miR-200c和miR-429靶向调节绵羊皮下脂肪细胞中SCD1表达的研究
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摘要
旨在预测并验证调节绵羊皮下脂肪细胞中SCD1基因表达的关键miRNAs。本研究利用4个生物信息学软件TargetScan、mirDB、StarBase和miRanda预测与该基因具有靶标关系的关键miRNAs;利用双荧光素酶报告系统验证SCD1基因与miRNAs的靶标关系;利用荧光定量PCR和Western-blotting技术,分别在mRNA和蛋白水平上检测miRNAs对SCD1 mRNA和蛋白质表达的影响。预测结果表明,miR-200c和miR-429是调控SCD1基因表达的关键miRNAs;双荧光素酶报告系统检测证明,2个miRNAs与SCD1基因都具有靶标关系;荧光定量PCR显示,miR-200c和miR-429显著抑制SCD1 mRNA的表达(P<0.05),且miR-200c作用更大;Western-blotting显示,miR-200c和miR-429极显著抑制SCD1蛋白质的表达(P<0.01);显微镜观察发现,miR-200c和miR-429抑制绵羊皮下脂肪细胞脂滴生成。由此证明,miR-200c和miR-429均靶向抑制SCD1基因的表达,进而抑制绵羊脂肪生成。
This study aimed to predict and validate the key miRNAs that regulated SCD1 gene expression in ovine subcutaneous adipocytes. miRNAs targeting SCD1 were predicted by bioinformatics softwares of TargetScan, mirDB, StarBase and miRanda. The dual luciferase reporter system was used to verify the target relationships between SCD1 gene and miRNAs. Fluorescent quantitative PCR and Western-blotting techniques were used to detect the effect of miRNAs on SCD1 expression at mRNA and protein levels. The prediction results indicated that SCD1 gene was targeted by both miR-200 c and miR-429. Dual luciferase reporter system assay demonstrated that there were target relationships between the 2 miRNAs and SCD1 gene. Fluorescent quantitative PCR analysis showed that miR-200 c and miR-429 significantly inhibited SCD1 mRNA expression(P<0.05), and miR-200 c was more effective. Western-blotting results showed that miR-200 c and miR-429 reduced SCD1 protein level(P<0.01). Microscopical observation showed that miR-200 c and miR-429 inhibited ovine subcutaneous lipid droplet formation. In conclusion, miR-200 c and miR-429 can down-regulate SCD1 gene expression, thus inhibit ovine lipogenesis.
This study aimed to predict and validate the key miRNAs that regulated SCD1 gene expression in ovine subcutaneous adipocytes. miRNAs targeting SCD1 were predicted by bioinformatics softwares of TargetScan, mirDB, StarBase and miRanda. The dual luciferase reporter system was used to verify the target relationships between SCD1 gene and miRNAs. Fluorescent quantitative PCR and Western-blotting techniques were used to detect the effect of miRNAs on SCD1 expression at mRNA and protein levels. The prediction results indicated that SCD1 gene was targeted by both miR-200 c and miR-429. Dual luciferase reporter system assay demonstrated that there were target relationships between the 2 miRNAs and SCD1 gene. Fluorescent quantitative PCR analysis showed that miR-200 c and miR-429 significantly inhibited SCD1 mRNA expression(P<0.05), and miR-200 c was more effective. Western-blotting results showed that miR-200 c and miR-429 reduced SCD1 protein level(P<0.01). Microscopical observation showed that miR-200 c and miR-429 inhibited ovine subcutaneous lipid droplet formation. In conclusion, miR-200 c and miR-429 can down-regulate SCD1 gene expression, thus inhibit ovine lipogenesis.
引文
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[23] 赵艳艳,乔利英,景炅婕,等.miR-142和miR-144靶向FoxO1基因调节绵羊前体脂肪细胞分化的研究[J].畜牧兽医学报,2018,49(4):675-684.ZHAO Y Y,QIAO L Y,JING J J,et al.Study on the differentiation regulation of miR-142 and miR-144 on ovine preadipocytes by targeting FoxO1 gene[J].Acta Veterinaria et Zootechnica Sinica,2018,49(4):675-684.(in Chinese)
[24] ZHANG H N,LIU J,QU D,et al.Inhibition of miR-200c restores endothelial function in diabetic mice through suppression of COX-2[J].Diabetes,2016,65(5):1196-1207.
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[26] YAO D,LUO J,HE Q,et al.SCD1 alters long-chain fatty acid (LCFA) composition and its expression is directly regulated by SREBP-1 and PPARγ1 in dairy goat mammary cells[J].J Cell Physiol,2017,232(3):635-649.
[27] IKEDA J,ICHIKI T,TAKAHARA Y,et al.PPARγ agonists attenuate palmitate-induced ER stress through up-regulation of SCD1 in macrophages[J].PLoS One,2015,10(6):e0128546.
[28] CHU K K,MIYAZAKI M,MAN W C,et al.Stearoyl-coenzyme A desaturase 1 deficiency protects against hypertriglyceridemia and increases plasma high-density lipoprotein cholesterol induced by liver X receptor activation[J].Mol Cell Biol,2006,26(18):6786-6798.
[29] ZHU D X,ZHU W,FANG C,et al.miR-181a/b significantly enhances drug sensitivity in chronic lymphocytic leukemia cells via targeting multiple anti-apoptosis genes[J].Carcinogenesis,2012,33(7):1294-1301.
[30] CHU X L,WANG Y Q,PANG L W,et al.miR-130 aggravates acute myocardial infarction-induced myocardial injury by targeting PPAR-γ[J].J Cell Biochem,2018,119(9):7235-7244.
[31] ILIOPOULOS D,DROSATOS K,HIYAMA Y,et al.microRNA-370 controls the expression of microRNA-122 and Cpt1α and affects lipid metabolism[J].J Lipid Res,2010,51(6):1513-1523.
[32] GUO J,FANG W W,SUN L B,et al.Reduced miR-200b and miR-200c expression contributes to abnormal hepatic lipid accumulation by stimulating JUN expression and activating the transcription of srebp1[J].Oncotarget,2016,7(24):36207-36219.
[33] XU Z H,BU Y W,CHITNIS N,et al.miR-216b regulation of c-Jun mediates GADD153/CHOP-dependent apoptosis[J].Nat Commun,2016,7:11422.
[2] DOBRZYN A,NTAMBI J M.The role of stearoyl-CoA desaturase in the control of metabolism[J].Prostaglandins Leukot Essent Fatty Acids,2005,73(1):35-41.
[3] ALJOHANI A M,SYED D N,NTAMBI J M.Insights into stearoyl-CoA desaturase-1 regulation of systemic metabolism[J].Trends Endocrinol Metab,2017,28(12):831-842.
[4] MAULUCCI G,COHEN O,DANIEL B,et al.Fatty acid-related modulations of membrane fluidity in cells:detection and implications[J].Free Radic Res,2016,50(S1):S40-S50.
[5] KAKUMA T,LEE Y,UNGER R H.Effects of leptin,troglitazone,and dietary fat on stearoyl CoA desaturase[J].Biochem Biophys Res Commun,2002,297(5):1259-1263.
[6] GIVEL A M,KIEFFER Y,SCHOLER-DAHIREL A,et al.miR200-regulated CXCL12β promotes fibroblast heterogeneity and immunosuppression in ovarian cancers[J].Nat Commun,2018,9(1):1056.
[7] HYUN S,LEE J H,JIN H,et al.Conserved microRNA miR-8/miR-200 and its target USH/FOG2 control growth by regulating PI3K[J].Cell,2009,139(6):1096-1108.
[8] LIU X L,GAO H X,WANG B C,et al.miR-192-5p regulates lipid synthesis in non-alcoholic fatty liver disease through SCD-1[J].World J Gastroenterol,2017,23(46):8140-8151.
[9] QIANG J,TAO Y F,HE J,et al.miR-29a modulates SCD expression and is regulated in response to a saturated fatty acid diet in juvenile genetically improved farmed tilapia (Oreochromis niloticus)[J].J Exp Biol,2017,220(8):1481-1489.
[10] GUO Y,YU J,WANG C,et al.miR-212-5p suppress lipid accumulation by targeting FAS and SCD1[J].J Mol Endocrinol,2017,59(3):205-217.
[11] CHENG X,XI Q Y,WEI S,et al.Critical role of miR-125b in lipogensis by targeting stearoyl-CoA desaturase-1 (SCD1)[J].J Anim Sci,2016,94(1):65-76.
[12] LEWIS B P,BURGE C B,BARTEL D P.Conserved seed pairing,often flanked by adenosines,indicates that thousands of human genes are microRNA targets[J].Cell,2005,120(1):15-20.
[13] WONG N,WANG X W.miRDB:an online resource for microRNA target prediction and functional annotations[J].Nucleic Acids Res,2015,43(D1):D146-D152.
[14] LI J H,LIU S,ZHOU H,et al.starBase v2.0:decoding miRNA-ceRNA,miRNA-ncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data[J].Nucleic Acids Res,2014,42(D1):D92-D97.
[15] JOHN B,ENRIGHT A J,ARAVIN A,et al.Human microRNA targets[J].PLoS Biol,2004,2(11):e363.
[16] FENG X L,WANG Z M,FILLMORE R,et al.miR-200,a new star miRNA in human cancer[J].Cancer Lett,2014,344(2):166-173.
[17] UHLMANN S,ZHANG J D,SCHW?GER A,et al.miR-200bc/429 cluster targets PLCγ1 and differentially regulates proliferation and EGF-driven invasion than miR-200a/141 in breast cancer[J].Oncogene,2010,29(30):4297-4306.
[18] TAO C,REN H Y,XU P,et al.Adipocyte miR-200b/a/429 ablation in mice leads to high-fat-diet-induced obesity[J].Oncotarget,2016,7(42):67796-67807.
[19] CHARTOUMPEKIS D V,ZARAVINOS A,ZIROS P G,et al.Differential expression of microRNAs in adipose tissue after long-term high-fat diet-induced obesity in mice[J].PLoS One,2012,7(4):e34872.
[20] ENDO K,WENG H C,KITO N,et al.miR-216a and miR-216b as markers for acute phased pancreatic injury[J].Biomed Res,2013,34(4):179-188.
[21] GOODWIN D,ROSENZWEIG B,ZHANG J,et al.Evaluation of miR-216a and miR-217 as potential biomarkers of acute pancreatic injury in rats and mice[J].Biomarkers,2014,19(6):517-529.
[22] 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.
[23] 赵艳艳,乔利英,景炅婕,等.miR-142和miR-144靶向FoxO1基因调节绵羊前体脂肪细胞分化的研究[J].畜牧兽医学报,2018,49(4):675-684.ZHAO Y Y,QIAO L Y,JING J J,et al.Study on the differentiation regulation of miR-142 and miR-144 on ovine preadipocytes by targeting FoxO1 gene[J].Acta Veterinaria et Zootechnica Sinica,2018,49(4):675-684.(in Chinese)
[24] ZHANG H N,LIU J,QU D,et al.Inhibition of miR-200c restores endothelial function in diabetic mice through suppression of COX-2[J].Diabetes,2016,65(5):1196-1207.
[25] WU T T,ZHOU H B,HONG Y F,et al.miR-30 family members negatively regulate osteoblast differentiation[J].J Biol Chem,2012,287(10):7503-7511.
[26] YAO D,LUO J,HE Q,et al.SCD1 alters long-chain fatty acid (LCFA) composition and its expression is directly regulated by SREBP-1 and PPARγ1 in dairy goat mammary cells[J].J Cell Physiol,2017,232(3):635-649.
[27] IKEDA J,ICHIKI T,TAKAHARA Y,et al.PPARγ agonists attenuate palmitate-induced ER stress through up-regulation of SCD1 in macrophages[J].PLoS One,2015,10(6):e0128546.
[28] CHU K K,MIYAZAKI M,MAN W C,et al.Stearoyl-coenzyme A desaturase 1 deficiency protects against hypertriglyceridemia and increases plasma high-density lipoprotein cholesterol induced by liver X receptor activation[J].Mol Cell Biol,2006,26(18):6786-6798.
[29] ZHU D X,ZHU W,FANG C,et al.miR-181a/b significantly enhances drug sensitivity in chronic lymphocytic leukemia cells via targeting multiple anti-apoptosis genes[J].Carcinogenesis,2012,33(7):1294-1301.
[30] CHU X L,WANG Y Q,PANG L W,et al.miR-130 aggravates acute myocardial infarction-induced myocardial injury by targeting PPAR-γ[J].J Cell Biochem,2018,119(9):7235-7244.
[31] ILIOPOULOS D,DROSATOS K,HIYAMA Y,et al.microRNA-370 controls the expression of microRNA-122 and Cpt1α and affects lipid metabolism[J].J Lipid Res,2010,51(6):1513-1523.
[32] GUO J,FANG W W,SUN L B,et al.Reduced miR-200b and miR-200c expression contributes to abnormal hepatic lipid accumulation by stimulating JUN expression and activating the transcription of srebp1[J].Oncotarget,2016,7(24):36207-36219.
[33] XU Z H,BU Y W,CHITNIS N,et al.miR-216b regulation of c-Jun mediates GADD153/CHOP-dependent apoptosis[J].Nat Commun,2016,7:11422.