SFRP4基因对牛前体脂肪细胞分化作用的影响

doi:10.11843/j.issn.0366-6964.2025.02.013

Abstract

Abstract:

This study aimed to explore the effects of the screted frizzled related protein 4 (SFRP4) gene on the differentiation of bovine preadipocytes, the Qinchuan cattle perirenal preadipocytes was used as the experimental subject. Both treatment and control groups had 3 biological replicates. The full-length protein-coding sequence of the SFRP4 gene was cloned and analyzed via bioinformatics. Quantitative real-time PCR assessed SFRP4 expression across various tissues and differentiation stages in perirenal preadipocytes. Effective bovine SFRP4 siRNA sequences were screened, and an adenoviral overexpression vector was constructed for transfection and differentiation induction in bovine preadipocytes. Oil Red O staining, qRT-PCR, and Western blotting were used to evaluate the function of SFRP4. Results demonstrated the CDS of SFRP4 gene was 1 041 bp; There were high homology between Qinchuan cattle SFRP4 amino acid sequences and those of sheep and goats, with more distant relationships to mice. This protein was classified as a hydrophilic protein and had a total of 24 phosphorylation modification sites. SFRP4 was broadly expressed across tissues, with higher expression in kidney and subcutaneous fat. Temporal expression patterns of SFRP4 during perirenal preadipocyte differentiation indicated an initial increase followed by a decrease. SFRP4 knockdown resulted in reduced lipid droplet accumulation in perirenal preadipocytes, with significant downregulation of adipogenic differentiation marker genes PPARγ, FABP4, PLIN2, and SCD1 (P < 0.05), and a highly significant reduction in CEBPβ expression (P < 0.01). PPARγ (P < 0.05) and CEBPβ (P < 0.01) protein levels were also downregulated. Conversely, SFRP4 overexpression led to significant upregulation of PPARγ (P < 0.01), CEBPβ (P < 0.01), FABP4 (P < 0.05), PLIN2 (P < 0.01), and SCD1 (P < 0.01) genes expression and increased PPARγ, CEBPβ, FABP4, and SCD1 protein levels (P < 0.05). These findings provide critical insights into the regulatory role of SFRP4 in bovine perirenal preadipocyte adipogenic differentiation and lipid deposition.

Key words: Qinchuan cattle, SFRP4, perirenal preadipocytes, adipogenic differentiation

CLC Number:

S823.2

ZHAO Gangkui, GAO Haixu, YIN Siqi, SUN Honghong, XIN Yiran, ZAN Linsen, ZHAO Chunping. The Effects of the SFRP4 Gene on Bovine Preadipocyte Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 611-620.

Figures/Tables 7

Table 1

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References 39

1	孟文翔, 游伟, 路一航, 等. 不同大理石花纹等级牛肉的质构特性与营养成分分析[J]. 中国畜牧杂志, 2024, 60 (6): 339- 343.
	MENG W X , YOU W , LU Y H , et al. Analysis of texture characteristics and nutritional components of beef with different marbling grades[J]. Chinese Journal of Animal Science, 2024, 60 (6): 339- 343.
2	吕亚宁, 叶文文, 兰旅涛. 牛肌内脂肪沉积影响因素及相关基因研究进展[J]. 中国草食动物科学, 2019, 39 (4): 55- 57.
	LÜ Y N , YE W W , LAN L T . Research progress on influencing factors and related genes of fat deposition in bovine muscle[J]. China Herbivore Science, 2019, 39 (4): 55- 57.
3	MORIGNY P , BOUCHER J , ARNER P , et al. Lipid and glucose metabolism in white adipocytes: pathways, dysfunction and therapeutics[J]. Nat Rev Endocrinol, 2021, 17 (5): 276- 295. doi: 10.1038/s41574-021-00471-8
4	KANAZAWA A , TSUKADA S , KAMIYAMA M , et al. Wnt5b partially inhibits canonical Wnt/β-catenin signaling pathway and promotes adipogenesis in 3T3-L1 preadipocytes[J]. Biochem Biophys Res Commun, 2005, 330 (2): 505- 510. doi: 10.1016/j.bbrc.2005.03.007
5	PARK J R , JUNG J W , LEE Y S , et al. The roles of Wnt antagonists Dkk1 and sFRP4 during adipogenesis of human adipose tissue-derived mesenchymal stem cells[J]. Cell Prolif, 2008, 41 (6): 859- 874. doi: 10.1111/j.1365-2184.2008.00565.x
6	HUANG A Q , HUANG Y L . Role of Sfrps in cardiovascular disease[J]. Ther Adv Chronic Dis, 2020, 11, 2040622320901990. doi: 10.1177/2040622320901990
7	RECINELLA L , ORLANDO G , FERRANTE C , et al. Adipokines: new potential therapeutic target for obesity and metabolic, rheumatic, and cardiovascular diseases[J]. Front Physiol, 2020, 11, 578966. doi: 10.3389/fphys.2020.578966
8	GUAN H , ZHANG J , LUAN J , et al. Secreted frizzled related proteins in cardiovascular and metabolic diseases[J]. Front Endocrinol, 2021, 12, 712217. doi: 10.3389/fendo.2021.712217
9	BOVOLENTA P , ESTEVE P , RUIZ J M , et al. Beyond Wnt inhibition: new functions of secreted Frizzled-related proteins in development and disease[J]. J Cell Sci, 2008, 121 (Pt 6): 737- 746.
10	CRUCIAT C M , NIEHRS C . Secreted and transmembrane wnt inhibitors and activators[J]. Cold Spring Harb Perspect Biol, 2013, 5 (3): a015081.
11	BARON R , KNEISSEL M . WNT signaling in bone homeostasis and disease: from human mutations to treatments[J]. Nat Med, 2013, 19 (2): 179- 192. doi: 10.1038/nm.3074
12	ZHANG Y L , GUAN H , FU Y , et al. Effects of SFRP4 overexpression on the production of adipokines in transgenic mice[J]. Adipocyte, 2020, 9 (1): 374- 383. doi: 10.1080/21623945.2020.1792614
13	BUKHARI S A , YASMIN A , ZAHOOR M A , et al. Secreted frizzled-related protein 4 and its implication in obesity and type-2 diabetes[J]. IUBMB Life, 2019, 71 (11): 1701- 1710. doi: 10.1002/iub.2123
14	VISWESWARAN M , SCHIEFER L , ARFUSO F , et al. Wnt antagonist secreted frizzled-related protein 4 upregulates adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells[J]. PLoS One, 2015, 10 (2): e0118005. doi: 10.1371/journal.pone.0118005
15	PARK J R , JUNG J W , LEE Y S , et al. The roles of Wnt antagonists Dkk1 and sFRP4 during adipogenesis of human adipose tissue-derived mesenchymal stem cells[J]. Cell Prolif, 2008, 41 (6): 859- 874. doi: 10.1111/j.1365-2184.2008.00565.x
16	GUAN H , ZHANG Y L , GAO S C , et al. Differential patterns of secreted frizzled-related protein 4 (SFRP4) in adipocyte differentiation: adipose depot specificity[J]. Cell Physiol Biochem, 2018, 46 (5): 2149- 2164. doi: 10.1159/000489545
17	尹思琦, 赵刚奎, 高海旭, 等. 秦川牛FADS基因家族克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 2024, 51 (3): 903- 915.
	YIN S Q , ZHAO G K , GAO H X , et al. Cloning, bioinformatics analysis and tissue expression of FADS gene family in Qinchuan cattle[J]. China Animal Husbandry & Veterinary Medicine, 2024, 51 (3): 903- 915.
18	陈星伊. Bta-miR-150调控秦川肉牛前体脂肪细胞分化的作用机制研究[D]. 杨凌: 西北农林科技大学, 2021.
	CHEN X Y. Mechanism of bta-miR-150 regulating differentiation of preadipocytes in Qinchuan beef cattle[D]. Yangling: Northwest A&F University, 2021. (in Chinese)
19	宋贵兵, 贾鸿儒, 蒋蕾, 等. 秦川牛LRRN1基因表达分析及其对成肌细胞增殖分化的影响[J]. 农业生物技术学报, 2023, 31 (7): 1419- 1429.
	SONG G B , JIA H R , JIANG L , et al. Expression analysis of Qinchuan cattle (Bos taurus) LRRN1 gene and its effect on proliferation and differentiation of myoblasts[J]. Journal of Agricultural Biotechnology, 2023, 31 (7): 1419- 1429.
20	JUNJVLIEKE Z , KHAN R , MEI C G , et al. Effect of ELOVL6 on the lipid metabolism of bovine adipocytes[J]. Genomics, 2020, 112 (3): 2282- 2290. doi: 10.1016/j.ygeno.2019.12.024
21	JASPARD B , COUFFINHAL T , DUFOURCQ P , et al. Expression pattern of mouse sFRP-1 and mWnt-8 gene during heart morphogenesis[J]. Mech Dev, 2000, 90 (2): 263- 267. doi: 10.1016/S0925-4773(99)00236-1
22	WONG V K , YAM J W , HSIAO W L . Cloning and characterization of the promoter region of the mouse frizzled-related protein 4 gene[J]. Biol Chem, 2003, 384 (8): 1147- 1154.
23	LEIMEISTER C , BACH A , GESSLER M . Developmental expression patterns of mouse sFRP genes encoding members of the secreted frizzled related protein family[J]. Mech Dev, 1998, 75 (1-2): 29- 42. doi: 10.1016/S0925-4773(98)00072-0
24	LIU M M , HEMBA-WADUGE R U S , LI X , et al. Wnt/Wingless signaling promotes lipid mobilization through signal-induced transcriptional repression[J]. Proc Natl Acad Sci U S A, 2024, 121 (28): e1972901175.
25	CHEN K X , XIONG J C , XU S , et al. Adipose-derived stem cells exosomes improve fat graft survival by promoting prolipogenetic abilities through Wnt/β-catenin pathway[J]. Stem Cells Int, 2022, 2022, 5014895.
26	ARDITO F , GIULIANI M , PERRONE D , et al. The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review)[J]. Int J Mol Med, 2017, 40 (2): 271- 280. doi: 10.3892/ijmm.2017.3036
27	LOW K O , MUHAMMAD M N , MD I R . Optimisation of signal peptide for recombinant protein secretion in bacterial hosts[J]. Appl Microbiol Biotechnol, 2013, 97 (9): 3811- 3826. doi: 10.1007/s00253-013-4831-z
28	CARMON K S , LOOSE D S . Development of a bioassay for detection of Wnt-binding affinities for individual frizzled receptors[J]. Anal Biochem, 2010, 401 (2): 288- 294. doi: 10.1016/j.ab.2010.03.009
29	ABU-JAWDEH G , COMELLA N , TOMITA Y , et al. Differential expression of frpHE: a novel human stromal protein of the secreted frizzled gene family, during the endometrial cycle and malignancy[J]. Lab Invest, 1999, 79 (4): 439- 447.
30	BÉRUBÉ M , ABEDINI A , LAPOINTE E , et al. SFRP4 promotes autophagy and blunts FSH responsiveness through inhibition of AKT signaling in ovarian granulosa cells[J]. Cell Commun Signal, 2024, 22 (1): 396. doi: 10.1186/s12964-024-01736-1
31	REN M M , YE X Q , OUYANG C , et al. JMJD2A mediates transcriptional activation of SFRP4 and regulates oxidative stress and mitochondrial dysfunction in heart failure[J]. Pathol Int, 2024, 74 (4): 210- 221. doi: 10.1111/pin.13413
32	YUE L , LU Z K , GUO T T , et al. Key genes and metabolites that regulate wool fibre diameter identified by combined transcriptome and metabolome analysis[J]. Genomics, 2024, 116 (5): 110886.
33	MAGANGA R , GILES N , ADCROFT K , et al. Secreted Frizzled related protein-4 (sFRP4) promotes epidermal differentiation and apoptosis[J]. Biochem Biophys Res Commun, 2008, 377 (2): 606- 611.
34	CHEN S , JIANG Y C , QI X Y , et al. Bioinformatics analysis to obtain critical genes regulated in subcutaneous adipose tissue after bariatric surgery[J]. Adipocyte, 2022, 11 (1): 550- 561.
35	冯雪, 赵金辉, 汪书哲, 等. 过表达circNMT1促进水牛脂肪细胞的成脂分化[J]. 畜牧兽医学报, 2022, 53 (4): 1077- 1088.
	FENG X , ZHAO J H , WANG S Z , et al. Overexpression of circNMT1 promotes adipogenic differentiation of buffalo adipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53 (4): 1077- 1088.
36	GUAN H , ZHENG H Y , ZHANG J , et al. Secreted frizzled-related protein 4 promotes brown adipocyte differentiation[J]. Exp Ther Med, 2021, 21 (6): 637.
37	WANG C H , LUNDH M , FU A , et al. CRISPR-engineered human brown-like adipocytes prevent diet-induced obesity and ameliorate metabolic syndrome in mice[J]. Sci Transl Med, 2020, 12 (558): eaaz8664.
38	VISWESWARAN M , SCHIEFER L , ARFUSO F , et al. Wnt antagonist secreted frizzled-related protein 4 upregulates adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells[J]. PLoS One, 2015, 10 (2): e0118005.
39	JANANI C , KUMARI B D R . PPAR gamma gene-a review[J]. Diabetes Metab Syndr: Res Rev, 2015, 9 (1): 46- 50.

引物 Primer	引物序列(5'→3') Sequence	扩增片段大小/bp Fragment size
β-actin-F	TCTAGGCGGACTGTTAGC	82
β-actin-R	CCATGCCAATCTCATCTCG
SFRP4-F	GCGCTCACGGATGATGCTTCT	135
SFRP4-R	CCTGCTGTTCGCTTCTTGTCCTG
PPARγ-F	GAGATCACAGAGTACGCCAAG	216
PPARγ-R	GGGCTCCATAAAGTCACCAA
CEBPβ-F	TTCCTCTCCGACCTCTTCTC	79
CEBPβ-R	CCAGACTCACGTAGCCGTACT
FABP4-F	GCTGCACTTCTTTCTCACCTTG	194
FABP4-R	ACCACACCCCCATTCAAACT
SCD1-F	CCATCAACCCCCGAGAGA	76
SCD1-R	AAGGTGTGGTGGTAGTTGTGGAA
PLIN2-F	AGTGAACTTGCCAGGAAGAATG	120
PLIN2-R	TTCATCTGTATCATCGTAGCCG
CTNNB1-F	AATCAGCTGGCCTGGTTTGA	146
CTNNB1-R	GCTTGGTTAGTGTGTCAGGC
Wnt7A-F	CGTCATAGGAGAAGGCTCGC	115
Wnt7A-R	TTTGAGCTCCTTGCCGAAGA

类别 Sort	不同二级结构占比/% Percentage of different secondary structures
类别 Sort	α-螺旋 α-helix	延伸链 Extended strand	无规则卷曲 Random coil	模棱两可状态 The ambiguous state of being
DSC	24.86	7.51	67.63	0
MLRC	32.37	9.25	58.38	0
PHD	28.90	8.67	62.43	0
Sec.Cons.	28.32	7.80	61.85	2.02

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The Effects of the SFRP4 Gene on Bovine Preadipocyte Differentiation

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