秦川牛Snail1克隆、表达特性分析及其对牛脂肪细胞增殖的作用研究

doi:10.3864/j.issn.0578-1752.2024.13.014

摘要/Abstract

摘要：

【目的】肉牛肌内脂肪沉积与牛肉的风味、多汁性和嫩度密切相关。脂肪沉积过程表现为脂肪细胞的增殖（数量增多）和分化（脂质生成），受到了多基因协同调控。前人研究发现，小鼠中Snail1可以参与肌肉发育和脂质稳态调控，但其在牛脂肪生成过程中的作用仍未知，有待进一步研究。【方法】以秦川牛为研究对象，克隆得到Snail1 CDS区序列，构建Snail1时空表达谱，运用生物信息学软件对其功能结构及靶基因进行预测。进一步，通过RNAi干扰结合CCK8、EdU、细胞流式及实时荧光定量PCR等方法探究Snail1对牛脂肪细胞增殖的影响。【结果】秦川牛Snail1与NCBI公布序列相比存在2处碱基同义突变，其在秦川牛新生牛肺、肾周脂肪、小肠呈现较高丰度表达；而在成年牛中，Snail1在肾周脂肪组织中的表达量最高，背最长肌中的表达量次之，肺脏组织中的表达量最低。生物信息学分析发现，Snail1 启动子区存在1个651 bp CpG岛及C/EBP、PPARα 等与脂肪生成相关的转录因子结合位点。CKⅠ(Ser92/96)、CKⅡ（Ser25/119，Thr89）、CDK1（Ser13/104/112/119/143/183/214/221）、CDK5(Ser105/107）等多个细胞周期相关激酶可能参与了Snail1蛋白的磷酸化修饰。通过对牛已注释基因启动子区提取、靶基因预测及 KEGG 动态网络构建发现，成脂相关的 MAPK、PI3K-Akt、mTOR 等信号通路为Snail1参与脂肪生成相关的潜在节点信号通路。进一步，通过RNAi干扰试验对其功能研究表明，Snail1下调促进了牛前体脂肪细胞的增殖，增加了复制期阳性细胞的比例（P<0.01）且促进了G1/S细胞周期转换。RT-qPCR和Western-blot检测表明，干扰Snail1显著上调了促增殖调控基因CCNB1、CCND2、CDK2、CDK4（P<0.05）和蛋白的表达。【结论】 Snail1在新生牛肾周脂肪及成年牛肾周脂肪和背最长肌中表达量相对较高。干扰Snail1促进了牛前体脂肪细胞的增殖、G1/S细胞周期转变和CCNB1、CCND2、CDK2、CDK4 等增殖相关基因表达；CKⅠ、CKⅡ、CDK1/5等多个细胞周期相关激酶可能通过磷酸化修饰Snail1蛋白进而参与细胞增殖调控，而MAPK、PI3K-Akt、mTOR 等为Snail1影响牛脂肪细胞增殖潜在的关键节点通路。研究结果为进一步探究Snail1参与牛脂肪生成作用机制奠定了基础。

关键词: 牛, Snail1, 脂肪沉积, KEGG动态网络, RNAi, 细胞增殖

Abstract:

【Objective】 The deposition of intramuscular fat in beef cattle is closely associated with the flavor, juiciness, and tenderness of beef, characterized by the proliferation (increased in number) and differentiation (lipid deposition) of fat cells, which is regulated by multiple genes. The previous studies have indicated that Snail1 is involved in muscle development and lipid homeostasis in mice; however, its role in bovine adipogenesis remains unknown, which warrants further investigation. 【Method】 Snail1 gene CDS region sequence was cloned from Qinchuan cattle, Snail1 gene temporal and spatial expression profile was constructed, and its functional structure and target gene were predicted by bioinformatics software. Subsequently, the impact of Snail1 on the proliferation of bovine adipocytes was explored through RNAi interference, along with CCK8, EdU, cell flow cytometry, and RT-qPCR methods. 【Result】 Compared with the sequence published by NCBI, the Snail1 gene in Qinchuan cattle had two synonymous mutations. The Snail1 gene exhibited higher abundance expression in newborn cattle lung, perirenal fat, and small intestine. However, in the adult cattle, the expression level of the Snail1 gene was the highest in perirenal fat tissue, followed by longissimus dorsi muscle, and lowest in lung tissue. Bioinformatics analysis revealed a 651 bp CpG island in the Snail1 promoter region, along with binding sites for transcription factors related to fat formation, such as C/EBP and PPARα. CKⅠ (Ser92/96), CKⅡ (Ser25/119, Thr89), CDK1 (Ser13/104/112/119/143/221/183/214), CDK5 (Ser105/107), and other cell cycle related kinases could participate in the Snail1 phosphorylation of proteins. By extracting promoter regions of bovine annotated genes, predicting target genes and constructing KEGG dynamic networks, it was found that lipid formation related signaling pathways, such as MAPK, PI3K-Akt and mTOR, were potential node signaling pathways involving in Snail1’s involvement in adipogenesis. Further functional studies through RNAi interference demonstrated that downregulation of the Snail1 gene promoted the proliferation of bovine preadipocytes, increased the proportion of cells in the S phase (P<0.01), and facilitated G1/S cell cycle transition. RT-qPCR and Western blot analysis indicated that interfering with the Snail1 gene significantly upregulated the expression of proliferation- regulating genes, including CCNB1, CCND2, CDK2, and CDK4 (P<0.05), at both the genes and protein levels. 【Conclusion】 The expression of Snail1 gene was relatively high in neonatal and adult perirenal fat and longissimus dorsi muscle. Interfering with the Snail1 gene promoted the proliferation of bovine preadipocytes and the G1/S cell cycle transition, as well as the expression of CCNB1, CCND2, CDK2, CDK4, and other proliferation-related genes. Cell cycle-related kinases, including CKⅠ, CKⅡ, and CDK1/5, could participate in the regulation of cell proliferation through the phosphorylation modification of Snail1 protein. Furthermore, the MAPK, PI3K-Akt, and mTOR pathways were potential node signaling pathways influenced by Snail1. This study established a foundation for further exploration of the mechanism by which Snail1 participated in bovine fat formation.

Key words: cattle, Snail1, fat deposition, KEGG dynamic network, RNAi, cell proliferation

朱炳霖, 于嘉莉, 陈嘉玥, 田媛, 万媛, 刘晨阳, 王晓宇, 王苗力, 成功. 秦川牛Snail1克隆、表达特性分析及其对牛脂肪细胞增殖的作用研究[J]. 中国农业科学, 2024, 57(13): 2674-2686.

ZHU BingLin, YU JiaLi, CHEN JiaYue, TIAN Yuan, WAN Yuan, LIU ChenYang, WANG XiaoYu, WANG MiaoLi, CHENG Gong. Cloning, Expression Characterization, and Functional Analysis of the Snail1 in Qinchuan Cattle and Its Impact on Proliferation of Bovine Adipocytes[J]. Scientia Agricultura Sinica, 2024, 57(13): 2674-2686.

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引物 Primer	引物序列 Primer sequences (5′-3′）	退火温度 Annealing temperature (℃)	产物长度 Product length (bp)	用途 Application
Snail1-cds	F: GCGGGTGGAGGGTTTTGC R: ACGAGGGGATTCTGTGGC	55	977	编码区克隆 Coding region cloning
Snail1	F：GCACGACTCTTCTCCAGA R: AGATGAGTGTCGGCAAGG	60	116	实时荧光定量PCR Real-time qPCR
CCNB1	F：TGATGGAACTAACTATGCTGG R：GCATAACAACGAGAAGGGATT		165
CCND2	F：GGGCAAGTTGAAATGGAA R：TCATCGACGGCGGGTAC		173
CDK2	F：GGGTCCCTGTTCGTACTTATAC R：CCACTGCTGTGGAGTAGTATTT		96
CDK4	AGACCTCGAGTGTATGGCG CCCTACAATCACAGAGGCTCAA		110
CCNA2	F：GCAGCCTTTCATTTAGCACTCT R：ATTGACTGTTGTGCGTGCTG		155
MAP4K4	F：ACACGTAAGAAAGAACCCACACT R：GCCAGTTTCCACAGATCGGA		259
PHLPP1	F：ACGGAGTGGACTTTCTCCCT R：TTTCGGCTTTCACAGACCCA		280
PHLPP2	F:CCTTGGCTGTATGCTCCGAT R:AAGATAGGTCTGCGCTTGGG		292
MAPKAP1	F:AGCAGCCACCACTACAAGTC R:TATTGCGTGACTGGGGCTTT		222
GAPDH	F:AGTTCAACGGCACAGTCAAGG R: ACCACATACTCAGCACCAGCA		124
β-actin	F: TCTAGGCGGACTGTTAGC R: CCATGCCAATCTCATCTCG		82

信号通路 Pathways	互作值 Degree	信号通路潜在靶基因 Potential target genes in the pathways
MAPK 信号通路 MAPK signaling pathway	9	PTP, MKP（MAP4K4）, HGK, JIP1/2 (MAPK8IP1/2), PP2CB, TAO, etc.
PI3K-Akt 信号通路 PI3K-Akt signaling pathway	8	PHLPP1/2, TCL1, etc.
mTOR 信号通路 mTOR signaling pathway	5	TBC1D7, MAPKAP1, RICTOR, FNIP, TTI1, CLIP1, etc.
癌症相关信号通路 Pathways in cancer	5	KNG1, ARHGEF12, PLEKHG5, etc.