关岭牛MEF2A基因干扰载体构建及其转染对成肌细胞的影响

doi:10.11843/j.issn.0366-6964.2023.02.016

摘要/Abstract

摘要： 旨在构建肌细胞增强因子MEF2A （myocyte enhancer factor 2A）基因的重组干扰载体，探究MEF2A基因对牛成肌细胞的影响。本研究选择3头健康的3日龄雌性关岭牛，体重约为21 kg，采集背最长肌组织成功培养成肌细胞。设计MEF2A基因的4对shRNA干扰序列和1对NC阴性对照序列，将其连接至pGPU6-GFP-Neo载体上，转染重组载体至关岭牛成肌细胞。采用qRT-PCR法筛选干扰效率最佳的载体，并检测干扰MEF2A基因对肌生成因子MEF2B、MEF2C、MEF2D，周期与凋亡因子CDK2、CCNA2、BCL2 mRNA表达水平的影响；随后利用流式细胞仪与酶标仪探究干扰载体对成肌细胞增殖生长的影响，各试验组别设置3个生物学重复。同时运用在线软件预测牛MEF2A蛋白理化性质与网络谱图。结果显示，本研究成功筛选出干扰效率最佳的shRNA-MEF2A-3载体（P<0.01）。MEF2A基因被抑制后，成肌细胞中MEF2B、MEF2C与MEF2D基因表达量均极显著上调（P<0.01）；CDK2与BCL2表达量皆显著下调（P<0.05），CCNA2表达量极显著下调（P<0.01）；与对照组相比，干扰组细胞周期明显延长，干扰组成肌细胞在6 h后的增殖活性均极显著低于对照组（P<0.01）。由此可初步推测，MEF2A干扰载体成功转染至牛成肌细胞后，可有效抑制MEF2A基因表达，改变基因表达模式，影响了关岭牛成肌细胞的分裂增殖，为进一步探究MEF2A基因对关岭牛肉质性状调控机制和挖掘地方种质资源提供数据支撑。

关键词: 关岭牛, MEF2A基因, 干扰, 成肌细胞, 细胞增殖

Abstract: The aim of this study was to construct a recombinant interference vector of myocyte enhancer factor 2A (MEF2A) gene to explore the effect of MEF2A on bovine myoblasts. In this study, 3 healthy 3-day-old female Guanling cattle, with the body weight of about 21 kg, were selected to culture myoblastasts from longissimus dorsi muscle tissue. Four shRNA interference sequences of MEF2A gene and one NC negative control sequence were designed and connected to pGPU6-GFP-Neo vector, and the recombinant vector was transfected into Guanling bovine myoblasts. qRT-PCR was used to screen the vector with the best interference efficiency, and the effects of interfering MEF2A gene on the mRNA expression levels of myogenic factors MEF2B, MEF2C, MEF2D, cycle and apoptotic factors CDK2, CCNA2 and BCL2 were detected. Subsequently, flow cytometry and enzyme marker were used to investigate the effect of interference vector on myoblast proliferation and growth. Three biological replicates were set in each experimental group. Online software was used to predict the physicochemical properties and network spectra of bovine MEF2A protein. The results showed that the shRNA-MEF2A-3 vector with the best interference efficiency was selected successfully (P<0.01). After interfering MEF2A gene, MEF2B, MEF2C and MEF2D gene expressions in myoblasts were significantly up-regulated (P<0.01); The expressions of CDK2 and BCL2 were significantly down-regulated (P<0.05), the expression of CCNA2 was significantly down-regulated (P<0.01); Compared with the control group, the cell cycle in the interference group was significantly prolonged, and the proliferative activity of the interfered myoblasts was significantly lower than that in the control group (P<0.01). It can be preliminarily speculated that the MEF2A interference vector successfully transfected into bovine myoblasts can effectively inhibit the expression of MEF2A gene, change the gene expression pattern, and affect the division and proliferation of Guanling cattle myoblasts, which provides data support for further exploring the regulatory mechanism of MEF2A gene on the meat quality traits of Guanling cattle and mining local germplasm resources.

Key words: Guanling cattle, MEF2A gene, interference, myoblasts, cell proliferation

中图分类号:

S823.81

孙金魁, 许厚强, 石鹏飞, 阮涌. 关岭牛MEF2A基因干扰载体构建及其转染对成肌细胞的影响[J]. 畜牧兽医学报, 2023, 54(2): 584-595.

SUN Jinkui, XU Houqiang, SHI Pengfei, RUAN Yong. Construction of MEF2A Gene Interference Vector and Effect of Its Transfection on Myoblasts in Guanling Cattle[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 584-595.

参考文献 43

[1]	刘鹏程.TBC1D7、GRB10基因表达调控及其与关岭牛生长性状相关性研究[D].贵阳:贵州大学, 2021.LIU P C.Expression regulation of TBC1D7 and GRB10 genes and correlation between two genes and growth traits of Guanling cattle[D].Guiyang:Guizhou University, 2021.(in Chinese)
[2]	彭梦阳, 王大会, 贺花, 等.贵州地方黄牛种业现状、存在问题及对策[J].中国牛业科学, 2019, 45(4):55-58.PENG M Y, WANG D H, HE H, et al.Current situation, problems and countermeasures of Guizhou local cattle breeding industry[J].China Cattle Science, 2019, 45(4):55-58.(in Chinese)
[3]	SHAHJAHAN M.Skeletal muscle development in vertebrate animals[J].Asian J Med Biol Res, 2015, 1(2):139-148.
[4]	GONG H J, XIE J, ZHANG N, et al.MEF2A binding to the glut4 promoter occurs via an AMPKα2-dependent mechanism[J].Med Sci Sports Exerc, 2011, 43(8):1441-1450.
[5]	叶涛, 杨华婷, 李辉, 等.兴义鸭MEF2C基因可变剪切体的组织表达差异研究[J].农业生物技术学报, 2021, 29(10):1990-1998.YE T, YANG H T, LI H, et al.Study on differential tissue expression of MEF2C splice Variants in Xingyi duck (Anas platyrhynchos)[J].Journal of Agricultural Biotechnology, 2021, 29(10):1990-1998.(in Chinese)
[6]	BRAND N J.Myocyte enhancer factor 2 (MEF2)[J].Int J Biochem Cell Biol, 1997, 29(12):1467-1470.
[7]	AKHTAR M W, KIM M S, ADACHI M, et al.In vivo analysis of MEF2 transcription factors in synapse regulation and neuronal survival[J].PLoS One, 2012, 7(4):e34863.
[8]	ZHU B F, CARMICHAEL R E, VALOIS L S, et al.The transcription factor MEF2A plays a key role in the differentiation/maturation of rat neural stem cells into neurons[J].Biochem Biophys Res Commun, 2018, 500(3):645-649.
[9]	PON J R, MARRA M A.MEF2 transcription factors:developmental regulators and emerging cancer genes[J].Oncotarget, 2016, 7(3):2297-2312.
[10]	WVST S, DRÖSE S, HEIDLER J, et al.Metabolic maturation during muscle stem cell differentiation is achieved by miR-1/133a-mediated inhibition of the DLK1-DIO3 mega gene cluster[J].Cell Metab, 2018, 27(5):1026-1039.e6.
[11]	BLACK B L, MOLKENTIN J D, OLSON E N.Multiple roles for the MyoD basic region in transmission of transcriptional activation signals and interaction with MEF2[J].Mol Cell Biol, 1998, 18(1):69-77.
[12]	CORRE S, GALIBERT M D.USF as a key regulatory element of gene expression[J].Med Sci (Paris), 2006, 22(1):62-67.
[13]	EDMONDSON D G, LYONS G E, MARTIN J F, et al.Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis[J].Development, 1994, 120(5):1251-1263.
[14]	CHEN X, GAO B, PONNUSAMY M, et al.MEF2 signaling and human diseases[J].Oncotarget, 2017, 8(67):112152-112165.
[15]	ESTRELLA N L, DESJARDINS C A, NOCCO S E, et al.MEF2 transcription factors reguLate distinct gene programs in mammalian skeletal muscle differentiation[J].J Biol Chem, 2015, 290(2):1256-1268.
[16]	WU R H, WANG J, YAO J, et al.MEF2A regulates Calpain 3 expression in L6 myoblasts[J].Gene, 2018, 668:204-210.
[17]	CLARK A L, NAYA F J.MicroRNAs in the myocyte enhancer factor 2 (MEF2)-regulated Gtl2-Dio3 noncoding RNA locus promote cardiomyocyte proliferation by targeting the transcriptional coactivator cited2[J].J Biol Chem, 2015, 290(38):23162-23172.
[18]	LIU B R, WANG L, JIANG W Y, et al.Myocyte enhancer factor 2A delays vascular endothelial cell senescence by activating the PI3K/p-Akt/SIRT1 pathway[J].Aging (Albany NY), 2019, 11(11):3768-3784.
[19]	BLIXT N, NORTON A, ZHANG A Q, et al.Loss of myocyte enhancer factor 2 expression in osteoclasts leads to opposing skeletal phenotypes[J].Bone, 2020, 138:115466.
[20]	宋林锦, 许厚强, 李永, 等.关岭牛IGF1基因干扰载体构建及其对成肌细胞的影响[J].农业生物技术学报, 2022, 30(5):896-907.SONG L J, XU H Q, LI Y, et al.Construction of IGF1 gene interference vector in guanling cattle (Bos taurus) and its effect on myogenic cells[J].Journal of Agricultural Biotechnology, 2022, 30(5):896-907.(in Chinese)
[21]	KUMAR V, KUNDU S, SINGH A, et al.Understanding the role of histone deacetylase and their inhibitors in neurodegenerative disorders:current targets and future perspective[J].Curr Neuropharmacol, 2022, 20(1):158-178.
[22]	YOKOTA T, LI J, HUANG J J, et al.p38 Mitogen-activated protein kinase regulates chamber-specific perinatal growth in heart[J].J Clin Invest, 2020, 130(10):5287-5301.
[23]	BLACK B L, OLSON E N.Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins[J].Annu Rev Cell Dev Biol, 1998, 14:167-196.
[24]	MCKINSEY T A, ZHANG C L, OLSON E N.MEF2:a calcium-dependent regulator of cell division, differentiation and death[J].Trends Biochem Sci, 2002, 27(1):40-47.
[25]	MEDRANO J L, NAYA F J.The transcription factor MEF2A fine-tunes gene expression in the atrial and ventricular chambers of the adult heart[J].J Biol Chem, 2017, 292(51):20975-20988.
[26]	王亚宁.MEF2A对秦川牛骨骼肌成肌细胞增殖和分化的调控作用及机理研究[D].杨凌:西北农林科技大学, 2019.WANG Y N.The roles of MEF2A in the regulation of skeletal muscle myoblasts proliferation and differentiation in Qinchuan beef cattle[D].Yangling:Northwest A&F University, 2019.(in Chinese)
[27]	VARGAS M A X, TIRNAUER J S, GLIDDEN N, et al.Myocyte enhancer factor 2 (MEF2) tethering to muscle selective A-kinase anchoring protein (mAKAP) is necessary for myogenic differentiation[J].Cell Signal, 2012, 24(8):1496-1503.
[28]	SNYDER C M, RICE A L, ESTRELLA N L, et al.MEF2A regulates the Gtl2-Dio3 microRNA mega-cluster to modulate WNT signaling in skeletal muscle regeneration[J].Development, 2013, 140(1):31-42.
[29]	JUSZCZUK-KUBIAK E, FLISIKOWSKI K, WICIŃSKA K.Nucleotide sequence and variations of the bovine myocyte enhancer factor 2C (MEF2C) gene promoter in Bos taurus cattle[J].Mol Biol Rep, 2011, 38(2):1269-1276.
[30]	杨涛.牛MEF2家族转录因子与MSTN启动子相互作用的机制研究[D].贵阳:贵州大学, 2019.YANG T.Study on interaction mechanism of MEF2 family transcription factors and MSTN promoter in cattle[D]. Guiyang:Guizhou University, 2019.(in Chinese)
[31]	LIU L, NELSON B R, BEZPROZVANNAYA S, et al.Requirement of MEF2A, C, and D for skeletal muscle regeneration[J].Proc Natl Acad Sci U S A, 2014, 111(11):4109-4114.
[32]	HOYECK M P, HADJ-MOUSSA H, STOREY K B.The role of MEF2 transcription factors in dehydration and anoxia survival in Rana sylvatica skeletal muscle[J].PeerJ, 2017, 5:e4014.
[33]	POTTHOFF M J, OLSON E N.MEF2:a central regulator of diverse developmental programs[J].Development, 2007, 134(23):4131-4140.
[34]	ZHAO X, LI X T, SHI X Y, et al.Diet-MEF2 interactions shape lipid droplet diversification in muscle to influence Drosophila lifespan[J].Aging Cell, 2020, 19(7):e13172.
[35]	WANG Y N, MEI C G, SU X T, et al.MEF2A regulates the MEG3-DIO3 miRNA mega cluster-targeted PP2A signaling in bovine skeletal myoblast differentiation[J].Int J Mol Sci, 2019, 20(11):2748.
[36]	HENNEBRY A, BERRY C, SIRIETT V, et al.Myostatinregulates fiber-type composition of skeletal muscle by regulating MEF2 and MyoD gene expression[J].Am J Physiol Cell Physiol, 2009, 296(3):C525-C534.
[37]	POTTHOFF M J, WU H, ARNOLD M A, et al.Histone deacetylase degradation and MEF2 activation promote the formation of slow-twitch myofibers[J].J Clin Invest, 2007, 117(9):2459-2467.
[38]	SCHIAFFINO S, SERRANO A L.Calcineurin signaling and neural control of skeletal musclefiber type and size[J]. Trends Pharmacol Sci, 2002, 23(12):569-575.
[39]	LI H, WANG F, GUO X Q, et al.Decreased MEF2A expression regulated by its enhancer methylation inhibits autophagy and may play an important role in the progression of Alzheimer's disease[J].Front Neurosci, 2021, 15:682247.
[40]	LU Y W, MARTINO N, GERLACH B D, et al.MEF2 (myocyte enhancer factor 2) is essential for endothelial homeostasis and the atheroprotective gene expression program[J].Arterioscl Thromb Vasc Biol, 2021, 41(3):1105-1123.
[41]	ZHANG M M, WEI Y L, LIU Y B, et al.Metastatic phosphatase PRL-3 induces ovarian cancer stem cell sub-population through phosphatase-independent deacetylation modulations[J].iScience, 2020, 23(1):100766.
[42]	HARASHIMA H, DISSMEYER N, SCHNITTGER A.Cell cycle control across the eukaryotic kingdom[J].Trends Cell Biol, 2013, 23(7):345-356.
[43]	WANG Y N, YANG W C, LI P W, et al.Myocyte enhancer factor 2A promotes proliferation and its inhibition attenuates myogenic differentiation via myozenin 2 in bovine skeletal muscle myoblast[J].PLoS One, 2018, 13(4):e0196255.