.Diagram of potential Snail1 binding sites in SFRP2 promoter; B.Luciferase detection of the effect of Snail1 concentration on the activity of SFRP2 promoter; C.pBasic-luc+ plasmid was used as negative control 图 7 SFRP2启动子区Snail1潜在结合位点和Snail1-SFRP2靶向关系的验证 Fig. 7 Verification of potential Snail1 binding sites and Snail1-SFRP2 targeting relationships in SFRP2 promoter region |
肉牛的脂肪沉积过程表现为脂肪细胞的增殖和分化过程，其受到了一系列转录因子的协同作用。近年来研究发现，Snail1在成脂分化过程中发挥重要作用。Snail1通过结合并抑制成脂分化关键基因PPARγ的表达，抑制小鼠3T3-L1细胞的分化。Snail1基因在3T3-F442A细胞中过表达导致其分化为成熟脂肪细胞的能力丧失[22]。此外，Snail1可以通过招募组蛋白去乙酰化酶(histone deacetylase, HDACs)抑制MyoD与其靶基因的结合，进而抑制肌源性分化[23]。上述研究表明，Snail1同时参与细胞脂肪生成和成肌细胞分化的调控，是影响动物脂肪沉积和肌肉发育的重要候选功能基因。然而，在细胞增殖方面Snail1的作用仍存在争议。目前多数研究表明Snail1促进了细胞的增殖，如Snail1可诱导癌症中的EMT过程从而促进肝癌细胞的增殖[24]；TRIM55可以通过UPP途径促进Snail1降解，进一步的抑制肿瘤的发展和肺癌细胞的增殖过程[25]。然而，也有研究表明Snail1对于细胞增殖具有抑制作用，如Snail1通过直接结合并抑制成脂基因Fasn表达，进而阻断由胰岛素刺激产生的产脂酶表达来抑制脂肪生成，同时抑制肝脏中的脂肪细胞增殖[26]。本研究发现，Snail1抑制了牛前脂肪细胞的增殖和分化过程。RNA-Seq、靶基因预测及荧光素酶报告试验等证实, Wnt/β-catenin信号通路关键拮抗蛋白基因SRFP2是Snail1的靶基因。Snail1可能通过靶向SRFP2介导的Wnt/β-catenin参与了脂肪细胞分化调控。
细胞增殖受到了细胞周期的调控，主要表现在DNA的复制及其随后的细胞分裂过程，其又可细分为4个不同的阶段(G0/G1期、S期、G2期和M期)[27]。本研究发现，Snail1过表达显著下调了促增殖相关基因如CCNB1、CCND2的mRNA水平和蛋白水平的表达。CCNB1是有丝分裂的主要调节因子，在调控周期蛋白依赖激酶1 (CDK1)中发挥关键作用，主要通过与CDK1形成复合物，磷酸化其底物，促进细胞周期由G2期向有丝分裂期过渡[28]。CCND2是一种D型细胞周期蛋白，与CDK4、CDK6结合参与G1/S检查点调控[29-30]。流式细胞术显示, Snail1基因过表达导致G1/S期阻滞，G2、M期细胞比例降低；EdU染色显示，过表达Snail1可显著降低复制期EdU阳性细胞比例；CCK-8结果也显示，Snail1过表达可降低细胞增殖速率。上述结果表明, Snail1基因抑制了牛前脂肪细胞增殖并导致细胞周期G1/S期阻滞。
前脂肪细胞能够在诱导剂的作用下分化为产生脂滴的成熟脂肪细胞，脂滴是成熟脂肪细胞的一个显著形态特征，其在脂肪细胞分化过程中逐渐积累[31]。本研究发现，Ad-Snail1抑制了脂肪细胞分化及脂滴的生成。通过对RNA-Seq差异基因的KEGG分析显示，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。PPAR信号通路是脂肪细胞分化的明星通路，该通路的下调会抑制脂肪细胞的分化过程[32]；尼古丁成瘾信号通路参与了脂肪的调控，尼古丁饲喂的小鼠体重降低并伴随着脂肪细胞体积减小[33]。也有研究表明，尼古丁促进了上皮细胞向间质细胞的转化，进而促进了肿瘤的形成[34]。本研究RNA-Seq测序也证实，Snail1过表达激活了尼古丁成瘾相关信号通路基因的表达，而Snail1及尼古丁成瘾信号通路基因均在上皮细胞向间质细胞转化及癌症的发生过程中发挥重要作用。
为了进一步探究Snail1参与脂肪细胞分化的潜在分子机制，对FIMO工具预测的靶基因及RNA-Seq等数据进行分析发现，SFRP2、AQP9、Serpine1、SCD、ATGL等为Snail1潜在靶基因。前人研究证实，Snail1可结合并靶向调节甘油三酯脂肪酶基因ATGL[35]。本研究通过荧光素酶试验表明，Wnt信号通路拮抗蛋白基因SFRP2是Snail1的潜在靶基因，而Wnt信号通路在PPI蛋白互作网络中被显著富集。Wnt/β-catenin属于经典的Wnt信号通路，主要由Wnt家族蛋白、糖原合成激酶3(GSK3)、轴突蛋白(Axin)、β-catenin和TCF/LEF家族转录因子组成。Wnt/β-catenin信号通路的激活抑制了GSK3β的磷酸化，进而抑制了C/EBPα和PPARγ的表达，抑制分化过程[36]。本研究表明，Snail1可通过结合并抑制Wnt信号通路拮抗蛋白基因SFRP2的表达，进而激活Wnt信号通路介导的成脂分化抑制作用。前人研究发现，Snail1可通过与β-catenin蛋白相互作用激活Wnt信号通路，参与调控癌细胞上皮间质转化，而Wnt信号通路的激活抑制了GSK3β活性，进一步提高了Snail1蛋白的稳定性[37]。那么可推测Wnt信号通路可能存在正反馈效应，通过“Snail1/SFRP2-Wnt/β-catenin-GSK3β”环[22]，进一步维持Snail1对Wnt信号的激活参与牛脂肪发育的调控，但其分子机制仍有待进一步研究。这些发现为进一步探究Snail1调节脂肪生成的机制提供了新的思路。
4 结论前人对于Snail1在牛前体脂肪细胞增殖和分化中的作用尚不明确。本研究通过CCK-8、EdU、流式细胞术及油红O、甘油三酯等试验发现, Snail1抑制了牛脂肪细胞的增殖和分化过程。通过转录组测序、靶基因预测及荧光素酶等试验证实，SFRP2是Snail1的潜在靶向基因，且Snail1可能通过“Snail1/SFRP2-Wnt/β-catenin-GSK3 β”正反馈环参与牛脂肪细胞分化调控。上述研究为进一步探索Snail1在脂肪生成中的机制奠定了基础，同时为通过分子育种提高牛肌内脂肪含量提供了思路。
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(编辑 郭云雁)