miR-17-3p靶向KCTD15调控延边黄牛前体脂肪细胞分化

doi:10.11843/j.issn.0366-6964.2020.11.008

摘要/Abstract

摘要： 旨在探究miR-17-3p是否可以通过靶向KCTD15调节延边黄牛前体脂肪细胞的分化。本研究使用生物信息学软件鉴定miR-17-3p的同源性，预测miR-17-3p的靶基因；通过在延边黄牛的前体脂肪细胞中转染miR-17-3p mimic或miR-17-3p inhibitor及阴性对照实现细胞内miR-17-3p过表达或抑制表达；采用双荧光素酶报告基因检测系统确定miR-17-3p与KCTD15的靶标关系；使用qRT-PCR和Western blot技术在mRNA和蛋白水平上检测miR-17-3p对KCTD15基因以及脂代谢标志基因PPARγ和C/EBPα表达水平的影响。结果表明，生物信息学软件预测KCTD15可作为miR-17-3p的靶基因，且miR-17-3p在哺乳动物中具有较高的同源性；转染miR-17-3p mimic后脂代谢标志基因PPARγ和C/EBPα的mRNA和蛋白表达量显著或极显著高于转染NC-mimic的对照组（P<0.05或P<0.01）；抑制miR-17-3p的表达后，PPARγ和C/EBPα的表达量则显著或极显著低于对照组（P<0.05或P<0.01）；双荧光素酶报告基因验证分析显示，过表达miR-17-3p极显著抑制了含有KCTD15 3'UTR片段的载体的荧光活性（P<0.01）；过表达miR-17-3p也会显著或极显著抑制候选靶基因KCTD15 mRNA和蛋白质的表达量（P<0.05或P<0.01）；而抑制miR-17-3p的表达则会显著提高靶基因KCTD15的mRNA和蛋白质的表达量（P<0.05）。这些结果说明，miR-17-3p可能通过抑制KCTD15的表达促进延边黄牛前体脂肪细胞分化。

关键词: miR-17-3p, 延边黄牛, 前体脂肪细胞分化, KCTD15

Abstract: The aim of this study was to explore whether miR-17-3p could target KCTD15 to regulate the preadipocytes differentiation of Yanbian Yellow cattle. Bioinformatics softwares were used to identify homology and predict target genes of miR-17-3p. miR-17-3p mimic or miR-17-3p inhibitor and their negative control were transfected into bovine preadipocytes to overexpress or inhibit the expression of miR-17-3p. The binding sites of miR-17-3p to KCTD15 were verified via double luciferase report system. qRT-PCR and Western blot were used to detect the effect of miR-17-3p on the expressions of PPARγ, C/EBPα and KCTD15 both at mRNA and protein levels. Bioinformatics software prediction showed that KCTD15 was the target gene of miR-17-3p, and miR-17-3p had high homology in mammals. The mRNA and protein expressions of adipogenic marker genes PPARγ and C/EBPα after transfection of miR-17-3p mimic were significantly or extremely significantly higher than those of the control group transfected with NC-mimic (P<0.05 or P<0.01). After the expression of miR-17-3p was inhibited, the expressions of PPARγ and C/EBPα were significantly or extremely significantly lower than that in the control group (P<0.05 or P<0.01). Through the dual-luciferase reporter assay verification, the overexpression of miR-17-3p extremely significantly inhibited the fluorescent activity of the luciferase reporter gene vector containing the 3'UTR fragment of KCTD15 (P<0.01). Overexpression of miR-17-3p could also significantly or extremely significantly inhibit the expression of KCTD15 mRNA and protein(P<0.05 or P<0.01). However, inhibition of miR-17-3p expression could significantly increase the expression of KCTD15 mRNA and protein(P<0.05). These results suggest that miR-17-3p may regulate the differentiation of preadipocytes in Yanbian Yellow cattle by inhibiting the expression of KCTD15.

Key words: miR-17-3p, Yanbian Yellow cattle, preadipocyte differentiation, KCTD15

中图分类号:

邵静, 张珈溯, 尹宝珍, 张洛萌, 夏广军. miR-17-3p靶向KCTD15调控延边黄牛前体脂肪细胞分化[J]. 畜牧兽医学报, 2020, 51(11): 2689-2698.

SHAO Jing, ZHANG Jiasu, YIN Baozhen, ZHANG Luomeng, XIA Guangjun. miR-17-3p Regulates Preadipocyte Differentiation by Targeting KCTD15 in Yanbian Yellow Cattle[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(11): 2689-2698.

参考文献 41

[1]	严昌国,王勇,朴圣哲,等.延边黄牛牛肉品质特性的研究[J].黄牛杂志,2004,30(3):5-7.YAN C G,WANG Y,PIAO S Z,et al.Study on the beef quality traits of Yanbian cattle[J].Journal Yellow Cattle Science,2004,30(3):5-7.(in Chinese)
[2]	HOEHNE A,NUERNBERG G,KUEHN C,et al.Relationships between intramuscular fat content,selected carcass traits,and fatty acid profile in bulls using a F2-population[J].Meat Sci,2012,90(3):629-635.
[3]	DANI C,SMITH A G,DESSOLIN S,et al.Differentiation of embryonic stem cells into adipocytes in vitro[J].J Cell Sci,1997,110:1279-1285.
[4]	PITTENGER M F,MACKAY A M,BECK S C,et al.Multilineage potential of adult human mesenchymal stem cells[J].Science,1999,284(5411):143-147.
[5]	GREGOIRE F M,SMAS C M,SUL H S.Under-standing adipocyte differentiation[J].Physiol Rev,1998,78(3):783-809.
[6]	OTTO T C,LANE M D.Adipose development:from stem cell to adipocyte[J].Crit Rev Biochem Mol Biol,2005, 40(4):229-242.
[7]	GREGOIRE F M.Adipocyte differentiation:from fibroblast to endocrine cell[J].Exp Biol Med,2001,226(11):997-1002.
[8]	LEE R C,FEINBAUM R L,AMBROS V.The C. elegans Heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J].Cell,1993,75(5):843-854.
[9]	BARTEL D P.MicroRNAs:target recognition and regulatory functions[J].Cell,2009,136(2):215-233.
[10]	MUDHASANI R,PURI V,HOOVER K,et al.Dicer is required for the formation of white but not brown adipose tissue[J].J Cell Physiol,2011,226(5):1399-1406.
[11]	XU J J,ZHANG L P,SHU G B,et al.microRNA-16-5p promotes 3T3-L1 adipocyte differentiation through regulating EPT1[J].Biochem Biophys Res Commun,2019,514(4):1251-1256.
[12]	LI G L,NING C Y,MA Y,et al.miR-26b promotes 3T3-L1 adipocyte differentiation through targeting PTEN[J].DNA Cell Biol,2017,36(8):672-681.
[13]	ZHANG Z,GAO Y,XU M Q,et al.miR-181a regulate porcine preadipocyte differentiation by tar-geting TGFBR1[J].Gene,2019,681:45-51.
[14]	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.
[15]	LIU S Y,SUN G J,YUAN B,et al.miR-375 negatively regulates porcine preadipocyte differen-tiation by targeting BMPR2[J].FEBS Lett,2016,590(10):1417-1427.
[16]	GAO Y,WANG Y Q,CHEN X C,et al.MiR-127 attenuates adipogenesis by targeting MAPK4 and HOXC6 in porcine adipocytes[J].J Cell Physiol,2019,234(12):21838-21850.
[17]	王冰冰.肉牛脂肪沉积MiRNA的筛选及MiR-26a在不同部位中的表达分析[D].延吉:延边大学,2018.WANG B B.Screening of fatty MiRNAs in beef cattle and the expression of MiR-26a in different parts[D].Yanji:Yanbian University,2018.(in Chinese)
[18]	庞喆.基于RNA-seq延边黄牛肌内脂肪候选基因的筛选与表达分析[D].延吉:延边大学,2018.PANG Z.Screening and expression analysis of intramuscular fat candidate genes in Yanbian Yellow cattle based on RNA-seq[D].Yanji:Yanbian University,2018.(in Chinese)
[19]	TRAN K V,GEALEKMAN O,FRONTINI A,et al.The vascular endothelium of the adipose tissue gives rise to both white and brown fat cells[J].Cell Metab,2012,15(2):222-229.
[20]	FERNYHOUGH M E,VIERCK J L,HAUSMAN G J,et al.Primary adipocyte culture:adipocyte purification methods may lead to a new understanding of adipose tissue growth and development[J].Cytotechnology,2004,46(2):163-172.
[21]	CARNEVALLI L S,MASUDA K,FRIGERIO F,et al.S6K1 plays a critical role in early adipocyte differentiation[J].Dev Cell,2010,18(5):763-774.
[22]	XI F X,WEI C S,XU Y T,et al.MicroRNA-214-3p targeting Ctnnb1 promotes 3T3-L1 preadipocyte differentiation by interfering with the Wnt/β-Catenin signaling pathway[J].Int J Mol Sci,2019,20(8):1816.
[23]	ZHU E D,ZHANG J J,ZHOU J,et al.miR-20a-5p promotes adipogenic differentiation of murine bone marrow stromal cells via targeting Kruppel-like factor 3[J].J Mol Endocrinol,2018,60(3):225-237.
[24]	SHEN L Y,LI Q,WANG J Y,et al.miR-144-3p promotes adipogenesis through releasing C/EBPα from Klf3 and CtBP2[J].Front Genet,2018,9:677.
[25]	王书芳,潘洋洋,任端阳,等.miR-200c和miR-429靶向调节绵羊皮下脂肪细胞中SCD1表达的研究[J].畜牧兽医学报, 2019,50(7):1347-1357.WANG S F,PAN Y Y,REN D Y,et al.Regulation of SCD1 gene expression by miR-200c and miR-429 in ovine subcutaneous adipocytes[J].Acta Veterinaria et Zootechnica Sinica,2019,50(7):1347-1357.(in Chinese)
[26]	SUN G R,LI F,MA X F,et al.gga-miRNA-18b-3p inhibits intramuscular adipocytes differentiation in chicken by targeting the ACOT13 gene[J].Cells,2019,8(6):556.
[27]	JANG S Y,CHAE M K,LEE J H,et al.MicroRNA-27 inhibits adipogenic differentiation in orbital fibroblasts from patients with Graves' orbitopathy[J].PLoS One,2019,14(8):e0221077.
[28]	SHI J,BEI Y H,KONG X Q,et al.miR-17-3p contributes to exercise-induced cardiac growth and protects against myocardial ischemia-reperfusion injury[J].Theranostics,2017,7(3):664-676.
[29]	LIU Z Y,ZHANG Z R,YAO J H,et al.Serum extracellular vesicles promote proliferation of H9C2 cardiomyocytes by increasing miR-17-3p[J].Biochem Biophys Res Commun,2018,499(3):441-446.
[30]	SAND M,HESSAM S,AMUR S,et al.Expression of oncogenic miR-17-92 and tumor suppressive miR-143-145 clusters in basal cell carcinoma and cutaneous squamous cell carcinoma[J].J Dermatol Sci,2017,86(2):142-148.
[31]	XU Z,ZHANG Y Y,DING J J,et al.miR-17-3p downregulates mitochondrial antioxidant enzymes and enhances the radiosensitivity of prostate cancer cells[J].Mol Ther Nucleic Acids,2018,13:64-77.
[32]	CURTI V,DI LORENZO A,ROSSI D,et al.Enantioselective modulatory effects of naringenin enantiomers on the expression levels of miR-17-3p involved in endogenous antioxidant defenses[J].Nutrients,2017,9(3):215.
[33]	WU D J,ZHANG H,JI F,et al.MicroRNA-17 promotes osteosarcoma cells proliferation and migration and inhibits apoptosis by regulating SASH1 expression[J].Pathol Res Pract,2019,215(1):115-120.
[34]	SKOBLOV M,MARAKHONOV A,MARAKASOVA E,et al.Protein partners of KCTD proteins provide insights about their functional roles in cell differentiation and vertebrate development[J].BioEssays,2013,35(7):586-596.
[35]	WILLER C J,SPELIOTES E K,LOOS R J F,et al.Six new loci associated with body mass index highlight a neuronal influence on body weight regulation[J].Nat Genet,2009,41(1):25-34.
[36]	LI S X,ZHAO J H,LUAN J A,et al.Cumulative effects and predictive value of common obesity-susceptibility variants identified by genome-wide association studies[J].Am J Clin Nutr,2010,91(1):184-190.
[37]	ELKS C E,LOOS R J F,HARDY R,et al.Adult obesity susceptibility variants are associated with greater childhood weight gain and a faster tempo of growth:the 1946 British Birth Cohort Study[J].Am J Clin Nutr,2012,95(5):1150-1156.
[38]	张鹏,金康宣,陈宇晟,等.表达谱芯片分析KCTD15对3T3-L1前体脂肪细胞的影响[J].第三军医大学学报,2014,36(9):937-940.ZHANG P,JIN K X,CHEN Y S,et al.Effect of KCTD15 gene on 3T3-L1 preadipocyte by global expression profile microarray[J].Journal of Third Military Medical University,2014,36(9):937-940.(in Chinese)
[39]	TAKAHASHI C,SUZUKI T,NISHIDA E,et al.Identification and characterization of Xenopus kctd15,an ectodermal gene repressed by the FGF pathway[J].Int J Dev Biol,2012,56(5):393-402.
[40]	PATEL N G,KUMAR S,EGGO M C.Essential role of fibroblast growth factor signaling in preadipoctye differentiation[J].J Clin Endocrinol Metab,2005,90(2):1226-1232.
[41]	DUTTA S,DAWID I B.KCTD15 inhibits neural crest formation by attenuating Wnt/β-catenin signaling output[J]. Development, 2010,137(18):3013-3018.