miR-150靶向AOC3调控绵羊前体脂肪细胞分化的研究

doi:10.11843/j.issn.0366-6964.2023.08.013

摘要/Abstract

摘要： 旨在通过探究miR-150与AOC3的靶标关系，揭示miR-150在广灵大尾羊前体脂肪细胞分化过程中的作用机制。本研究以3只5日龄发育正常且健康的广灵大尾羊公羊为试验材料，采用qPCR方法检测广灵大尾羊皮下、肾周和尾部脂肪组织中miR-150的表达量；采集广灵大尾羊的尾部脂肪组织并分离绵羊前体脂肪细胞；通过细胞免疫荧光技术鉴定细胞纯度；利用生物信息学软件预测和双荧光素酶报告试验验证miR-150与AOC3的靶标关系；转染miR-150 mimics和inhibitors并检测AOC3和成脂分化标志基因的表达情况；构建并转染绵羊AOC3基因的过表达和干扰载体至绵羊前体脂肪细胞，利用qPCR和Western blotting检测miR-150与AOC3在广灵大尾羊前体脂肪细胞分化中的表达规律；油红O染色检测成脂能力，上述试验方法均设立3个生物学重复。结果表明，miR-150在3个不同部位脂肪组织中尾部脂肪表达量最高，差异极显著（P<0.001）。与对照组相比，过表达miR-150后，AOC3基因和成脂标志基因的表达显著下调（P<0.05），且脂滴生成减少，抑制了绵羊前体脂肪细胞分化；抑制miR-150后，结果相反。miR-150与AOC3的3'-UTR存在结合位点，miR-150极显著降低了AOC3野生型的相对荧光活性（P<0.001）。过表达AOC3后，成脂分化标志基因的表达极显著上调（P<0.01），脂肪细胞产生了更多脂滴，AOC3促进了绵羊前体脂肪的分化；干扰AOC3后，结果相反。本研究表明，miR-150与AOC3的3'-UTR存在结合位点，在绵羊前体脂肪细胞分化的过程中，miR-150与AOC3的表达量呈负相关性，miR-150通过靶向AOC3抑制脂肪分化，研究结果为microRNAs （miRNAs）在分子水平上的研究提供了一定的理论依据。

关键词: 广灵大尾羊, miR-150, AOC3, 脂肪细胞分化

Abstract: The aim of this study was to reveal the action mechanism of miR-150 during the differentiation of preadipocytes in Guangling Large-Tailed sheep by investigating the relationship between miR-150 and target gene AOC3. In this study, three 5-day-old Guangling Large-Tailed male sheep with normal development and health were used as the research material. The expression level of miR-150 in subcutaneous, perirenal and tail adipose tissues of Guangling Large-Tailed sheep was detected by qPCR. Tail adipose tissue of Guangling large-tailed sheep was collected to isolate ovine preadipocytes. Cell purity was identified by cell immunofluorescence. Bioinformatics software was used to predict the target relationship between miR-150 and AOC3, and double luciferase reporter system was used to verify this relationship. miR-150 mimics and inhibitors were transfected into preadipocyte and the expression of AOC3 and adipogenic marker genes were detected. The overexpression and interference vector of ovine AOC3 were constructed and transfected into ovine preadipocytes. qPCR and Western blotting were used to detect the expression patterns of miR-150 and AOC3 during the differentiation of preadipocytes in Guangling Large-Tailed sheep. Lipid accumulation ability was detected by Oil Red O staining. Three biological replicates were set up for each of the above tests. The results showed that the expression of miR-150 was the highest in tail fat, and the difference was significant (P<0.001). After overexpression of miR-150, the expression of AOC3 and adipogenic marker genes were significantly down-regulated (P<0.05), lipid droplet accumulation decreased. These results implied that miR-150 inhibits the differentiation of ovine preadipocytes. The results were opposite after inhibition of miR-150. There was a binding site between miR-150 and the 3'-UTR of AOC3, and miR-150 was extremely significant decreased the relative fluorescence activity of wild type AOC3 (P<0.001). After overexpressing AOC3, the expression of adipogenic marker genes were significantly up-regulated (P<0.01) and more lipid droplets were produced, indicating that AOC3 promotes the differentiation of ovine preadipocytes. After interfering with AOC3, the result was reversed. In conclusion, there is a binding site between miR-150 and the 3'-UTR of AOC3. The expression level of miR-150 was negatively correlated with that of AOC3 during the differentiation of ovine preadipocyte. miR-150 inhibits the differentiation of ovine preadipocytes by targeting AOC3. The results provide a theoretical basis for the research of microRNAs (miRNAs) at the molecular level.

Key words: Guangling Large-Tailed sheep, miR-150, AOC3, adipocyte differentiation

中图分类号:

S826.2

张寒月, 赵丹, 梁煜, 赵弼时, 范蒙丹, 乔利英, 刘建华, 杨凯捷, 潘洋洋, 刘文忠. miR-150靶向AOC3调控绵羊前体脂肪细胞分化的研究[J]. 畜牧兽医学报, 2023, 54(8): 3262-3274.

ZHANG Hanyue, ZHAO Dan, LIANG Yu, ZHAO Bishi, FAN Mengdan, QIAO Liying, LIU Jianhua, YANG Kaijie, PAN Yangyang, LIU Wenzhong. miR-150 Regulates Ovine Preadipocyte Differentiation by Targeting AOC3[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3262-3274.

参考文献 41

[1]	SULIMAN G M, AL-OWAIMER A N, EL-WAZIRY A M, et al.A Comparative study of sheep breeds:fattening performance, carcass characteristics, meat chemical composition and quality attributes[J].Front Vet Sci, 2021, 8:647192.
[2]	ZHANG X Y, LIU C Y, KONG Y Y, et al.Effects of intramuscular fat on meat quality and its regulation mechanism in Tan sheep[J].Front Nutr, 2022, 9:908355.
[3]	SCHUMACHER M, DELCURTO-WYFFELS H, THOMSON J, et al.Fat deposition and fat effects on meat quality-a review[J]. Animals (Basel), 2022, 12(12):1550.
[4]	WANG J H, ZHANG X X, WANG X J, et al.Polymorphism and expression of the HMGA1 gene and association with tail fat deposition in Hu sheep[J/OL].Animal Biotechnology, 2021, doi:10.1080/10495398.2021.1998093.
[5]	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 of tail fat deposition in sheep[J].Modern Animal Husbandry, 2022(4):16-18.(in Chinese)
[6]	於建国.新疆地方品种绵羊尾脂利用现状与展望[J].中国畜禽种业, 2021, 17(10):117-118.YU J G.Current situation and prospect of utilization of tail fat of local breed sheep in Xinjiang[J].The Chinese Livestock and Poultry Breeding, 2021, 17(10):117-118.(in Chinese)
[7]	YOU L H, WANG Y, GAO Y, et al.The role of microRNA-23b-5p in regulating brown adipogenesis and thermogenic program[J]. Endocr Connect, 2020, 9(5):457-470.
[8]	SARJEANT K, STEPHENS J M.Adipogenesis[J].Cold Spring Harb Perspect Biol, 2012, 4(9):a008417.
[9]	LU T X, ROTHENBERG M E.MicroRNA[J].J Allergy Clin Immunol, 2018, 141(4):1202-1207.
[10]	TREIBER T, TREIBER N, MEISTER G.Regulation of microRNA biogenesis and its crosstalk with other cellular pathways[J]. Nat Rev Mol Cell Biol, 2019, 20(1):5-20.
[11]	LIZ J, ESTELLER M.lncRNAs and microRNAs with a role in cancer development[J].Biochim Biophys Acta, 2016, 1859(1):169-176.
[12]	MENS M M J, GHANBARI M.Cell cycle regulation of stem cells by MicroRNAs[J].Stem Cell Rev Rep, 2018, 14(3):309-322.
[13]	KINSER H E, PINCUS Z.MicroRNAs as modulators of longevity and the aging process[J].Hum Genet, 2020, 139(3):291-308.
[14]	YING W, TSENG A, CHANG R C A, et al.miR-150 regulates obesity-associated insulin resistance by controlling B cell functions[J].Sci Rep, 2016, 6:20176.
[15]	DESGAGNÉ V, GUÉRIN R, GUAY S P, et al.Changes in high-density lipoprotein-carried miRNA contribution to the plasmatic pool after consumption of dietary trans fat in healthy men[J].Epigenomics, 2017, 9(5):669-688.
[16]	DESJARLAIS M, DUSSAULT S, DHAHRI W, et al.MicroRNA-150 modulates ischemia-induced neovascularization in atherosclerotic conditions[J].Arterioscler Thromb Vasc Biol, 2017, 37(5):900-908.
[17]	CHEN X Y, RAZA S H A, MA X H, et al.Bovine pre-adipocyte adipogenesis is regulated by bta-miR-150 through mTOR signaling[J].Front Genet, 2021, 12:636550.
[18]	MATZ A J, QU L L, KARLINSEY K, et al.MicroRNA-regulated B cells in obesity[J].Immunometabolism (Cobham), 2022, 4(3):e00005.
[19]	ANDRÉS N, LIZCANO J M, RODRÍGUEZ M J, et al.Tissue activity and cellular localization of human semicarbazide-sensitive amine oxidase[J].J Histochem Cytochem, 2001, 49(2):209-217.
[20]	SHEN S H, WERTZ D L, KLINMAN J P.Implication for functions of the ectopic adipocyte copper amine oxidase (AOC3) from purified enzyme and cell-based kinetic studies[J].PLoS One, 2012, 7(1):e29270.
[21]	BOUR S, CASPAR-BAUGUIL S, IFFIÚ-SOLTÉSZ Z, et al.Semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 deficiency reduces leukocyte infiltration into adipose tissue and favors fat deposition[J].Am J Pathol, 2009, 174(3):1075-1083.
[22]	张恬, 张龙超, 王立刚, 等.猪脂肪沉积候选基因AOC3、PPARG1及SOD3 DNA甲基化差异研究[J].中国畜牧兽医, 2016, 43(11):2820-2825.ZHANG T, ZHANG L C, WANG L G, et al.Study on methylation difference of fat deposition candidate genes AOC3, PPARG1 and SOD3 in pig[J].China Animal Husbandry & Veterinary Medicine, 2016, 43(11):2820-2825.(in Chinese)
[23]	史明月, 张雪莲, 杨晓奋, 等.NR1H3基因调控猪前体脂肪细胞分化的研究[J].畜牧兽医学报, 2022, 53(7):2094-2103.SHI M Y, ZHANG X L, YANG X F, et al.Study on NR1H3 gene regulating differentiation of porcine preadipocyte[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(7):2094-2103.(in Chinese)
[24]	MADHUMITA M, PAUL S.A review on methods for predicting miRNA-mRNA regulatory modules[J].J Integr Bioinform, 2022, 19(3):20200048.
[25]	CHAVA S, REYNOLDS C P, PATHANIA A S, et al.miR-15a-5p, miR-15b-5p, and miR-16-5p inhibit tumor progression by directly targeting MYCN in neuroblastoma[J].Mol Oncol, 2020, 14(1):180-196.
[26]	LONG J M, MALONEY B, ROGERS J T, et al.Novel upregulation of amyloid-β precursor protein (APP) by microRNA-346 via targeting of APP mRNA 5'-untranslated region:implications in Alzheimer's disease[J].Mol Psychiatry, 2019, 24(3):345-363.
[27]	ZHANG J T, ZHOU W L, LIU Y Y, et al.Oncogenic role of microRNA-532-5p in human colorectal cancer via targeting of the 5'UTR of RUNX3[J].Oncol Lett, 2018, 15(5):7215-7220.
[28]	NIU Y, JIN Y, DENG S C, et al.MiRNA-646-mediated reciprocal repression between HIF-1α and MIIP contributes to tumorigenesis of pancreatic cancer[J].Oncogene, 2018, 37(13):1743-1758.
[29]	TIAN W H, HAO X, NIE R X, et al.Integrative analysis of miRNA and mRNA profiles reveals that gga-miR-106-5p inhibits adipogenesis by targeting the KLF15 gene in chickens[J].J Anim Sci Biotechnol, 2022, 13(1):81.
[30]	WANG Z, ZHAO Q S, LI X Q, et al.MYOD1 inhibits avian adipocyte differentiation via miRNA-206/KLF4 axis[J].J Anim Sci Biotechnol, 2021, 12(1):55.
[31]	WEN F Y, AN C Q, WU X T, et al.MiR-34a regulates mitochondrial content and fat ectopic deposition induced by resistin through the AMPK/PPARα pathway in HepG2 cells[J].Int J Biochem Cell Biol, 2018, 94:133-145.
[32]	ZHANG P W, LI X R, ZHANG S H, et al.miR-370-3p regulates adipogenesis through targeting Mknk[J].Molecules, 2021, 26(22):6926.
[33]	LIU X F, HE Y, FENG Z L, et al.miR-345-5p regulates adipogenesis via targeting VEGF-B[J].Aging (Albany NY), 2020, 12(17):17114-17121.
[34]	KARKENI E, BONNET L, MARCOTORCHINO J, et al.Vitamin D limits inflammation-linked microRNA expression in adipocytes in vitro and in vivo:a new mechanism for the regulation of inflammation by vitamin D[J].Epigenetics, 2018, 13(2):156-162.
[35]	YAO Y F, WANG H, XI X Q, et al.miR-150 and SRPK1 regulate AKT3 expression to participate in LPS-induced inflammatory response[J].Innate Immun, 2021, 27(4):343-350.
[36]	LI J, ZHANG S H.microRNA-150 inhibits the formation of macrophage foam cells through targeting adiponectin receptor 2[J].Biochem Biophys Res Commun, 2016, 476(4):218-224.
[37]	刘莉, 彭金富, 郭成贤, 等.miR-150对药物代谢酶CYP3A4的调控作用[J].中国临床药理学与治疗学, 2018, 23(7):749-754.LIU L, PENG J F, GUO C X, et al.Regulative effects of miR-150 on CYP3A4[J].Chinese Journal of Clinical Pharmacology and Therapeutics, 2018, 23(7):749-754.(in Chinese)
[38]	FERREIRA M V, CABRAL E T, COROADINHA A S.Progress and perspectives in the development of lentiviral vector producer cells[J].Biotechnol J, 2021, 16(1):2000017.
[39]	SCHWEIZER M, MERTEN O W.Large-scale production means for the manufacturing of lentiviral vectors[J].Curr Gene Ther, 2010, 10(6):474-486.
[40]	IFFIÚ-SOLTÉSZ Z, WANECQ E, PRÉVOT D, et al.Histamine oxidation in mouse adipose tissue is controlled by the AOC3 gene-encoded amine oxidase[J].Inflamm Res, 2010, 59(S2):227-229.
[41]	AGHA G, HOUSEMAN E A, KELSEY K T, et al.Adiposity is associated with DNA methylation profile in adipose tissue[J].Int J Epidemiol, 2015, 44(4):1277-1287.