Wnt10b对山羊前体脂肪细胞分化相关基因表达的影响

doi:10.11843/j.issn.0366-6964.2018.04.004

摘要/Abstract

摘要：

旨在利用RNA干扰技术，明确Wnt10b基因对山羊前体脂肪细胞分化的影响。设计合成山羊Wnt10b siRNA序列，利用胶原酶消化法获得山羊皮下和肌内前体脂肪细胞。利用有效的siRNA序列干扰山羊皮下和肌内前体脂肪细胞Wnt10b基因表达后，检测其对细胞分化及脂肪细胞分化标志基因表达的影响。结果表明，筛选获得有效的山羊Wnt10b siRNA序列，转染山羊皮下和肌内前体脂肪细胞后，Wnt10b被显著干扰，其mRNA表达量分别下调63%（P<0.01）和67%（P<0.01）；油红O染色结果显示，干扰Wnt10b基因可明显抑制山羊皮下和肌内脂肪细胞的脂滴积聚；且干扰Wnt10b基因后，在皮下前体脂肪细胞分化过程中C/EBPβ、PPARγ、SREBP1和AP2基因出现极显著下调（P<0.01），LPL出现显著下调（P<0.05）；而在肌内前体脂肪细胞分化过程中，AP2和LPL基因出现极显著下调（P<0.01），C/EBPβ出现显著下调（P<0.05），Pref1出现极显著上调（P<0.01）。本试验发现，Wnt10b基因可能通过调控C/EBPβ、PPARγ、SREBP1和LPL的表达来促进山羊皮下前体脂肪细胞分化，通过调控AP2、LPL、C/EBPβ和Pref1的表达来促进山羊肌内前体脂肪细胞分化。

关键词: 山羊, Wnt10b基因, RNA干扰, 皮下脂肪细胞, 肌内脂肪细胞

Abstract:

The aim of this study was to investigate the effect of Wnt10b gene on the precursor adipocytes differentiation of goat by RNA interference. The goat Wnt10b siRNA sequence was designed and compounded,and the goat subcutaneous and intramuscular precursor adipocytes were obtained by collagenase digestion. The effect of Wnt10b gene expression on the cell differentiation and adipocyte differentiation marker genes expression were detected by using the effective siRNA sequence to interfere the expression of Wnt10b gene in goat subcutaneous and intramuscular precursor adipocytes.The results showed that an effective goat Wnt10b siRNA sequence was obtained,and the expression of Wnt10b was down-regulated by 63% (P<0.01) and 67% (P<0.01), respectively after transfection in goat subcutaneous and intramuscular precursor adipocytes. Results of Oil red O staining showed that the accumulation of lipid droplets were inhibited significantly by Wnt10b RNAi in goat subcutaneous and intramuscular adipocytes. The C/EBPβ, PPARγ,SREBP1 and Pref1 genes were extremely significantly down-regulated in the subcutaneous precursor adipocyte differentiation after Wnt10b RNAi (P<0.01), LPL was significantly down-regulated (P<0.05). The AP2 and LPL were extremely significantly down-regulated (P<0.01), C/EBPβ was significantly down-regulated (P<0.05), and Pref1 was extremely significantly up-regulated (P<0.01) in intramuscular precursor adipocytes differentiation. In this study, it was found that Wnt10b gene could promote the differentiation of goat subcutaneous precursor adipocytes by regulating the expression of C/EBPβ, PPARγ, SREBP1 and LPL, and promote the differentiation of goat intramuscular precursor adipocytes by regulating the expression of AP2, LPL, C/EBPβ and Pref1.

Key words: goat, Wnt10b gene, RNA interference, subcutaneous adipocytes, intramuscular adipocytes

中图分类号:

S827.2

林森, 林亚秋, 朱江江, 许晴, 赵越, 池永东, 王永. Wnt10b对山羊前体脂肪细胞分化相关基因表达的影响[J]. 畜牧兽医学报, 2018, 49(4): 685-692.

LIN Sen, LIN Ya-qiu, ZHU Jiang-jiang, XU Qing, ZHAO Yue, CHI Yong-dong, WANG Yong. Effect of Wnt10b on the Expression of Precursor Adipocytes Differentiation Related Genes in Goat[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(4): 685-692.

参考文献

[1] NOVAKOFSKI J. Adipogenesis:usefulness of in vitro and in vivo experimental models[J]. J Anim Sci, 2004, 82(3):905-915.
[2] HARPER G S, PETHICK D W. How might marbling begin?[J]. Aust J Exp Agric, 2004, 44(7):653-662.
[3] DU M, WANG B, FU X, et al. Fetal programming in meat production[J]. Meat Sci, 2015, 109:40-47.
[4] JO J, SHREIF Z, PERIWAL V. Quantitative dynamics of adipose cells[J]. Adipocyte, 2012, 1(2):80-88.
[5] NUSSE R, CLEVERS H. Wnt/β-catenin signaling, disease, and emerging therapeutic modalities[J]. Cell, 2017, 169(6):985-999.
[6] TANAKA K, KITAGAWA Y, KADOWAKI T. Drosophila segment polarity gene product porcupine stimulates the posttranslational N-glycosylation of wingless in the endoplasmic reticulum[J]. J Biol Chem, 2012, 277(15):12816-12823.
[7] LONGO K A, WRIGHT W S, KANG S, et al. Wnt10b inhibits development of white and brown adipose tissues[J]. J Biol Chem, 2004, 279(34):35503-35509.
[8] KUEM N, SONG S J, YU R N, et al. Oleuropein attenuates visceral adiposity in high-fat diet-induced obese mice through the modulation of WNT10b-and galanin-mediated signalings[J]. Mol Nutr Food Res, 2014, 58(11):2166-2176.
[9] KARCZEWSKA-KUPCZEWSKA M, STEFANOWICZ M, Matulewicz N, et al. Wnt signaling genes in adipose tissue and skeletal muscle of humans with different degrees of insulin sensitivity[J]. J Clin Endocrinol Metab, 2016, 101(8):3079-3087.
[10] WRIGHT W S, LONGO K A, DOLINSKY V W, et al. Wnt10b inhibits obesity in ob/ob and agouti mice[J]. Diabetes, 2007, 56(2):295-303.
[11] LUO H F, WEI H K, HUANG F R, et al. The effect of linseed on intramuscular fat content and adipogenesis related genes in skeletal muscle of pigs[J]. Lipids, 2009, 44(11):999-1010.
[12] JEONG J Y, KIM J S, NGUYEN T H, et al. Wnt/β-catenin signaling and adipogenic genes are associated with intramuscular fat content in the longissimus dorsi muscle of Koreancattle[J]. Anim Genet, 2013, 44(6):627-635.
[13] WANG Y H, BYRNE K A, REVERTER A, et al. Transcriptional profiling of skeletal muscle tissue from two breeds of cattle[J]. Mamm Genome, 2005, 16(3):201-210.
[14] 曹怡玮, 郭熙志. Wnt信号通路调控脂肪发育与代谢的研究进展[J]. 中国科学:生命科学, 2017, 47(4):355-362.
CAO Y W, GUO X Z. Wnt signaling regulates adipose tissue formation and metabolism[J]. Scientia Sinica Vitae, 2017, 47(4):355-362. (in Chinese)
[15] ROSS S E, HEMATI N, LONGO K A, et al. Inhibition of adipogenesis by Wnt signaling[J]. Science, 2000, 289(5481):950-953.
[16] CAWTHORN W P, BREE A J, YAO Y, et al. Wnt6, Wnt10a and Wnt10b inhibit adipogenesis and stimulate osteoblastogenesis through a β-catenin-dependent mechanism[J]. Bone, 2012, 50(2):477-489.
[17] BIRSOY K, CHEN Z, FRIEDMAN J. Transcriptional regulation of adipogenesis by KLF4[J]. Cell Metab, 2008, 7(4):339-347.
[18] PARK Y K, WANG L M, GIAMPIETRO A, et al. Distinct roles of transcription factors KLF4, Krox20 and PPARγ in adipogenesis[J]. Mol Cell Biol, 2017, 37(2):e00554-16.
[19] JIANG S Z, WEI H K, SONG T X, et al. KLF13 promotes porcine adipocyte differentiation through PPARγ activation[J]. CellBiosci, 2015, 5:28.
[20] FARMER S R. Transcriptional control of adipocyte formation[J]. Cell Metab, 2006, 4(4):263-273.
[21] TANG Q Q, ZHANG J W, LANE M D. Sequential gene promoter interactions of C/EBPβ, C/EBPα, and PPARγ during adipogenesis[J]. Biochem Biophys Res Commun, 2004, 319(1):235-239.
[22] CHMURZYN'SKA A. The multigene family of fatty acid-binding proteins(FABPs):function, structure and polymorphism[J]. J Appl Genet, 2006, 47(1):39-48.
[23] JEON T I, OSBORNE T F. SREBPs:metabolic integrators in physiology and metabolism[J]. Trends Endocrinol Metab, 2012, 23(2):65-72.
[24] MEAD J R, IRVINE S A, RAMJI D P. Lipoprotein lipase:structure, function, regulation, and role in disease[J]. J Mol Med, 2002, 80(12):753-769.
[25] 王刚, 曾勇庆, 武英, 等. 猪肌肉组织LPL基因表达的发育性变化及其与肌内脂肪沉积关系的研究[J]. 畜牧兽医学报, 2013, 38(3):253-257.
WANG G, ZENG Y Q, WU Y, et al. The developmental changes of LPL mRNA expression in muscle and their association with intramuscular fat for pigs[J]. Acta Veterinaria et Zootechnica Sinica,2013, 38(3):253-257. (in Chinese)
[26] JEONG J, KWON E G, IM S K, et al. Expression of fat deposition and fat removal genes is associated with intramuscular fat content in longissimus dorsi muscle of Korean cattle steers[J]. J Anim Sci, 2012, 90(6):2044-2053.
[27] 乔永, 黄治国, 李齐发, 等. 绵羊肌肉LPL基因表达的发育性变化及其对肌内脂肪含量的影响[J]. 中国农业科学, 2007, 40(10):2323-2330.
QIAO Y, HUANG Z G, LI Q F, et al. Developmental changes of the LPL mRNA expression and the effect on IMF content in sheep muscle[J]. Scientia Agricultura Sinica, 2007, 40(10):2323-2330. (in Chinese)
[28] 王晶. 广西三黄鸡肉质性状分析及LPL、H-FABP基因表达与肌内脂肪含量的相关研究[D]. 南宁:广西大学, 2013.
WANG J. Studies on the meat quality traits and the relationship between LPL and H-FABP genes expression with intramuscular fat content in chicken[D]. Nanning:Guangxi University, 2013.(in Chinese)
[29] WANG Y H, KIM KA, KIM J H, et al. Pref-1, a preadipocyte secreted factor that inhibits adipogenesis[J]. J Nutr, 2006, 136(12):2953-2956.
[30] CHARALAMBOUS M, DA ROCHA S T, RADFORD E J, et al. DLK1/PREF1 regulates nutrient metabolism and protects from steatosis[J]. Proc Natl Acad Sci U S A, 2014, 111(45):16088-16093.
[31] TISCHENDORF F, SCHÖNE F, KIRCHHEIM U, et al. Influence of a conjugated linoleic acid mixture on growth, organ weights, carcass traits and meat quality in growing pigs[J]. J Anim Physiol Anim Nutr(Berl), 2002, 86(3-4):117-128.
[32] 张罕星, 朱晓彤, 束刚, 等. 猪肌内脂肪前体细胞与皮下脂肪前体细胞分化过程中基因差异表达分析[J]. 中国农业科学, 2008, 41(11):3760-3768.
ZHANG H X, ZHU X T, SHU G, et al. Differential mRNA expression profiles of porcine intramuscular preadipocytes compared with subcutaneous preadipocytes during differentiation[J]. Scientia Agricultura Sinica, 2008, 41(11):3760-3768. (in Chinese)
[33] SUZUKI K, IRIE M, KADOWAKI H, et al. Genetic parameter estimates of meat quality traits in Duroc pigs selected for average daily gain, longissimus muscle area, backfat thickness, and intramuscular fat content[J]. J Anim Sci, 2005, 83(9):2058-2065.
[34] KOUBA M, BONNEAU M, NOBLET J. Relative development of subcutaneous, intermuscular, and kidney fat in growing pigs with different body compositions[J]. J Anim Sci, 1999, 77(3):622-629.