miR-196a-5p对3T3-L1前脂肪细胞增殖和分化的影响效应
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摘要
目的:探究miR-196a-5p对小鼠前体脂肪细胞增殖、分化的影响及其潜在的分子机制。方法:(1)构建小鼠肥胖模型,RT-PCR检测脂肪组织中miR-196a-5p表达量;(2)鸡尾酒法诱导3T3-L1前脂肪细胞分化,RT-PCR检测分化过程中miR-196a-5p的表达变化;(3)合成miR-196a-5p mimics和inhibitors转染3T3-L1细胞,以CCK8、EdU试剂盒检测miR-196a-5p对3T3-L1前脂肪细胞增殖的影响作用;(4)运用油红O染色、甘油三酯测定评估miR-196a-5p对3T3-L1细胞分化的影响;(5) RT-PCR检测miR-196a-5p对前脂肪细胞增殖、分化相关基因的影响;(6)结合前人文献,运用生物信息软件、萤光素酶报告系统对miR-196a-5p调控脂肪细胞分化的靶基因进行筛选和验证。结果:(1) miR-196a-5p在肥胖小鼠脂肪组织中高表达,在3T3-L1前脂肪细胞分化过程中先升高后下降;(2)与阴性对照组相比,mimics转染抑制了3T3-L1细胞增殖,inhibitors转染促进了3T3-L1细胞增殖;(3)与阴性对照组相比,mimics组积累了大量油红着色的脂滴,甘油三酯含量增多,而inhibitors组的脂滴少而小,甘油三酯含量相对降低;(4)与阴性对照组相比,mimics转染抑制了增殖标志基因Cyclin D1、Cyclin E、CDK2和CDK4表达,促进了分化标志基因PPARγ、C/EBPα、LPL、aP2等的表达,inhibitors转染则表现出与mimics转染相反的作用;(5) miR-196a-5p可显著抑制野生型MAP4K3和MAPK1 3'UTR萤光素酶活性,而突变绑定位点可废除该抑制效应。结论:miR-196a-5p不仅可抑制3T3-L1前脂肪细胞增殖,还可促进其诱导分化、沉积脂滴;miR-196a-5p可能通过靶向调节MAP4K3和MAPK1来介导3T3-L1前脂肪细胞分化。
Objective: To investigate the effect of miR-196a-5p on proliferation and differentiation of mouse adipocyte,and explore its potential molecular mechanisms. Methods:(1) Utilizing RT-PCR,miR-196a-5p expression levels in adipose tissues from obese or normal mice were measured;(2) The miR-196a-5p expression level during preadipocyte differentiation were measured by RT-PCR method,after 3T3-L1 cells were induced to differentiate by cocktail method;(3)After miR-196a-5p mimics or inhibitors were transfected into 3 T3-L1 cells,CCK8 and EdU detection were performed to evaluate the effect of miR-196 a-5 p on its proliferation;(4) Measuring the effect of miR-196a-5 p on 3T3-L1 cells differentiation by Oil red O staining and triglyceride assay;(5) Detecting the effect of miR-196 a-5 p on the expression levels of 3T3-L1 cells proliferation and differentiation related genes;(6) Based on previous reports,using bioinformatics and luciferase reporter assays to identify targets that miR-196a-5p regulates preadipocyte differentiation. Result:(1)miR-196a-5p not only were highly expressed in adipose tissues of obese mice,but also were expressed dynamically during 3T3-L1 cells differentiation;(2)When compared with negative control,mimics transfection inhibited 3T3-L1 cells proliferation,inhibitors transfection promoted its proliferation;(3)When compared with negative control,mimics or inhibitors transfection increased or decreased lipid accumulation and triglyceride content,respectively;(4)When compared with negative control,mimics transfection repressed proliferation related markers( Cyclin D1,Cyclin E,CDK2 and CDK4) and promoted differentiation related markers( PPARγ,C/EBPα,LPL and aP2),however,inhibitors transfection had an opposite effect than that of mimics transfection;(5) The miR-196 a-5 p mimics significantly suppressed a luciferase reporter gene whose expression was regulated by the mouse MAP4 K3 and MAPK1 mRNA 3'UTR,whereas mutation of the miR-196a-5p binding site in murine MAP4K3 and MAPK1 3' UTR completely abolished this response. Conclusions: miR-196 a-5 p might inhibit 3T3-L1 preadipocyte proliferation, and enhance its differentiation. The regulation of preadipocyte differentiation may be mediated by targeting MAP4K3 and MAPK1.
Objective: To investigate the effect of miR-196a-5p on proliferation and differentiation of mouse adipocyte,and explore its potential molecular mechanisms. Methods:(1) Utilizing RT-PCR,miR-196a-5p expression levels in adipose tissues from obese or normal mice were measured;(2) The miR-196a-5p expression level during preadipocyte differentiation were measured by RT-PCR method,after 3T3-L1 cells were induced to differentiate by cocktail method;(3)After miR-196a-5p mimics or inhibitors were transfected into 3 T3-L1 cells,CCK8 and EdU detection were performed to evaluate the effect of miR-196 a-5 p on its proliferation;(4) Measuring the effect of miR-196a-5 p on 3T3-L1 cells differentiation by Oil red O staining and triglyceride assay;(5) Detecting the effect of miR-196 a-5 p on the expression levels of 3T3-L1 cells proliferation and differentiation related genes;(6) Based on previous reports,using bioinformatics and luciferase reporter assays to identify targets that miR-196a-5p regulates preadipocyte differentiation. Result:(1)miR-196a-5p not only were highly expressed in adipose tissues of obese mice,but also were expressed dynamically during 3T3-L1 cells differentiation;(2)When compared with negative control,mimics transfection inhibited 3T3-L1 cells proliferation,inhibitors transfection promoted its proliferation;(3)When compared with negative control,mimics or inhibitors transfection increased or decreased lipid accumulation and triglyceride content,respectively;(4)When compared with negative control,mimics transfection repressed proliferation related markers( Cyclin D1,Cyclin E,CDK2 and CDK4) and promoted differentiation related markers( PPARγ,C/EBPα,LPL and aP2),however,inhibitors transfection had an opposite effect than that of mimics transfection;(5) The miR-196 a-5 p mimics significantly suppressed a luciferase reporter gene whose expression was regulated by the mouse MAP4 K3 and MAPK1 mRNA 3'UTR,whereas mutation of the miR-196a-5p binding site in murine MAP4K3 and MAPK1 3' UTR completely abolished this response. Conclusions: miR-196 a-5 p might inhibit 3T3-L1 preadipocyte proliferation, and enhance its differentiation. The regulation of preadipocyte differentiation may be mediated by targeting MAP4K3 and MAPK1.
引文
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[29]Siersbk R,Nielsen R,Mandrup S.PPARγin adipocyte differentiation and metabolism-novel insights from genome-wide studies.Febs Letters,2010,584(15):3242-3249.
[30]Choi S K,Park S,Jang S,et al.Cascade regulation of PPARγ(2)and C/EBPαsignaling pathways by celastrol impairs adipocyte differentiation and stimulates lipolysis in 3T3-L1adipocytes.Metabolism Clinical&Experimental,2016,65(5):646-654.
[31]Zhou J,Guo F,Wang G,et al.miR-20a regulates adipocyte differentiation by targeting lysine-specific demethylase 6b and transforming growth factor-βsignaling.International Journal o Obesity,2015,39(8):1282-1291.
[32]Rosen E D,Hsu C H,Wang X,et al.C/EBPαinduces adipogenesis through PPARγ:a unified pathway.Genes&Development,2002,16(1):22-26.
[33]Bushati N,Cohen S M.microRNA functions.Annual Review of Cell&Developmental Biology,2007,23(23):175-205.
[34]Bost F,Aouadi M,Caron L,et al.The role of MAPKs in adipocyte differentiation and obesity.Biochimie,2005,87(1):51-56.
[35]Machinalquélin F,DieudonnéM N,Leneveu M C,et al.Proadipogenic effect of leptin on rat preadipocytes in vitro:activation of MAPK and STAT3 signaling pathways.American Journal Physiol Cell Physiol,2002,282(4):c853-c863.
[2]Kahn S E,Hull R L,Utzschneider K M,et al.Mechanisms linking obesity to insulin resistance and type 2 diabetes.Nature,2007,444(7121):840-846.
[3]Mayer J,Thomas D W.Regulation of food intake and obesity.Science,1967,156(3773):328-337.
[4]Li J.MicroRNAs:target recognition and regulatory functions.Cell,2009,136(2):215-233.
[5]Ambros V.The function of animal MicroRNAs.Nature,2004,431(7006):350-355.
[6]Du T,Zamore P D.Microprimer:the biogenesis and function of microRNA.Development,2005,132(21):4645-4652.
[7]Perri R,Nares S,Zhang S,et al.MicroRNA modulation in obesity and periodontitis.Journal of Dental Research,2012,91(1):33-38.
[8]Kajimoto K,Naraba H,Iwai N.MicroRNA and 3T3-L1 preadipocyte differentiation.Rna-a Publication of the Rna Society,2006,12(9):1626-1632.
[9]Berthold S,Kovacs P,Fasshauer M,et al.MicroRNA expression in human omental and subcutaneous adipose tissue.PLo S One,2009,4(3):e4699.
[10]Karbiener M,Fischer C,Nowitsch S,et al.MicroRNA miR-27b impairs human adipocyte differentiation and targets PPARγ.Biochemical and Biophysical Research Communications,2009,390(2):247-251.
[11]Peng Y,Xiang H,Chen C,et al.MiR-224 impairs adipocyte early differentiation and regulates fatty acid metabolism.International Journal of Biochemistry&Cell Biology,2013,45(8):1585-1593.
[12]Lv S,Ma M,Sun Y,et al.MicroRNA-129-5p inhibits 3T3-L1preadipocyte proliferation by targeting G3BP.Animal Cells&Systems the Official Publication of the Zoological Society of Korea,2017,21(4):269-277.
[13]Pan J,Li X,Wu W,et al.Long non-coding RNA UCA1promotes cisplatin/gemcitabine resistance through CREBmodulating miR-196a-5p in bladder cancer cells.Cancer Letters,2016,382(1):64-76.
[14]Zhao X,Liu Y,Zheng J,et al.GAS5 suppresses malignancy of human glioma stem cells via a miR-196a-5p/FOXO1 feedback loop.Biochimica Et Biophysica Acta.2017,1864(10):1605-1617.
[15]赵华路,姚南,魏雪菊,等.miR-196a-5p抑制小鼠胚胎干细胞的自我更新.基础医学临床,2014,34(12):1645-1649.Zhao H L,Yao N,Wei X J,et al.miR-196a-5p suppresses selfrenewal of mouse embryonic stem cells.Basic&Clinical Medicine,2014,34(12):1645-1649.
[16]Huang N,Wang J,Xie W,et al.MiR-378a-3p enhances adipogenesis by targeting mitogen-activated protein kinase 1.Biochemical&Biophysical Research Communications,2015,457(1):37-42.
[17]黃筠雅.Map4k3基因对脂肪細胞分化的影响.花莲:慈济大学生命科学研究所,2012.Huang J Y.The effect of Map4k3 on adipocytes differentiation.Hualian:Dissertation of Institute of Life Science,Tzu Chi University,2012.
[18]Chen J F,Mandel E M,Thomson J M,et al.The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation.Nature Genetics,2006,38(2):228-233.
[19]Taylor D D,Gercel-Taylor C.MicroRNA signatures of tumorderived exosomes as diagnostic biomarkers of ovarian cancer.Gynecologic Oncology,2008,110(1):13-21.
[20]Delaloy C,Liu L,Lee J A,et al.MicroRNA-9 coordinates proliferation and migration of human embryonic stem cell-derived neural progenitors.Cell Stem Cell,2010,6(4):323-335.
[21]Gaudet A D,Fonken L K,Gushchina L V,et al.miR-155deletion in female mice prevents diet-induced obesity.Scientific Reports,2016,6(22862):1-10.
[22]Du J,Cheng X,Shen L,et al.Methylation of miR-145a-5p promoter mediates adipocytes differentiation.Biochemical and Biophysical Research Communications,2016,475(1):140-148.
[23]Tan Z,Du J,Shen L,et al.miR-199a-3p affects adipocytes differentiation and fatty acid composition through targeting SCD.Biochemical and Biophysical Research Communications,2017,492(1):82-88.
[24]Du J,Xu Y,Zhang P.et al.MicroRNA-125a-5p affects adipocytes proliferation,differentiation and fattyacid composition of porcine intramuscular fat.International Journal of Molecular Sciences,2018,19(2):1-3.
[25]Baldin V,Lukas J,Marcote M J,et al.Cyclin D1 is a nuclear protein required for cell cycle progression in G1.Genes&Development.1993,7(5):812-821.
[26]Stacey D W.Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells.Current Opinion in Cell Biology.2003,15(2):158-163.
[27]Koff A,Dulic V,Lees E,et al.Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle:Science 257,1689-1694.Trends in Cell Biology,1992,2(12):362-362.
[28]Meyer C A,Jacobs H W,Lehner C F.Cyclin D-cdk4 is not a master regulator of cell multiplication in Drosophila embryos.Current Biology,2002,12(8):661-666.
[29]Siersbk R,Nielsen R,Mandrup S.PPARγin adipocyte differentiation and metabolism-novel insights from genome-wide studies.Febs Letters,2010,584(15):3242-3249.
[30]Choi S K,Park S,Jang S,et al.Cascade regulation of PPARγ(2)and C/EBPαsignaling pathways by celastrol impairs adipocyte differentiation and stimulates lipolysis in 3T3-L1adipocytes.Metabolism Clinical&Experimental,2016,65(5):646-654.
[31]Zhou J,Guo F,Wang G,et al.miR-20a regulates adipocyte differentiation by targeting lysine-specific demethylase 6b and transforming growth factor-βsignaling.International Journal o Obesity,2015,39(8):1282-1291.
[32]Rosen E D,Hsu C H,Wang X,et al.C/EBPαinduces adipogenesis through PPARγ:a unified pathway.Genes&Development,2002,16(1):22-26.
[33]Bushati N,Cohen S M.microRNA functions.Annual Review of Cell&Developmental Biology,2007,23(23):175-205.
[34]Bost F,Aouadi M,Caron L,et al.The role of MAPKs in adipocyte differentiation and obesity.Biochimie,2005,87(1):51-56.
[35]Machinalquélin F,DieudonnéM N,Leneveu M C,et al.Proadipogenic effect of leptin on rat preadipocytes in vitro:activation of MAPK and STAT3 signaling pathways.American Journal Physiol Cell Physiol,2002,282(4):c853-c863.