microRNA-425-5p对3T3-L1前体脂肪细胞增殖、分化的影响
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摘要
为了探究miR-425-5p对小鼠3T3-L1前体脂肪细胞增殖、分化的影响效应,本试验采用实时荧光定量PCR检测miR-425-5p组织和细胞表达水平;运用CCK8、EdU、油红染色、甘油三酯含量分析等分别检测miR-425-5p对前体脂肪细胞增殖、分化的影响;利用生物信息学软件和双荧光素酶报告试验分别预测、验证miR-425-5p调控前体脂肪细胞分化的靶基因。结果表明,miR-425-5p在肥胖小鼠脂肪组织中低表达,在前体脂肪细胞增殖、分化过程中动态表达;与阴性对照相比,过表达miR-425-5p可促进前体脂肪细胞增殖,抑制脂肪细胞分化标志基因(PPARγ、C/EBPα、FAS等)表达,减少脂滴和甘油三酯积累;抑制miR-425-5p表达可抑制前体脂肪细胞增殖,阻止前体脂肪细胞诱导分化。在前体脂肪细胞分化过程中,过表达或抑制miR-425-5p可分别抑制或促进IGF1基因表达;与阴性对照相比,过表达miR-425-5p可抑制IGF1基因3′-UTR荧光活性,而突变miR-425-5p种子序列与IGF1基因3′-UTR的绑定位点可解除该抑制效果。综上所述,miR-425-5p可促进3T3-L1前体脂肪细胞增殖,并可直接靶向IGF1负向调控其分化。
In order to explore the effect of miR-425-5 p on the proliferation and differentiation of3 T3-L1 preadipocyte,the expression of miR-425-5 p in tissues and cells were measured by Realtime PCR,the effect of miR-425-5 p on the proliferation and differentiation of preadipocyte were measured by CCK8,EdU,oil red O staining and triglyceride detection methods,respectively.The target genes of miR-425-5 p regulation preadipocyte differentiation were predicted and identified using bioinformatics softwares and dual luciferase reporter assays,respectively.The results showed that miR-425-5 p was expressed at lower levels in adipose tissues of obese mice,and dynamically expressed during preadipocyte proliferation and differentiation.Compared with negativecontrol,miR-425-5 p overexpression promoted preadipocyte proliferation,inhibited marker genes related to adipocyte differentiation such as PPARγ,C/EBPαand FASgenes,and reduced lipid droplets and triglyceride accumulation.Inhibition of miR-425-5 p expression inhibited the proliferation and prevented the differentiation of preadipocyte.Further analysis found that overexpression or inhibition of miR-425-5 p decreased or increased IGF1 gene expression in preadipocyte differentiation,respectively.Compared with negative control,miR-425-5 p overexpression inhibited the fluorescence activity of IGF1 gene 3′-UTR,whereas mutation of the miR-425-5 p binding site in IGF1 gene 3′-UTR completely abolished this response.This results indicated that miR-425-5 p might promote 3 T3-L1 preadipocyte proliferation,whereas negatively regulates its differentiation by targeting IGF1.
In order to explore the effect of miR-425-5 p on the proliferation and differentiation of3 T3-L1 preadipocyte,the expression of miR-425-5 p in tissues and cells were measured by Realtime PCR,the effect of miR-425-5 p on the proliferation and differentiation of preadipocyte were measured by CCK8,EdU,oil red O staining and triglyceride detection methods,respectively.The target genes of miR-425-5 p regulation preadipocyte differentiation were predicted and identified using bioinformatics softwares and dual luciferase reporter assays,respectively.The results showed that miR-425-5 p was expressed at lower levels in adipose tissues of obese mice,and dynamically expressed during preadipocyte proliferation and differentiation.Compared with negativecontrol,miR-425-5 p overexpression promoted preadipocyte proliferation,inhibited marker genes related to adipocyte differentiation such as PPARγ,C/EBPαand FASgenes,and reduced lipid droplets and triglyceride accumulation.Inhibition of miR-425-5 p expression inhibited the proliferation and prevented the differentiation of preadipocyte.Further analysis found that overexpression or inhibition of miR-425-5 p decreased or increased IGF1 gene expression in preadipocyte differentiation,respectively.Compared with negative control,miR-425-5 p overexpression inhibited the fluorescence activity of IGF1 gene 3′-UTR,whereas mutation of the miR-425-5 p binding site in IGF1 gene 3′-UTR completely abolished this response.This results indicated that miR-425-5 p might promote 3 T3-L1 preadipocyte proliferation,whereas negatively regulates its differentiation by targeting IGF1.
引文
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[30]XIONG Y,HANNON G J,ZHANG H,et al.p21is a universal inhibitor of cyclin kinases[J].Nature,1993,366(6456):701-704.
[31]PU Y,VEIGA-LOPEZ A.PPARγagonist through the terminal differentiation phase is essential foradipogenic differentiation of fetal ovine preadipocytes[J].Cellular&Molecular Biology Letters,2017,22:6.
[32]ROSEN E D,HSU C,WANG X,et al.C/EBP alpha induces adipogenesis through PPAR gamma:A unified pathway[J].Genes&Development,2002,16(1):22-26.
[33]ZHAO P,DENG Y,GU P,et al.Insulin-like growth factor 1promotes the proliferation and adipogenesis of orbital adipose-derived stromal cells in thyroidassociated ophthalmopathy[J].Experimental Eye Research,2013,107(1):65-73.
[34]胡丽.IGF1通过PPARγ-Axin2-Wnt信号通路促进内源性脂肪干/前体细胞成脂分化[D].武汉:华中科技大学,2014.HU L.IGF1 promotes adipogenesis of endogenous adipose stem/progenitor cells via PPARγ-Axin2-Wnt pathways[D].Wuhan:Huazhong University of Science&Tchnology,2014.(in Chinese)
[2]MUST A,SPADANO J,COAKLEY E H,et al.The disease burden associated with overweight and obesity[J].JAMA,1999,282(16):1523-1529.
[3]VAFEIADI M,ROUMELIOTAKI T,MYRIDAKIS A,et al.Association of early life exposure to bisphenol Awith obesity and cardiometabolic traits in childhood[J].Environmental Research,2016,146(2):379-389.
[4]BAEK Y S,YANG P S,KIM T H,et al.Associations of abdominal obesity and new-onset atrial fibrillation in the general population[J].Journal of the American Heart Association,2017,6(6):e004705.
[5]CHAKRABORTY D,BENHAM V,BULLARD B,et al.Fibroblast growth factor receptor is a mechanistic link between visceral adiposity and cancer[J].Oncogene,2017,36(48):6668-6679.
[6]GHORPADE D S,OZCAN L,ZHENG Z,et al.Hepatocyte-secreted DPP4in obesity promotes adipose inflammation and insulin resistance[J].Nature,2018,555(7698):673-677.
[7]CIMMINO A,CALIN G A,FABBRI M,et al.miR-15and miR-16induce apoptosis by targeting BCL2[J].Proceedings of the National Academy of Sciences,2005,102(39):13944-13949.
[8]苗志国,张金洲,张晓建,等.microRNA在猪中的研究进展[J].中国畜牧兽医,2018,45(6):1626-1632.MIAO Z G,ZHANG J Z,ZHANG X J,et al.Research progress on microRNA in pigs[J].China Animal Husbandry&Veterinary Medicine,2018,45(6):1626-1632.(in Chinese)
[9]张抒,张强,张萌,等.miRNA对乳腺发育及泌乳调节作用的研究进展[J].中国畜牧兽医,2015,42(3):663-667.ZHANG S,ZHANG Q,ZHANG M,et al.Research progress on regulation effect of microRNA on mammary gland development and lactation[J].China Animal Husbandry&Veterinary Medicine,2015,42(3):663-667.(in Chinese)
[10]GUO Y,CHEN Y,YUN Z,et al.Up-regulated miR-145expression inhibits porcine preadipocytes differentiation by targeting IRS1[J].International Journal of Biological Sciences,2012,8(10):1408-1417.
[11]DU J,XU Y,ZHANG P,et al.microRNA-125a-5p affects adipocytes proliferation,differentiation and fatty acid composition of porcine intramuscular fat[J].International Journal of Molecular Sciences,2018,19(2):E501.
[12]CHEN L,DAI Y M,JI C B,et al.miR-146bis a regulator of human visceral preadipocyte proliferation and differentiation and its expression is altered in human obesity[J].Molecular&Cellular Endocrinology,2014,393(1-2):65-74.
[13]GAO Y,YIN Y,XING X,et al.Arsenic-induced antiangiogenesis via miR-425-5p-regulated CCM3[J].Toxicology Letters,2016,254:22-31.
[14]FANG F,SONG T,ZHANG T,et al.miR-425-5p promotes invasion and metastasis of hepatocellular carcinoma cells through SCAI-mediated dysregulation of multiple signaling pathways[J].Oncotarget,2017,8(19):31745-31757.
[15]ZHANG Z,WEN M,GUO J,et al.Clinical value of miR-425-5p detection and its association with cell proliferation and apoptosis of gastric cancer[J].Pathology Research&Practice,2017,213(8):929-937.
[16]CHEN F F,XIONG Y,PENG Y,et al.miR-425-5p inhibits differentiation and proliferation in porcine intramuscular preadipocytes[J].International Journal of Molecular Sciences,2017,18(10):E2101.
[17]DU J,ZHANG Y,SHEN L,et al.Effect of miR-143-3p on C2C12 myoblast differentiation[J].Bioscience Biotechnology&Biochemistry,2016,80(4):706-711.
[18]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using Real-time quantitative PCR and the 2(-delta delta C(T))method[J].Methods,2001,25(4):402-408.
[19]师涛,闫晓茹,潘洋洋,等.miR-132-3p靶向UCP2调节绵羊前体脂肪细胞分化的研究[J].畜牧兽医学报,2017,48(11):2059-2067.SHI T,YAN X R,PAN Y Y,et al.Study on theregulation of miR-132-3p in ovine preadipocyte differentiation by targeting UCP2[J].Acta Veterinaria et Zootechnica Sinica,2017,48(11):2059-2067.(in Chinese)
[20]ZOU G,LIU T,GUO L,et al.microRNA-32silences WWP2expression to maintain the pluripotency of human amniotic epithelial stem cells andβislet-like cell differentiation[J].International Journal of Molecular Medicine,2018,41(4):1983-1991.
[21]SASSI Y,AVRAMOPOULOS P,RAMANUJAM D,et al.Cardiac myocyte miR-29promotes pathological remodeling of the heart by activating Wnt signaling[J].Nature Communications,2017,8(1):1614.
[22]ZHAO S,LIU H,LIU Y,et al.miR-143inhibits glycolysis and depletes stemness of glioblastoma stemlike cells[J].Cancer Letters,2013,333(2):253-260.
[23]YAN Y F,GONG F M,WANG B S,et al.miR-425-5p promotes tumor progression via modulation of CYLDin gastric cancer[J].European Review for Medical&Pharmacological Sciences,2017,21(9):2130-2136.
[24]SUN L,JIANG R,LI J,et al.micoRNA-425-5p is a potential prognostic biomarker for cervical cancer[J].Annals of Clinical Biochemistry,2017,54(1):127-133.
[25]ESAU C,KANG X,PERALTA E,et al.microRNA-143regulates adipocyte differentiation[J].Journal of Biological Chemistry,2004,279(50):52361-52365.
[26]DU J,CHENG X,SHEN L,et al.Methylation of miR-145a-5p promoter mediates adipocytes differentiation[J].Biochemical&Biophysical Research Communications,2016,475(1):140-148.
[27]TAN Z,DU J,SHEN L,et al.miR-199a-3p affects adipocytes differentiation and fatty acid composition through targeting SCD[J].Biochemical and Biophysical Research Communications,2017,492(1):82-88.
[28]GAO S Y,LI J,QU X Y,et al.Downregulation of Cdk1and cyclinB1expression contributes to oridonininduced cell cycle arrest at G2/M phase and growth inhibition in SGC-7901gastric cancer cells[J].Asian Pacific Journal of Cancer Prevention,2014,15(15):6437-6441.
[29]ZEITLER H,KO Y,GLODNY B,et al.Cell-cycle arrest in G0/G1phase of growth factor-induced endothelial cell proliferation by various calcium channel blockers[J].Cancer Detection&Prevention,1997,21(4):332-339.
[30]XIONG Y,HANNON G J,ZHANG H,et al.p21is a universal inhibitor of cyclin kinases[J].Nature,1993,366(6456):701-704.
[31]PU Y,VEIGA-LOPEZ A.PPARγagonist through the terminal differentiation phase is essential foradipogenic differentiation of fetal ovine preadipocytes[J].Cellular&Molecular Biology Letters,2017,22:6.
[32]ROSEN E D,HSU C,WANG X,et al.C/EBP alpha induces adipogenesis through PPAR gamma:A unified pathway[J].Genes&Development,2002,16(1):22-26.
[33]ZHAO P,DENG Y,GU P,et al.Insulin-like growth factor 1promotes the proliferation and adipogenesis of orbital adipose-derived stromal cells in thyroidassociated ophthalmopathy[J].Experimental Eye Research,2013,107(1):65-73.
[34]胡丽.IGF1通过PPARγ-Axin2-Wnt信号通路促进内源性脂肪干/前体细胞成脂分化[D].武汉:华中科技大学,2014.HU L.IGF1 promotes adipogenesis of endogenous adipose stem/progenitor cells via PPARγ-Axin2-Wnt pathways[D].Wuhan:Huazhong University of Science&Tchnology,2014.(in Chinese)