CTCF调控绵羊前体脂肪细胞分化的功能研究

doi:10.11843/j.issn.0366-6964.2025.12.017

摘要/Abstract

摘要：

旨在研究CTCF对绵羊前体脂肪细胞分化的作用及其调控机制。本研究采集4只8月龄的广灵大尾羊不同部位脂肪组织，实时荧光定量PCR(qRT-PCR)检测CTCF的表达水平；1只3日龄健康广灵大尾羊中分离原代前体脂肪细胞，在前体脂肪细胞内过表达或干扰CTCF基因，每个处理组设置3个重复。采用qRT-PCR、蛋白质印迹法(Western blotting)和油红O染色技术检测该基因对绵羊前体脂肪细胞分化的调控作用；对过表达CTCF的绵羊前体脂肪细胞进行转录组测序分析，筛选差异基因和通路，探索CTCF调控脂代谢相关信号通路的分子机制。结果表明，CTCF在尾部脂肪中的表达量显著高于肾周和皮下脂肪组织(P < 0.05)；与对照组相比，过表达CTCF后，成脂标志基因的表达水平较对照组显著升高(P < 0.05)，细胞内脂滴生成量增加；干扰CTCF后结果相反。转录组分析结果显示，两组间筛选出1 079个差异表达基因，这些差异表达基因富集到一些脂肪代谢相关通路，包括鞘脂信号通路、FoxO信号通路和AMPK信号通路等，筛选出CCND2、ITGA7、BRCA1、LAMA5和CCNE2等影响脂肪代谢的候选基因。本研究结果表明，CTCF能够促进绵羊前体脂肪细胞的分化，并揭示出影响CTCF脂肪代谢的相关通路及候选基因。

关键词: 绵羊, CTCF基因, 前体脂肪细胞, 分化, 转录组测序

Abstract:

This study aimed to reveal the regulating role of CTCF in the differentiation of ovine preadipocytes and its regulatory mechanism. In this study, adipose tissues from different parts of 4 8-month-old Guangling Large-tailed sheep were collected, and the expression level of CTCF was detected by quantitative real-time fluorescent quantitative PCR (qRT-PCR). Primary preadipocytes were isolated from 1 healthy 3-day-old Guangling Large-tailed sheep. CTCF gene was overexpressed or interfered in preadipocytes, with 3 replicates set for each treatment group. The regulatory effect of CTCF gene on the differentiation of sheep preadipocytes was detected using qRT-PCR, Western blotting and Oil Red O staining techniques. On this basis, transcriptomic sequencing analysis on sheep preadipocytes with CTCF overexpression was performed to explore the molecular mechanisms by which CTCF regulates lipid metabolism-related signaling pathways. The results showed that the mRNA expression of CTCF gene in the tail adipose of Guangling Large-tailed sheep was significantly higher than that in the perirenal and subcutaneous adipose tissues (P < 0.05). After overexpression of CTCF, the expression level of adipogenic marker genes was significantly increased (P < 0.05), and the amount of intracellular lipid droplets was increased. The opposite was true when the CTCF is interfered with. Transcriptome results showed that there were 1 079 differentially expressed genes between two groups. These differentially expressed genes were enriched in several pathways related to fat metabolism, including the sphingolipid signaling pathway, the FoxO signaling pathway, and the AMPK signaling pathway. Candidate genes affecting fat metabolism, such as CCND2, ITGA7, BRCA1, LAMA5, and CCNE2, were screened out. In summary, this study results shows that CTCF can promote the differentiation of sheep preadipocytes, and reveals the related pathways and candidate genes affecting CTCF-mediated fat metabolism.

Key words: sheep, CTCF gene, preadipocytes, differentiation, transcriptome sequencing

中图分类号:

S826.2

乔利英, 徐常松, 张莉, 丁毅, 潘洋洋, 杨凯捷, 刘建华, 刘文忠. CTCF调控绵羊前体脂肪细胞分化的功能研究[J]. 畜牧兽医学报, 2025, 56(12): 6130-6144.

QIAO Liying, XU Changsong, ZHANG Li, DING Yi, PAN Yangyang, YANG Kaijie, LIU Jianhua, LIU Wenzhong. Study on Function of CTCF Regulating the Differentiation of Ovine Preadipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6130-6144.

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参考文献 39

1	KALDS P , LUO Q , SUN K , et al. Trends towards revealing the genetic architecture of sheep tail patterning: promising genes and investigatory pathways[J]. Anim Genet, 2021, 52 (6): 799- 812. doi: 10.1111/age.13133
2	DENG J , XIE XL , WANG DF , et al. Paternal origins and migratory episodes of domestic sheep[J]. Curr Biol, 2020, 30 (20): 4085- 4095. doi: 10.1016/j.cub.2020.07.077
3	赵有璋. 羊生产学[M]. 北京: 中国农业出版社, 2002: 98.
	ZHAO Y Z . Sheep production[M]. Beijing: China Agriculture Press, 2002: 98.
4	LEE J E , SCHMIDT H , LAI B , et al. Transcriptional and epigenomic regulation of adipogenesis[J]. Mol Cell Biol, 2019, 39 (11): e00601- e00618.
5	BARQUISSAU V , GHANDOUR R A , AILHAUD G , et al. Control of adipogenesis by oxylipins, GPCRs and PPARs[J]. Biochimie, 2017, 136, 3- 11. doi: 10.1016/j.biochi.2016.12.012
6	LI Y , JIN D , XIE W , et al. PPAR-γ and Wnt regulate the differentiation of MSCs into adipocytes and osteoblasts respectively[J]. Curr Stem Cell Res Ther, 2018, 13 (3): 185- 192. doi: 10.2174/1574888X12666171012141908
7	YANG W , YANG C , LUO J , et al. Adiponectin promotes preadipocyte differentiation via the PPARγ pathway[J]. Mol Med Rep, 2018, 17 (1): 428- 435.
8	ZHANG M , SHAO Y , GAO B , et al. Erchen decoction mitigates lipid metabolism disorder by the regulation of PPARγ and LPL gene in a high-fat diet c57b/6 mice model[J]. Evid based Complement Alternat Med, 2020, 2020, 9102475. doi: 10.1155/2020/9102475
9	KLENOVA E M , NICOLAS R H , PATERSON H F , et al. CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms[J]. Mol Cell Biol, 1993, 13 (12): 7612- 7624.
10	ARZATE-MEJÍA R G , RECILLAS-TARGA F , CORCES V G . Developing in 3D: the role of CTCF in cell differentiation[J]. Development, 2018, 145 (6): dev137729. doi: 10.1242/dev.137729
11	KIM S , YU N K , KAANG B K . CTCF as a multifunctional protein in genome regulation and gene expression[J]. Exp Mol Med, 2015, 47 (6): e166. doi: 10.1038/emm.2015.33
12	ONG C T , CORCES V G . CTCF: an architectural protein bridging genome topology and function[J]. Nat Rev Genet, 2014, 15 (4): 234- 246. doi: 10.1038/nrg3663
13	DUBOIS-CHEVALIER J , OGER F , DEHONDT H , et al. A dynamic CTCF chromatin binding landscape promotes DNA hydroxymethylation and transcriptional induction of adipocyte differentiation[J]. Nucleic Acids Res, 2014, 42 (17): 10943- 10959. doi: 10.1093/nar/gku780
14	CHEN Y , HE R , HAN Z , et al. Cooperation of ATF4 and CTCF promotes adipogenesis through transcriptional regulation[J]. Cell Biol Toxicol, 2022, 38 (5): 741- 763. doi: 10.1007/s10565-021-09608-x
15	TOWBIN H , STAEHELIN T , GORDON J . Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications[J]. Proc Natl Acad Sci USA, 1979, 76 (9): 4350- 4354. doi: 10.1073/pnas.76.9.4350
16	李涛, 陈卫林, 卢岩, 等. 哈萨克羊不同部位脂肪特性的研究[J]. 中国油脂, 2018, 43 (7): 32-35, 40.
	LI T , CHEN W L , LU Y , et al. Fat characteristics in different parts of Kazak sheep[J]. China Oils and Fats, 43 (7): 32-35, 40.
17	张越, 曹贵方. 绵羊尾脂沉积的研究进展[J]. 当代畜禽养殖业, 2022 (4): 16- 18.
	ZHANG Y , CAO G F . Research progress on tail fat deposition in sheep[J]. Journal of Agricultural and Livestock Products Processing, 2022 (4): 16- 18.
18	QIN H , HAN Z , ZHANG W , et al. CTCF modulates adipocyte lipolysis via directly regulating the expression of Beclin 1 with the cooperation of PPARγ[J]. Cellular Signal, 2024 (113): 110968.
19	CHEN Y , HE R , HAN Z , et al. Cooperation of ATF4 and CTCF promotes adipogenesis through transcriptional regulation[J]. Cell Biol Toxicol, 2021, 38 (5): 1- 23.
20	RAICHUR S , WANG S T , CHAN P W , et al. CerS2 haploinsufficiency inhibits β-oxidation and confers susceptibility to diet-induced steatohepatitis and insulin resistance[J]. Cell Metab, 2014, 20 (5): 919. doi: 10.1016/j.cmet.2014.10.007
21	YANG G , BADEANLOU L , BIELAWSKI J , et al. Central role of ceramide biosynthesis in body weight regulation, energy metabolism, and the metabolic syndrome[J]. Am J Physiol endocrinol Metab, 2009, 297 (1): E211- E224. doi: 10.1152/ajpendo.91014.2008
22	QIAO L , ZHAO B , LIU X , et al. TPT1 promotes the adipogenic differentiation of stromal vascular fractions via the PI3K/AKT pathway and FOXO1 in sheep[J]. J Appl Anim Res, 2023, 51 (1): 388- 396. doi: 10.1080/09712119.2023.2203745
23	SONG Y , ZHANG J , JIANG C , et al. FOXO1 regulates the formation of bovine fat by targeting CD36 and STEAP4[J]. Int J Biol Macromol, 2023, 248, 126025. doi: 10.1016/j.ijbiomac.2023.126025
24	ARMONI M , HAREL C , KARNI S , et al. FOXO1 represses peroxisome proliferator-activated receptor-gamma1 and -gamma2 gene promoters in primary adipocytes. A novel paradigm to increase insulin sensitivity[J]. J Biol Chem, 2006, 281 (29): 19881- 19891. doi: 10.1074/jbc.M600320200
25	LI Y , XU S , MIHAYLOVA M M , et al. AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice[J]. Cell Metab, 2011, 13 (4): 376- 388. doi: 10.1016/j.cmet.2011.03.009
26	袁弯弯. Asprosin通过TLR4-cAMP-AMPK-ULK1通路促进脂肪细胞自噬抑制白色脂肪褐色化参与肥胖的发生[D]. 南昌: 南昌大学, 2022.
	YUAN W W. Asprosin inhibits browning by promoting the autophagy ofadipocyte via TLR4-cAMP-AMPK-ULK1 pathway in white adiposetissue[D]. Nanchang: Nanchang University, 2022. (in Chinese)
27	SUN T , HAO Z , MENG F , et al. The effects of sika deer antler peptides on 3T3-L1 preadipocytes and C57BL/6 mice via activating AMPK signaling and gut microbiota[J]. Molecules, 2025, 30 (5): 1173. doi: 10.3390/molecules30051173
28	SICINSKI P , DONAHER JL , GENG Y , et al. Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis[J]. Nature, 1996, 384 (6608): 470- 474. doi: 10.1038/384470a0
29	GENG Y , YU Q , SICINSKA E , et al. Cyclin E ablation in the mouse[J]. Cell, 2003, 114 (4): 431- 443. doi: 10.1016/S0092-8674(03)00645-7
30	YAO C C , ZIOBER B L , SQUILLACE R M , et al. Alpha7 integrin mediates cell adhesion and migration on specific laminin isoforms[J]. J Biol Chem, 1996, 271 (41): 25598- 25603. doi: 10.1074/jbc.271.41.25598
31	AUMAILLEY M , BRUCKNER-TUDERMAN L , CARTER W , et al. A simplified laminin nomenclature[J]. Matrix Biol, 2005, 24 (5): 326- 332. doi: 10.1016/j.matbio.2005.05.006
32	CHEN H J , YAN X Y , SUN A , et al. High-fat-diet-induced extracellular matrix deposition regulates integrin - FAK signals in adipose tissue to promote obesity[J]. Mol Nutr Food Res, 2022, 66 (7): e2101088. doi: 10.1002/mnfr.202101088
33	JIAO H , KULYTÉ A , NÄSLUND E , et al. Whole-exome sequencing suggests LAMB3 as a susceptibility gene for morbid obesity[J]. Diabetes, 2016, 65 (10): 2980- 2989. doi: 10.2337/db16-0522
34	王思元, 刘迪, 张伟红, 等. 基于转录组测序分析牛不同脂肪组织的脂肪沉积差异研究[J]. 西北农业学报, 2021, 30 (12): 1755- 1766.
	WANG S Y , LIU D , ZHANG W H , et al. Analysis of the difference of fat deposition in different adipose tissues of cattle based on transcriptome sequencing[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2021, 30 (12): 1755- 1766.
35	ZHAO R , KAAKATI R , LIU X , et al. CRISPR/Cas9-mediated BRCA1 knockdown adipose stem cells promote breast cancer progression[J]. Plast Reconstr Surg, 2019, 143 (3): 747- 756. doi: 10.1097/PRS.0000000000005316
36	ORTEGA F J , MORENO-NAVARRETE J M , MAYAS D , et al. Breast cancer 1 (BrCa1) may be behind decreased lipogenesis in adipose tissue from obese subjects[J]. PLoS One, 2012, 7 (5): e33233. doi: 10.1371/journal.pone.0033233
37	WENG M , ZHU X . Thrombospondin-2 induces M2 macrophage polarization through fatty acid metabolism to drive lung adenocarcinoma proliferation[J]. Anticancer Drugs, 2025, 36 (6): 459- 467. doi: 10.1097/CAD.0000000000001713
38	欧阳翱镕. 肿瘤相关脂肪细胞促进前列腺癌骨转移及其分子机制的初步研究[D]. 广州: 南方医科大学, 2024.
	OUYANG A R. A preliminary study of tumor-associated adipocytespromoting bone metastasis of prostate cancer and itsmolecular mechanism[D]. Guangzhou: Southern Medical University, 2024. (in Chinese)
39	HAO J , LIU Z , JU W , et al. Role and mechanism of FLT4 in high-fat diet-induced obesity in mice[J]. Biocheml Biophys Res Commun, 2023, 675, 61- 70. doi: 10.1016/j.bbrc.2023.06.025

名称 Name	GenBank登录号 GenBank accession number	序列(5′→3′) Sequence	产物大小/bp Products length
CTCF正向引物CTCF forward primer	XM_027978089.3	TGCTGAGCCAGACTTGGATG	150
CTCF反向引物CTCF reverse primer		TGTTTGGGCTGGTTGGTTCT
β-actin正向引物β-actin forward primer	NM_001009784.3	TGATGATATTGCTGCGCTCG	194
β-actin正向引物β-actin reverse primer		GGGTCAGGATGCCTCTCTTG

名称Name	体积/μL Volume
TB Green预混型Ex Taq Ⅱ酶(2倍浓度)TB Green Premix Ex Taq Ⅱ(2×)	10
引物(上下游)primer(forward + reverse)	2
DNA模板DNA template	100 ng
无酶水RNase-Free Water	6
总体系Total volume	补充至20

名称 Name	GenBank登录号 GenBank accession number	序列(5′→3′) Sequence	产物大小/bp Products length
PPARγ正向引物PPARγ forward primer	NM_001100921.1	ATCTTGACGGGAAAGACGAC	156
PPARγ反向引物PPARγ reverse primer		AAACTGACACCCCTGGAAGAT
C/EBPα正向引物C/EBPα forward primer	NM_001308574.1	TCCGTGGACAAGAACAGCAA	137
C/EBPα反向引物C/EBPα reverse primer		TCATTGTCACTGGTCAGCTCC
FABP4正向引物FABP4 forward primer	NM_001114667.1	AAACTGGGATGGGAAATCAACC	109
FABP4反向引物FABP4 reverse primer		TGCTCTCTCGTAAACTCTGGTAGC
Adiponectin正向引物Adiponectin forward primer	NM_001308565.1	ATCCCCGGGCTGTACTACTT	129
Adiponectin反向引物Adiponectin reverse primer		CTGGTCCACGTTCTGGTTCT

样品 Sample	原始读数 Raw Reads	原始碱基数/G Raw Bases	高质量读数 Clean Reads	高质量碱基数/G Clean Bases	GC碱基/% GC	Q20/%	Q30/%
NC1	47 491 428	7.12	45 464 734	6.82	49.89	99.15	96.14
NC2	42 400 148	6.36	39 968 312	6.00	49.94	99.16	96.33
NC3	40 446 960	6.07	37 627 358	5.64	51.64	99.09	96.05
CTCF1	48 650 008	7.30	48 493 692	7.27	53.34	98.97	95.47
CTCF2	47 789 778	7.17	47 701 964	7.16	53.17	99.09	95.95
CTCF3	43 215 118	6.48	39 365 356	5.90	52.47	99.10	96.16

ID	功能描述 Description	上调基因数 Up_Gene	下调基因数 Down_Gene
KEGG：oas04071	鞘脂信号通路Sphingolipid signaling pathway	42	33
KEGG：oas04140	动物中的自噬Autophagy - animal	58	51
KEGG：oas04068	FoxO信号通路FoxO signaling pathway	42	33
KEGG：oas04152	AMPK信号通路AMPK signaling pathway	44	30
KEGG：oas04920	脂肪细胞因子信号通路Adipocytokine signaling pathway	31	13
KEGG：oas04668	TNF信号通路TNF signaling pathway	42	24
KEGG：oas04012	ErbB信号通路ErbB signaling pathway	29	20
KEGG：oas04340	Hedgehog信号通路Hedgehog signaling pathway	20	15
KEGG：oas04910	胰岛素信号通路Insulin signaling pathway	47	35
KEGG：oas04151	PI3K/AKT信号通路PI3K-Akt signaling pathway	104	73