干扰Smad3促进山羊脂肪细胞分化

doi:10.11843/j.issn.0366-6964.2020.03.008

Abstract

Abstract: The aim of this study was to clone goat Smad3, to clarify its tissue and cell expression profiles, and finally to elucidate the effect of interfering Smad3 gene on the differentiation of goat intramuscular and subcutaneous adipocytes. In this study, 5 one-week-old Jianzhou Big-ear goats in good physical condition were selected, slaughtered after fasted for 24 h, and the corresponding tissues and cells were sampled for testing. The cDNA sequence of goat Smad3 gene was cloned by RT-PCR and analyzed by bioinformatics. Real-time quantitative PCR (qPCR) was used to detect the tissue and cell temporal expression of Smad3 gene. siRNA targeting to Smad3 was designed and synthesized. Oil red O staining was used to detect the effect of interfering Smad3 on adipogenic differentiation of goat preadipocytes, and qPCR was used to detect the effect of interfering Smad3 on the expression levels of adipocyte differentiation marker genes (C/EBPα, C/EBPβ, LPL, SREBP1, AP2, PPARγ, Pref-1, KLF3, KLF4, KLF6, KLF7, KLF8, KLF9, KLF10 and KLF15), Smads-related genes (Smad2, Smad4, Smad7) and TGF-β1 mRNA, with exploring the possible mechanism. The results showed that the goat Smad3 gene was 1 449 bp in length, the CDS region sequence was 1 278 bp, encoding 425 amino acids. Smad3 was widely expressed in goat tissues with the highest expression level in the kidney (P<0.01); Smad3 expression was lowest in goat intramuscular and subcutaneous adipocytes at 36 h of induced differentiation, which was significantly lower than that in undifferentiated precursor adipocytes (P<0.01). The interference of Smad3 significantly promoted the accumulation of lipid droplets in intramuscular and subcutaneous adipocytes of goats, the expression level of adipocyte differentiation marker genes, KLF3, KLF4, KLF8, KLF9, KLF10 and KLF15 were significantly increased (P<0.05), and the relative expression level of Pref-1 was extremely significantly decreased (P<0.01). At the same time, the interference of Smad3 gene down-regulated the relative expression levels of Smad2, Smad4 and Smad7 genes (P<0.05). The results indicate that interference of Smad3 promotes the differentiation of goat adipocytes by regulating the expression of adipocyte differentiation marker genes LPL, SREBP1, AP2, C/EBPα, C/EBPβ, Pref-1, KLF3, KLF4, KLF8, KLF9, KLF10 and KLF15 cooperating with Smad2, Smad4 and Smad7.

Key words: goat, Smad3, gene cloning, RNA interference, intramuscular adipocytes, subcutaneous adipocytes

CLC Number:

CUI Sheng, LIN Yaqiu, XU Qing, ZHU Jiangjiang, WANG Yong. Interfering Smad3 Promotes Goat Adipocyte Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 475-489.

References 48

[1]	ANDERSON F,PANNIER L,PETHICK D W,et al.Intramuscular fat in lamb muscle and the impact of selection for improved carcass lean meat yield[J].Animal,2015,9(6):1081-1090.
[2]	EATON S,FUKUMOTO K,DURAN N P,et al.Carnitine palmitoyl transferase I and the control of myocardial β-oxidation flux[J].Biochem Soc Trans,2001,29(2):245-250.
[3]	WANG Y,LI H,ZHANG Y D,et al.Analysis on association of a SNP in the chicken OBR gene with growth and body composition traits[J].Asian-Australas J Anim Sci,2006,19(12):1706-1710.
[4]	WON K K,CHOI H R,PARK S G,et al.Myostatin inhibits brown adipocyte differentiation via regulation of Smad3-mediated β-catenin stabilization[J].Int J Biochem Cell Biol,2012,44(2):327-334.
[5]	TANG L Y,HELLER M,MENG Z J,et al.TGF-β directly activates the JAK1-STAT3 axis to induce hepatic fibrosis in coordination with SMAD pathway[J].J Biol Chem, 2017,292(10):4302-4312.
[6]	LI Y,LIU H X,LIANG Y Z,et al.DKK3 regulates cell proliferation,apoptosis and collagen synthesis in keloid fibroblasts via TGF-β1/Smad signaling pathway[J].Biomed Pharmacother,2017,91:174-180.
[7]	GAARENSTROOM T,HILL C S.TGF-β signaling to chromatin:how Smads regulate transcription during self-renewal and differentiation[J].Semin Cell Dev Biol,2014,32:107-118.
[8]	YADAV H,QUIJANO C,KAMARAJU A K,et al.Protection from obesity and diabetes by blockade of TGF-β/Smad3 signaling[J].Cell Metab,2011,14(1):67-79.
[9]	LI X,MCFARLAND D C,VELLEMAN S G.Effect of Smad3-mediated transforming growth factor-β1 signaling on satellite cell proliferation and differentiation in chickens[J].Poult Sci,2008,87(9):1823-1833.
[10]	YANG Y A,ZHANG G M,FEIGENBAUM L,et al.Smad3 reduces susceptibility to hepatocarcinoma by sensitizing hepatocytes to apoptosis through downregulation of Bcl-2[J].Cancer Cell,2006,9(6):445-457.
[11]	ASHCROFT G S,YANG X,GLICK A B,et al.Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response[J].Nat Cell Biol,1999,1(5):260-266.
[12]	HU B,WU Z,PHAN S H.Smad3 mediates transforming growth factor-β induced α-smooth muscle actin expression[J]. Am J Respir Cell Mol Biol,2003,29(3):397-404.
[13]	CHOY L,SKILLINGTON J,DERYNCK R.Roles of autocrine TGF-β Receptor and Smad signaling in adipocyte differentiation[J].J Cell Biol,2000,149(3):667-682.
[14]	SPIEGELMAN B M,FLIER J S.Adipogenesis and obesity:rounding out the big picture[J].Cell,1996,87(3):377-389.
[15]	HOOT K E,LIGHTHALL J,HAN G,et al.Keratinocyte-specific Smad2 ablation results in increased epithelial-mesenchymal transition during skin cancer formation and progression[J].J Clin Invest,2008,118(8):2722-2732.
[16]	MENG X M,HUANG X R,CHUNG A C K,et al.Smad2 protects against TGF-β/Smad3-mediated renal fibrosis[J].J Am Soc Nephrol,2010,21(9):1477-1487.
[17]	FENG X H,DERYNCK R.Specificity and versatility in TGF-β signaling through Smads[J].Annu Rev Cell Dev Biol,2005,21:659-693.
[18]	ZHANG L,NING Y,LI P W,et al.Tissue expression analysis and characterization of Smad3 promoter in bovine myoblasts and preadipocytes[J].DNA Cell Biol,2018,37(6):551-559.
[19]	ISLAM S S,MOKHTARI R B,EL HOUT Y,et al.TGF-β1 induces EMT reprogramming of porcine bladder urothelial cells into collagen producing fibroblasts-like cells in a Smad2/Smad3-dependent manner[J].J Cell Commun Signal,2014,8(1):39-58.
[20]	LIN C H,SHIH C H,LIN Y C,et al.MEKK1,JNK,and SMAD3 mediate CXCL12-stimulated connective tissue growth factor expression in human lung fibroblasts[J].J Biomed Sci,2018,25(1):19.
[21]	LU C P,YIN Y P,CUI Y L,et al.1,25(OH)2D3 improves blood lipid metabolism,liver function,and atherosclerosis by constraining the TGF-β/Smad signaling pathway in rats with hyperlipidemia[J].Cell Cycle,2019,18(22):3111-3124.
[22]	许晴,林森,朱江江,等.山羊肌内前体脂肪细胞诱导分化过程中内参基因的表达稳定性分析[J].畜牧兽医学报,2018,49(5):907-918.XU Q,LIN S,ZHU J J,et al.The expression stability analysis of reference genes in the process of goat intramuscular preadipocytes differentiation in goat[J].Acta Veterinaria et Zootechnica Sinica,2018,49(5):907-918.(in Chinese)
[23]	LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the method[J].Methods,2001,25(4):402-408.
[24]	LIU D,BLACK B L,DERYNCK R.TGF-β inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3[J].Genes Dev,2001,15(22):2950-2966.
[25]	OLSEN O E,WADER K F,HELLA H,et al.Activin a inhibits BMP-signaling by binding ACVR2A and ACVR2B[J].Cell Commun Signal,2015,13(1):27.
[26]	MITSUI K,SHIRAKATA M,PATERSON B M.Phosphorylation inhibits the DNA-binding activity of MyoD homodimers but not MyoD-E12 heterodimers[J].J Biol Chem,1993,268(32):24415-24420.
[27]	程萱,徐晓玲,沈善祥,等.小鼠Smad3基因的克隆及其在小鼠组织中的表达[J].中国生物化学与分子生物学报,2002,18(1):14-18.CHENG X,XU X L,SHEN S X,et al.Cloning of murine Smad3 gene and its expression pattern in mouse tissues[J].Chinese Journal of Biochemistry and Molecular Biology,2002,18(1):14-18.(in Chinese)
[28]	刘育林.鸡Smad3的克隆表达及其与Myostatin关系的研究[D].哈尔滨:哈尔滨工业大学,2004.LIU Y L.Newly cloned chicken Smad3 isdownregulated by myostatin[D].Harbin:Harbin Institute of Technology,2004.(in Chinese)
[29]	TSURUTANI Y,FUJIMOTO M,TAKEMOTO M,et al.The roles of transforming growth factor-β and Smad3 signaling in adipocyte differentiation and obesity[J] Biochem Biophys Res Commun,2011,407(1):68-73.
[30]	ZHOU S H,LECHPAMMER S,GREENBERGER J S,et al.Hypoxia inhibition of Adipocytogenesis in Human bone marrow stromal cells requires transforming growth factor-β/Smad3 signaling[J].J Biol Chem,2005, 280(24):22688-22696.
[31]	GUNARATNE A,CHAN E,EL-CHABIB T H,et al.aPKC alters the TGFβ response in NSCLC cells through both Smad-dependent and Smad-independent pathways[J].J Cell Sci,2015,128(3):487-498.
[32]	TONTONOZ P,HU E D,SPIEGELMAN B M.Stimulation of adipogenesis in fibroblasts by PPARγ2,a lipid-activated transcription factor[J].Cell,1994,79(7):1147-1156.
[33]	ZHANG F J,PAN T L,NIELSEN L D,et al.Lipogenesis in fetal rat lung importance of C/EBPα,SREBP-1c,and stearoyl-CoA desaturase[J].Am J Respir Cell Mol Biol,2004,30(2):174-183.
[34]	HALDAR S M,IBRAHIM O A,JAIN M K.Kruppel-like Factors (KLFs) in muscle biology[J].J Mol Cell Cardiol, 2007, 43(1):1-10.
[35]	BREY C W,NELDER M P,HAILEMARIAM T,et al.Krüppel-like family of transcription factors:an emerging new frontier in fat biology[J].Int J Biol Sci,2009,5(6):622-636.
[36]	王翠喆,哈晓丹,李伟,等.KLF4、KLF7在大鼠网膜脂肪组织中的表达与炎症的相关性研究[J].免疫学杂志,2016,32(3):204-209.WANG C Z,HA X D,LI W,et al.The correlation between the KLF4,KLF7 expression and inflammatory in the omental adipose tissue of rats[J].Immunolo-gical Journal,2016,32(3):204-209.(in Chinese)
[37]	LI D,YEA S,LI S D,et al.Krüppel-like Factor-6 promotes preadipocyte differentiation through Histone Deacetylase 3-dependent repression of DLK1[J].J Biol Chem,2005,280(29):26941-26952.
[38]	徐嘉威,贺花,张静,等.转录因子KLF8基因结构及其功能研究进展[J].中国生物工程杂志,2018,38(4):90-95.XU J W,HE H,ZHANG J,et al.Research progress on the structure and function of transcription factor KLF8 gene[J].China Biotechnology,2018,38(4):90-95.(in Chinese)
[39]	SUN G R,ZHANG M,SUN J W,et al.Krüppel-like factor KLF9 inhibits chicken intramuscular preadipocyte differentiation[J].Br Poult Sci,2019,60(6):790-797.
[40]	YANG Y J,ZHU Z,WANG D T,et al.Tanshinol alleviates impaired bone formation by inhibiting adipogenesis via KLF15/PPARγ2 signaling in GIO rats[J].Acta Pharmacol Sin,2018,39(4):633-641.
[41]	GUO H F,KHAN R,RAZA S H A,et al.KLF15 promotes transcription of KLF3 gene in bovine adipocytes[J]. Gene,2018,659:77-83.
[42]	YUAN L,PENG W Q,GUO Y Y,et al.Krüppel-like factor 10(KLF10) is transactivated by the transcription factor C/EBPβ and involved in early 3T3-L1 preadipocyte differentiation[J].J Biol Chem,2018,293(36):14012-14021.
[43]	LIU N,LI Z,PEI D Q,et al.Zfyve9a regulates the proliferation of hepatic cells during zebrafish embryogenesis[J].Int J Dev Biol,2013,57(9-10):773-778.
[44]	INMAN G J,NICOLÁS F J,HILL C S.Nucleocytoplasmic shuttling of Smads 2,3,and 4 permits sensing of TGF-β receptor activity[J].Mol Cell,2002,10(2):283-294.
[45]	XU L,KANG Y B,ÇÖL S,et al.Smad2 nucleocytoplasmic shuttling by nucleoporins CAN/Nup214 and Nup153 feeds TGFβ signaling complexes in the cytoplasm and nucleus[J].Mol Cell,2002,10(2):271-282.
[46]	DE KROON L M G,NARCISI R,VAN DEN AKKER G G H,et al.SMAD3 and SMAD4 have a more dominant role than SMAD2 in TGFβ-induced chondrogenic differentiation of bone marrow-derived mesenchymal stem cells[J].Sci Rep,2017,7:43164.
[47]	马松林,赵秋,龚勇,等.纤维化胰腺组织中TGF-β1、Smad3、Smad7的表达及意义[J].世界华人消化杂志,2007,15(2):185-188.MA S L,ZHAO Q,GONG Y,et al.Expression of transforming growth factor-β1,Smad3 and Smad7 and their significances in pancreatic fibrosis[J].World Chinese Journal of Digestology,2007,15(2):185-188.(in Chinese)
[48]	SCHMIERER B,HILL C S.Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth β-dependent nuclear accumulation of Smads[J].Mol Cell Biol,2005,25(22):9845-9858.

Interfering Smad3 Promotes Goat Adipocyte Differentiation

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 48

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	DONG Shucan, MAO Shuaixiang, WU Cuiying, LI Yaokun, SUN Baoli, GUO Yongqing, DENG Ming, LIU Dewu, LIU Guangbin. The Effect of the Androgen Receptor Inhibitor Enzalutamide on Proliferation and Apoptosis of Goat Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2022-2031.
[2]	LI Qiuyun, TIAN Xinyuan, LIAO Wensheng, ZHANG Huanrong, REN Yupeng, YANG Falong, ZHU Jiangjiang, XIANG Hua. Effects of SOCS2 on Proliferation, Cycle and Apoptosis of Turbinate Bone Cells in Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2226-2240.
[3]	LI Pengfei, GAO Guiqin, ZHOU Guangqing, WU Jinyan, YAN Xinmin, CAO Xiaoan, HE Jijun, YUAN Ligang, SHANG Youjun. Establishment and Application of TaqMan Fluorescence Quantitative RT-PCR Detection Method for Enzootic Nasal Tumor Virus of Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2259-2266.
[4]	PENG Peiya, CHEN Yuhan, YANG Long, WANG Ming, ZHAO Ruiting, HE Jun, YIN Yulong, LIU Mei. Research Progress of Copy Number Variation in Livestock [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1356-1369.
[5]	HUANG Xianpeng, XING Jiayi, BAI Yuanyuan, JIANG Yuting, MA Zhiwei, FU Wei, LAN Daoliang. Cloning of Six Pluripotent Related Transcription Factors OSKMNL in Yak and Construction of Polycistron Lentiviral Vector [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1579-1591.
[6]	KANG Jia, DUAN Xiangru, YIN Xuejiao, YANG Ruochen, LI Taichun, SHAN Xinyu, CHEN Meijing, ZHANG Yingjie, LIU Yueqin. Effects of Cysteine and Methionine on Secondary Hair Follicle Growth and Hair Dermal Papilla Cell Proliferation in vitro in Cashmere Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 515-527.
[7]	SONG Yan, YUAN Yongfeng, QIAN Hongyu, LI Xincan, LUO Hongyan, WANG Zhiying, ZHOU Zuoyong. Identification and Partial Biological Characteristics Analysis of Corynebacterium pseudotuberculosis Isolated from Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 680-687.
[8]	YAN Xiaochun, XI Haijiao, LI Jinquan, WANG Zhiying, SU Rui. Study on Estimates of Genomic Breeding Value of Fleece Traits in Inner Mongolia Cashmere Goats [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 120-128.
[9]	ZHANG Chenjian, LI Yinxia, DING Qiang, LIU Weijia, WANG Huili, HE Nan, WU Jiashun, CAO Shaoxian. Efficient Preparation of CRISPR/Cas9-mediated Goat SOCS2 Gene Edited Embryos [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 129-141.
[10]	ZHANG De'an, YANG Ruozhu, LIU Jie, LIU Dewu, DENG Ming, LIU Guangbin, SUN Baoli, GUO Yongqing, LI Yaokun. Expression Analysis of Transcriptome in the Liver of Chuanzhong Black Goats Fed with Silage Neolamarckia Cadamba Substitute for Silage Corn [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 232-244.
[11]	TANG Yinmei, LI Qi, LI Haiyang, LIN Yaqiu, WANG Yong, XIANG Hua, HUANG Lian, ZHU Jiangjiang. Cloning of the Goat FATP2 Gene and Its Regulatory Effect on Lipid Deposition in Precursor Adipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3642-3652.
[12]	SHAO Peng, TANG Yinmei, LIN Yaqiu, WANG Yong, XIANG Hua, HUANG Lian, ZHU Jiangjiang. Regulation Effect of PSMD9 on Lipid Deposition in Goat Precursor Adipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3653-3663.
[13]	LIU Xinhuan, YUN Jialei, MAO Li, LI Jizong, HAO Fei, HE Miaofeng, YANG Leilei, ZHANG Wenwen, CHENG Zilong, SUN Min, LIU Maojun, WANG Shaohui, BAI Juan, LI Wenliang. Isolation, Identification, Virulence Genes and Drug Resistance Analysis of Escherichia coli Isolated from Diarrheal Goat and Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3445-3454.
[14]	ZHANG Renbao, ZHOU Donghui, ZHOU Lisheng, GAO Xiaoxiao, LIU Nan, HE Jianning. Analysis of Genetic Structure of Conservation Population in Jining Gray Goats Based on 70 K SNP Chip [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2836-2847.
[15]	SHAO Yuexin, ZHANG Xinyu, GE Liyan, SHI Huaiping. Cloning and RNA Interference Analysis of ATF4 Gene in Xinong Saanen Dairy Goat [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2353-2364.