Tribbles同源蛋白3抑制人脂肪间充质干细胞成脂向分化

doi:10.19723/j.issn.1671-167X.2020.01.001

摘要/Abstract

摘要：

目的:研究Tribbles同源蛋白3(Tribbles pseudokinase 3, TRIB3)在人脂肪间充质干细胞(human adipose-derived mesenchymal stem cells, hASCs)成脂分化中的调控作用,为提高hASCs成脂向分化能力、并为基于hASCs的脂肪组织工程与软组织缺损修复提供新靶点与新思路。方法:通过慢病毒转染建立TRIB3稳定敲低(TRIB3敲低组)和过表达(TRIB3过表达组)的hASCs细胞系,通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)和蛋白质印迹实验(Western blot)检测慢病毒转染hASCs的转染效率和效果。通过油红O染色和定量、qRT-PCR等实验方法,检测敲低、过表达TRIB3对hASCs成脂分化能力的影响。结果:TRIB3敲低慢病毒转染后,TRIB3敲低组hASCs中TRIB3 mRNA表达量较对照组下降84.3%(P<0.01), TRIB3蛋白表达也显著下降,表明成功构建了TRIB3敲低的hASCs细胞系;TRIB3过表达慢病毒转染后,TRIB3过表达组TRIB3 mRNA表达量较对照组增高约1.6倍(P<0.01),TRIB3蛋白表达也显著升高,表明成功构建了TRIB3过表达的hASCs细胞系。在此基础上,油红O染色显示,TRIB3敲低的hASCs成脂诱导后胞浆内红色脂滴明显增多,定量结果表明TRIB3敲低组油红O着色较对照组显著增多(P<0.01)。与之相反,TRIB3过表达的hASCs成脂诱导后胞浆内红色脂滴明显减少,定量结果表明TRIB3敲低组油红O着色较对照组显著减少(P<0.01)。qRT-PCR结果显示,敲低TRIB3的hASCs成脂诱导后,成脂分化相关基因过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor γ, PPARγ)、CCAAT增强子结合蛋白α(CCAAT/enhancer binding protein α, C/EBPα)和白细胞分化抗原36(cluster of differentiation 36, CD36)mRNA水平显著升高(P<0.01);TRIB3过表达的hASCs成脂诱导后成脂分化相关基因PPARγ、CD36和脂蛋白酯酶(lipoprotein lipase, LPL)mRNA水平明显下降。结论:TRIB3能够调控hASCs成脂分化能力,敲低TRIB3促进hASCs成脂分化,过表达TRIB3抑制hASCs成脂分化。

关键词: 人脂肪间充质干细胞, Tribbles同源蛋白3, 成脂分化

Abstract:

Objective: To identify the role of Tribbles pseudokinase 3 (TRIB3) during the process of adipogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs), and to provide a new target and a novel idea for the application of hASCs in adipose tissue engineering and soft tissue regeneration. Methods: TRIB3-knockdown hASCs (shTRIB3) and TRIB3-overexpression hASCs (TRIB3-over) were established using lentivirus transfection technique. The transfection effect was estimated by the visible presence of green fluorescence as the expression of green fluorescent protein (GFP) in the transfected hASCs. The lentiviral transfection efficiency was examined by quantitative real-time poly-merase chain reaction (qRT-PCR) and Western blot. After adipogenic induction, Oil Red staining and quantification, as well as qRT-PCR about several specific adipogenic markers were used to evaluate the adipogenic differentiation ability of hASCs. Results: In TRIB3-knockdown hASCs, the TRIB3 mRNA expression level decreased by about 84.3% compared with the control group (P<0.01), and the TRIB3 protein level also showed obvious reduction. Oppositely, in TRIB3-overexpression hASCs, the TRIB3 mRNA expression level increased by approximately 160% compared with the control group (P<0.01), and the TRIB3 protein level also showed a significant increase. These results indicated a successful construction of TRIB3-knockdown hASCs and TRIB3-overexpression hASCs. The Oil Red staining results showed that the down-regulation of TRIB3 significantly promoted lipid droplets formation in hASCs, consistent with Oil Red quantification. On the other hand, the up-regulation of TRIB3 suppressed lipid droplets formation in hASCs, consistent with Oil Red quantification. After adipogenic induction, adipogenesis-related genes, including peroxisome proliferator-activated receptor γ (PPARγ), cluster of differentiation 36 (CD36) and CCAAT/enhancer binding protein α (C/EBPα), were increased significantly in TRIB3-knockdown hASCs compared with the control group (P<0.01). Oppositely, PPARγ, CD36 and lipoprotein lipase (LPL) were significantly decreased in TRIB3-overexpression hASCs compared with the control group (P<0.01). Conclusion: TRIB3 inhibited the adipogenic differentiation of hASCs. Knockdown of TRIB3 promoted the ability of adipogenesis of hASCs, while overexpression of TRIB3 inhibited the adipogenic differentiation of hASCs. Considering the important role of PPARγ in the adipogenis process, the molecular mechanism of the regulatory function of TRIB3 may be related with PPARγ signal pathway.

Key words: Human adipose-derived mesenchymal stem cells, Tribbles pseudokinase 3, Adipogenic differentiation

中图分类号:

R329.2

白向松,吕珑薇,周永胜. Tribbles同源蛋白3抑制人脂肪间充质干细胞成脂向分化[J]. 北京大学学报（医学版）, 2020, 52(1): 1-9.

Xiang-song BAI,Long-wei LV,Yong-sheng ZHOU. Tribbles pseudokinase 3 inhibits the adipogenic differentiation of human adipose-derived mesenchymal stem cells[J]. Journal of Peking University(Health Sciences), 2020, 52(1): 1-9.

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参考文献 39

[1]	Rubin JP, Marra KG . Adipose stem cell therapy for soft tissue reconstruction[J]. Lancet, 2013,382(9898):1077-1079.
[2]	Patrick CJ, Chauvin PB, Hobley J , et al. Preadipocyte seeded PLGA scaffolds for adipose tissue engineering[J]. Tissue Eng, 1999,5(2):139-151.
[3]	Paschos NK, Brown WE, Eswaramoorthy R , et al. Advances in tissue engineering through stem cell-based co-culture[J]. J Tissue Eng Regen Med, 2015,9(5):488-503.
[4]	Cossu G, Birchall M, Brown T , et al. Lancet Commission: Stem cells and regenerative medicine[J]. Lancet, 2018,391(10123):883-910.
[5]	Zuk PA, Zhu M, Mizuno H , et al. Multilineage cells from human adipose tissue: Implications for cell-based therapies[J]. Tissue Eng, 2001,7(2):211-228.
[6]	Zuk PA, Zhu M, Ashjian P , et al. Human adipose tissue is a source of multipotent stem cells[J]. Mol Biol Cell, 2002,13(12):4279-4295.
[7]	Gentile P, Orlandi A, Scioli MG , et al. Concise review: Adipose-derived stromal vascular fraction cells and platelet-rich plasma: Basic and clinical implications for tissue engineering therapies in regenerative surgery[J]. Stem Cells Transl Med, 2012,1(3):230-236.
[8]	Lv LW, Liu YS, Zhang P , et al. Transcriptomics and functional analysis of graphene-guided osteogenic differentiation of mesenchymal stem cells[J]. Chin J Dent Res, 2018,21(2):101-111.
[9]	Eyers PA, Keeshan K, Kannan N . Tribbles in the 21st Century: The evolving roles of tribbles pseudokinases in biology and disease[J]. Trends Cell Biol, 2017,27(4):284-298.
[10]	Ord T, Ord T . Mammalian pseudokinase TRIB3 in normal physi-ology and disease: Charting the progress in old and new avenues[J]. Curr Protein Pept Sci, 2017,18(8):819-842.
[11]	Prudente S, Sesti G, Pandolfi A , et al. The mammalian tribbles homolog TRIB3, glucose homeostasis, and cardiovascular diseases[J]. Endocr Rev, 2012,33(4):526-546.
[12]	Liu G, Jin S, Hu Y , et al. Disease status affects the association between rs4813620 and the expression of Alzheimer’s disease susceptibility gene TRIB3[J]. Proc Natl Acad Sci USA, 2018,115(45):E10519-E10520.
[13]	Hua F, Li K, Yu JJ , et al. The TRIB3-SQSTM1 interaction mediates metabolic stress-promoted tumorigenesis and progression via suppressing autophagic and proteasomal degradation[J]. Auto-phagy, 2015,11(10):1929-1931.
[14]	Mondal D, Mathur A, Chandra PK . Tripping on TRIB3 at the junction of health, metabolic dysfunction and cancer[J]. Biochimie, 2016,124:34-52.
[15]	Kusminski CM, Bickel PE, Scherer PE . Targeting adipose tissue in the treatment of obesity-associated diabetes[J]. Nat Rev Drug Discov, 2016,15(9):639-660.
[16]	Picon-Ruiz M, Morata-Tarifa C, Valle-Goffin JJ , et al. Obesity and adverse breast cancer risk and outcome: Mechanistic insights and strategies for intervention[J]. CA Cancer J Clin, 2017,67(5):378-397.
[17]	Bezy O, Vernochet C, Gesta S , et al. TRB3 blocks adipocyte differentiation through the inhibition of C/EBPbeta transcriptional activity[J]. Mol Cell Biol, 2007,27(19):6818-6831.
[18]	Wang X, Gao L, Han Y , et al. Silicon-enhanced adipogenesis and angiogenesis for vascularized adipose tissue engineering[J]. Adv Sci (Weinh), 2018,5(11):1800776.
[19]	Levi B, Longaker MT . Concise review: Adipose-derived stromal cells for skeletal regenerative medicine[J]. Stem Cells, 2011,29(4):576-582.
[20]	Zhu Y, Zhang P, Gu RL , et al. Origin and clinical applications of neural crest-derived dental stem cells[J]. Chin J Dent Res, 2018,21(2):89-100.
[21]	Lu Z, Yuan Y, Gao J , et al. Adipose tissue extract promotes adipose tissue regeneration in an adipose tissue engineering chamber model[J]. Cell Tissue Res, 2016,364(2):289-298.
[22]	Volz AC, Huber B, Kluger PJ . Adipose-derived stem cell differentiation as a basic tool for vascularized adipose tissue engineering[J]. Differentiation, 2016,92(1/2):52-64.
[23]	Gomillion CT, Burg KJ . Stem cells and adipose tissue engineering[J]. Biomaterials, 2006,27(36):6052-6063.
[24]	Scott MA, Nguyen VT, Levi B , et al. Current methods of adipogenic differentiation of mesenchymal stem cells[J]. Stem Cells Dev, 2011,20(10):1793-1804.
[25]	Saltiel AR . Putting the brakes on insulin signaling[J]. N Engl J Med, 2003,349(26):2560-2562.
[26]	Qi L, Heredia JE, Altarejos JY , et al. TRB3 links the E3 ubi-quitin ligase COP1 to lipid metabolism[J]. Science, 2006,312(5781):1763-1766.
[27]	Brazil DP, Hemmings BA . Ten years of protein kinase B signalling: A hard Akt to follow[J]. Trends Biochem Sci, 2001,26(11):657-664.
[28]	Cho H, Mu J, Kim JK , et al. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta)[J]. Science, 2001,292(5522):1728-1731.
[29]	Du K, Herzig S, Kulkarni RN , et al. TRB3: A tribbles homolog that inhibits Akt/PKB activation by insulin in liver[J]. Science, 2003,300(5625):1574-1577.
[30]	Park JS, Kim M, Song NJ , et al. A reciprocal role of the Smad4-Taz axis in osteogenesis and adipogenesis of mesenchymal stem cells[J]. Stem Cells, 2019,37(3):368-381.
[31]	Hyvari L, Ojansivu M, Juntunen M , et al. Focal adhesion kinase and ROCK signaling are switch-like regulators of human adipose stem cell differentiation towards osteogenic and adipogenic lineages[J]. Stem Cells Int, 2018, 9(2018-09-12)[2018-12-12]. .
[32]	Liu X, He J, Zhang S , et al. Adipose stem cells controlled by surface chemistry[J]. J Tissue Eng Regen Med, 2013,7(2):112-117.
[33]	Furuhata Y, Yoshitomi T, Kikuchi Y , et al. Osteogenic lineage commitment of adipose-derived stem cells is predetermined by three-dimensional cell accumulation on micropatterned surface[J]. ACS Appl Mater Interfaces, 2017,9(11):9339-9347.
[34]	Takahashi Y, Ohoka N, Hayashi H , et al. TRB3 suppresses adipocyte differentiation by negatively regulating PPARgamma transcriptional activity[J]. J Lipid Res, 2008,49(4):880-892.
[35]	Chan MC, Hilyard AC, Wu C , et al. Molecular basis for antagonism between PDGF and the TGFbeta family of signalling pathways by control of miR-24 expression[J]. EMBO J, 2010,29(3):559-573.
[36]	Roy L, Bikorimana E, Lapid D , et al. MiR-24 is required for hematopoietic differentiation of mouse embryonic stem cells[J]. PLoS Genet, 2015,11(1):e1004959.
[37]	Kim S, Hata A, Kang H . Down-regulation of miR-96 by bone morphogenetic protein signaling is critical for vascular smooth muscle cell phenotype modulation[J]. J Cell Biochem, 2014,115(5):889-895.
[38]	Eom HJ, Chatterjee N, Lee J , et al. Integrated mRNA and micro RNA profiling reveals epigenetic mechanism of differential sensiti-vity of Jurkat T cells to AgNPs and Ag ions[J]. Toxicol Lett, 2014,229(1):311-318.
[39]	Liu X, Zhao J, Liu Q , et al. MicroRNA-124 promotes hepatic triglyceride accumulation through targeting tribbles homolog 3[J]. Sci Rep, 2016,6:37170.

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Gene	Forward primer (5' to 3')	Reverse primer (5' to 3')
GAPDH	CGGACCAATACGACCAAATCCG	AGCCACATCGCTCAGACACC
TRIB3	GGGTCTGTTTTGCATGCGAGC	AGCTCGTTTCTGGACGGGAC
PPARγ	GAGGAGCCTAAGGTAAGGAG	GTCATTTCGTTAAAGGCTGA
C/EBPα	CGCAAGAGCCGAGATAAAGC	CACGGCTCAGCTGTTCCA
LPL	CGGATTAACATTGGAGAAGCTATCCG	AGCTGGTCCACATCTCCAAGTC
CD36	CGATTAACATAAGTAAAGTTGCCATAATCG	CGCAGTGACTTTCCCAATAGGAC