microRNA-425-5p对3T3-L1前体脂肪细胞增殖、分化的影响

doi:10.16431/j.cnki.1671-7236.2019.02.004

摘要/Abstract

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。

关键词: miRNA-425-5p; 前体脂肪细胞; 增殖; 分化; IGF1基因

Abstract:

In order to explore the effect of miR-425-5p on the proliferation and differentiation of 3T3-L1 preadipocyte,the expression of miR-425-5p in tissues and cells were measured by Real-time PCR,the effect of miR-425-5p 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-425-5p regulation preadipocyte differentiation were predicted and identified using bioinformatics softwares and dual luciferase reporter assays,respectively.The results showed that miR-425-5p was expressed at lower levels in adipose tissues of obese mice,and dynamically expressed during preadipocyte proliferation and differentiation.Compared with negative control,miR-425-5p overexpression promoted preadipocyte proliferation,inhibited marker genes related to adipocyte differentiation such as PPARγ,C/EBPα and FAS genes,and reduced lipid droplets and triglyceride accumulation.Inhibition of miR-425-5p expression inhibited the proliferation and prevented the differentiation of preadipocyte.Further analysis found that overexpression or inhibition of miR-425-5p decreased or increased IGF1 gene expression in preadipocyte differentiation,respectively.Compared with negative control,miR-425-5p overexpression inhibited the fluorescence activity of IGF1 gene 3'-UTR,whereas mutation of the miR-425-5p binding site in IGF1 gene 3'-UTR completely abolished this response.This results indicated that miR-425-5p might promote 3T3-L1 preadipocyte proliferation,whereas negatively regulates its differentiation by targeting IGF1.

Key words: miRNA-425-5p; preadipocyte; proliferation; differentation; IGF1 gene

中图分类号:

Q591.5

杨琼, 辜浩, 堵晶晶, 刘进远, 张顺华, 朱砺. microRNA-425-5p对3T3-L1前体脂肪细胞增殖、分化的影响[J]. 《中国畜牧兽医》, 2019, 46(2): 354-364.

YANG Qiong, GU Hao, DU Jingjing, LIU Jinyuan, ZHANG Shunhua, ZHU Li. Effect of miR-425-5p on Proliferation and Differentiation of 3T3-L1 Preadipocyte[J]. , 2019, 46(2): 354-364.

参考文献

[1] UAUY R,DANGOUR A D.Fat and fatty acid requirements and recommendations for infants of 0-2 years and children of 2-18 years[J].Annals of Nutrition & Metabolism,2009,55(1-3):76-96.
[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 A with 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 DPP4 in 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-15 and miR-16 induce 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-145 expression 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-146b is 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 anti-angiogenesis 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-32 silences WWP2 expression to maintain the pluripotency of human amniotic epithelial stem cells and β islet-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-29 promotes 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-143 inhibits glycolysis and depletes stemness of glioblastoma stem-like 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 CYLD in 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-143 regulates 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 Cdk1 and cyclinB1 expression contributes to oridonin-induced cell cycle arrest at G2/M phase and growth inhibition in SGC-7901 gastric 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/G1 phase 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.p21 is a universal inhibitor of cyclin kinases[J].Nature,1993,366(6456):701-704.
[31] PU Y,VEIGA-LOPEZ A.PPARγ agonist 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 1 promotes the proliferation and adipogenesis of orbital adipose-derived stromal cells in thyroid-associated 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)