miR-33a靶向Lipin1和IRS2调节绵羊前体脂肪细胞分化的研究

doi:10.11843/j.issn.0366-6964.2020.04.006

摘要/Abstract

摘要： 旨在揭示miR-33a在绵羊前体脂肪细胞分化中的生物学功能。本研究以15日龄雄性绵羊背部皮下前体脂肪细胞为试验材料，所有的试验均设立3个重复；利用生物信息学软件预测miR-33a的靶基因，并通过双荧光素酶报告试验对预测的潜在靶基因进行验证；用qPCR和Western blotting分别检测miR-33a、Lipin1和IRS2及其编码蛋白的表达，以揭示miR-33a与其靶基因在绵羊前体脂肪细胞分化中的表达规律；慢病毒介导实现miR-33a的过表达和干扰后，检测Lipin1、IRS2和成脂标志基因的表达，并用油红O染色检测脂滴沉积能力，以解析miR-33a对其靶基因的调节机制。生物信息学分析发现，miR-33a与Lipin1和IRS2 3'-UTR都存在结合位点，miR-33a显著下调Lipin1和IRS2野生型双荧光质粒的相对荧光活性（P<0.05）；在绵羊前体脂肪细胞分化中，miR-33a与Lipin1和IRS2的表达趋势相反；过表达miR-33a后，显著下调了Lipin1（P<0.01）和IRS2（P<0.05）及其编码蛋白以及成脂标志基因的表达；干扰miR-33a后，这些基因和蛋白的表达则显著上调；过表达miR-33a减少了脂滴沉积，干扰miR-33a促进了脂滴沉积。在绵羊前体脂肪细胞分化中，miR-33a与Lipin1和IRS2的表达呈负相关。miR-33a靶向Lipin1和IRS2的3'-UTR抑制绵羊前体脂肪细胞分化和脂滴沉积。

关键词: miR-33a, Lipin1, IRS2, 绵羊, 脂肪细胞分化

Abstract: The aim of this study was to reveal the biological functions of miR-33a during the differentiation of ovine preadipocytes. In this study, preadipocytes from the back fat of 15-day-old male lambs were used as research material. All the experiments in this study had 3 replicates. Bioinformatics software was used to predict the target genes of miR-33a. The potential target genes were verified by double luciferase reporter system. In order to reveal the expression pattern of miR-33a and its targeting genes during the differentiation of ovine preadipocytes, qPCR and Western blotting were used to detect the expressions of miR-33a, Lipin1, and IRS2 and proteins. In order to elucidate the regulatory mechanism of miR-33a on its target genes, miR-33a was overexpressed or interfered using lentivirus-mediated method. The expressions of Lipin1, IRS2 and adipogenic marker genes in ovine preadipocytes were detected. The lipid droplet deposition ability was measured by Oil Red O staining. The binding sites of miR-33a with 3'-UTR of Lipin1 and IRS2 were found using bioinformatics prediction. miR-33a significantly down-regulated the relative fluorescence activity of Lipin1 and IRS2 wild-type double fluorescent plasmids (P<0.05). The expression levels of miR-33a and Lipin1, IRS2 exhibited opposite trends during ovine preadipocyte differentiation. The overexpression of miR-33a significantly down-regulated the expressions of Lipin1 (P<0.01), IRS2 (P<0.05) and their encoded proteins, and adipogenic marker genes. After the miR-33a was interfered, the expressions of these genes and proteins were significantly up-regulated. Overexpression of miR-33a reduced lipid droplet deposition, and interference of miR-33a promoted lipid droplet deposition. In conclusion, the expressions of miR-33a and Lipin1, IRS2 were negative correlated during ovine preadipocyte differentiation. miR-33a negatively regulates the differentiation of ovine preadipocytes and the lipid droplets deposition by targeting the 3'-UTR of Lipin1 and IRS2.

Key words: miR-33a, Lipin1, IRS2, sheep, adipocyte differentiation

中图分类号:

S826.2

王强, 潘洋洋, 乔利英, 刘建华, 赵弼时, 刘旭莹, 王凤, 梁煜, 刘文忠. miR-33a靶向Lipin1和IRS2调节绵羊前体脂肪细胞分化的研究[J]. 畜牧兽医学报, 2020, 51(4): 701-712.

WANG Qiang, PAN Yangyang, QIAO Liying, LIU Jianhua, ZHAO Bishi, LIU Xuying, WANG Feng, LIANG Yu, LIU Wenzhong. miR-33a Regulates Ovine Preadipocyte Differentiation by Targeting Lipin1 and IRS2[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(4): 701-712.

参考文献 41

[1]	CHUSYD D E,WANG D H,HUFFMAN D M,et al.Relationships between rodent white adipose fat pads and human white adipose fat depots[J].Front Nutr,2016,3:10.
[2]	PAN Y,JING J,QIAO L,et al.MiRNA-seq reveals that miR-124-3p inhibits adipogenic differentiation of the stromal vascular fraction in sheep via targeting C/EBPα[J].Domest Anim Endocrinol,2018,65:17-23.
[3]	MARQUART T J,ALLEN R M,ORY D S,et al.miR-33 links SREBP-2 induction to repression of sterol transporters[J].Proc Natl Acad Sci U S A,2010,107(27):12228-12232.
[4]	PRICE N L,SINGH A K,ROTLLAN N,et al.Genetic ablation of miR-33 increases food intake,enhances adipose tissue expansion,and promotes obesity and insulin resistance[J].Cell Rep,2018,22(8):2133-2145.
[5]	PÉTERFY M,PHAN J,XU P,et al.Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin[J].Nat Genet,2001,27(1):121-124.
[6]	REUE K,PÉTERFY M.Mouse models of lipodystrophy[J].Curr Atheroscler Rep,2000,2(5):390-396.
[7]	REUE K,XU P,WANG X P,et al.Adipose tissue deficiency,glucose intolerance,and increased atherosclerosis result from mutation in the mouse fatty liver dystrophy (fld) gene[J].J Lipid Res,2000,41(7):1067-1076.
[8]	PHAN J,REUE K.Lipin,a lipodystrophy and obesity gene[J].Cell Metab,2005,1(1):73-83.
[9]	CSAKI L S,DWYER J R,FONG L G,et al.Lipins,lipinopathies,and the modulation of cellular lipid storage and signaling[J].Prog Lipid Res,2013,52(3):305-316.
[10]	HAN G S,WU W I,CARMAN G M.The Saccharomyces cerevisiae Lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme[J].J Biol Chem,2006,281(14):9210-9218.
[11]	FINCK B N,GROPLER M C,CHEN Z J,et al.Lipin 1 is an inducible amplifier of the hepatic PGC-1α/PPARα regulatory pathway[J].Cell Metab,2006,4(3):199-210.
[12]	HU M,WANG F M,LI X,et al.Regulation of hepatic lipin-1 by ethanol:role of AMP-activated protein kinase/sterol regulatory element-binding protein 1 signaling in mice[J].Hepatology,2012,55(2):437-446.
[13]	CHENG X Y,LIU J D,LU X Y,et al.miR-203 inhibits alcohol-induced hepatic steatosis by targeting Lipin1[J].Front Pharmacol, 2018,9:275.
[14]	ZHANG J J,LIU F.Tissue-specific insulin signaling in the regulation of metabolism and aging[J].IUBMB Life,2014, 66(7):485-495.
[15]	LEE S,DONG H H.FoxO integration of insulin signaling with glucose and lipid metabolism[J].J Endocrinol, 2017, 233(2):R67-R79.
[16]	PAYANKAULAM S,RAICU A M,ARNOSTI D N.Transcriptional regulation of INSR,the insulin receptor gene[J].Genes (Basel),2019,10(12):984.
[17]	MIKI H,YAMAUCHI T,SUZUKI R,et al.Essential role of insulin receptor substrate 1(IRS-1) and IRS-2 in adipocyte differentiation[J]. Mol Cell Biol,2001,21(7):2521-2532.
[18]	WANG Y C,WANG S H,WANG S H,et al.Target gene identification and functional characterization of miR-1704 in chicken[J]. Anim Biotechnol,2019,30:1-8.
[19]	WANG Y L,YANG L,LIU X F,et al.miR-431 inhibits adipogenic differentiation of human bone marrow-derived mesenchymal stem cells via targeting insulin receptor substance 2[J].Stem Cell Res Ther,2018,9(1):231.
[20]	PU Y,VEIGA-LOPEZ A.PPARγ agonist through the terminal differentiation phase is essential for adipogenic differentiation of fetal ovine preadipocytes[J].Cell Mol Biol Lett,2017,22:6.
[21]	DENG K P,REN C F,LIU Z F,et al.Characterization of RUNX1T1,an adipogenesis regulator in ovine preadipocyte differentiation[J]. Int J Mol Sci,2018,19(5):1300.
[22]	DENG K P,REN C F,FAN Y X,et al.YAP1 regulates PPARG and RXR alpha expression to affect the proliferation and differentiation of ovine preadipocyte[J].J Cell Biochem,2019,120(12):19578-19589.
[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]	郭红芳,昝林森,孙永刚.牛前体脂肪细胞的分离培养及诱导分化[J].西北农林科技大学学报:自然科学版,2014,42(2):1-6,12.GUO H F,ZAN L S,SUN Y G.Primary culture and differentiation of bovine preadipocytes[J].Journal of Northwest A&F University:Natural Science Edition,2014,42(2):1-6,12.(in Chinese)
[25]	YU H,EMONT M,JUN H,et al.Isolation and differentiation of murine primary brown/beige preadipocytes[M]//BUNNELL B A,GIMBLE J M.Adipose-Derived Stem Cells:Methods and Protocols.New York:Humana Press,2018:273-282.
[26]	GAO W,KONG X X,YANG Q.Isolation,primary culture,and differentiation of preadipocytes from mouse brown adipose tissue[M]//WU J.Thermogenic Fat:Methods and Protocols.New York:Humana Press,2017:3-8.
[27]	DING F,LI Q Q,LI L,et al.Isolation,culture and differentiation of duck (Anas platyrhynchos) preadipocytes[J]. Cytotechnology, 2015,67(5):773-781.
[28]	SHI T,YAN X R,QIAO L Y,et al.MiR-330-5p negatively regulates ovine preadipocyte differentiation by targeting branched-chain aminotransferase 2[J].Anim Sci J,2018,89(6):858-867.
[29]	RAMÍREZ-ZACARÍAS J L,CASTRO-MUÑOZLEDO F,KURI-HARCUCH W.Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O[J].Histochemistry,1992,97(6):493-497.
[30]	RIVA G,VILLANOVA M,CIMA L,et al.Oil Red O is a useful tool to assess donor liver steatosis on frozen sections during transplantation[J].Transplant Proc,2018,50(10):3539-3543.
[31]	MEHLEM A,HAGBERG C E,MUHL L,et al.Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease[J].Nat Protoc,2013,8(6):1149-1154.
[32]	PAN Y Y,JING J J,QIAO L Y,et al.miR-124-3p affects the formation of intramuscular fat through alterations in branched chain amino acid consumption in sheep[J].Biochem Biophys Res Commun,2018,495(2):1769-1774.
[33]	PÉTERFY M,PHAN J,REUE K.Alternatively spliced lipin isoforms exhibit distinct expression pattern,subcellular localization, and role in adipogenesis[J].J Biol Chem,2005,280(38):32883-32889.
[34]	SUVIOLAHTI E,REUE K,CANTOR R M,et al.Cross-species analyses implicate Lipin 1 involvement in human glucose metabolism[J]. Hum Mol Genet,2006,15(3):377-386.
[35]	KANG E S,PARK S E,HAN S J,et al.LPIN1 genetic variation is associated with rosiglitazone response in type 2 diabetic patients[J]. Mol Genet Metab,2008,95(1-2):96-100.
[36]	RYU D,OH K J,JO H Y,et al.TORC2 regulates hepatic insulin signaling via a mammalian phosphatidic acid phosphatase, LIPIN1[J]. Cell Metab,2009,9(3):240-251.
[37]	TANG T,HU Y,PENG M,et al.Effects of high-fat diet on growth performance,lipid accumulation and lipid metabolism-related MicroRNA/gene expression in the liver of grass carp (Ctenopharyngodon idella)[J].Comp Biochem Physiol Part B:Biochem Mol Biol, 2019, 234:34-40.
[38]	KIM H E,BAE E,JEONG D Y,et al.Lipin1 regulates PPARγ transcriptional activity[J].Biochem J,2013,453(1):49-60.
[39]	BOU KHALIL M,BLAIS A,FIGEYS D,et al.Lipin-the bridge between hepatic glycerolipid biosynthesis and lipoprotein metabolism[J]. Biochim Biophys Acta,2010,1801(12):1249-1259.
[40]	魏琳琳,高晋生,王景霖,等.绵羊LPIN1基因的克隆和mRNA表达研究[J].畜牧兽医学报,2013,44(9):1371-1379.WEI L L,GAO J S,WANG J L,et al.Study on the cloning and ontogenetic mRNA expression of ovine LPIN1 gene[J].Acta Veterinaria et Zootechnica Sinica,2013,44(9):1371-1379.(in Chinese)
[41]	CHEN Z,SHI H P,SUN S,et al.MicroRNA-181b suppresses TAG via target IRS2 and regulating multiple genes in the Hippo pathway[J].Exp Cell Res,2016,348(1):66-74.