m6A甲基化在鸡肌肉生长发育中的表达研究

doi:10.16431/j.cnki.1671-7236.2021.05.003

摘要/Abstract

摘要： 试验旨在研究RNA m6A修饰相关基因去甲基化酶Alk B同源蛋白5（Alk B homologue 5，ALKBH5）、去甲基化酶肥胖相关蛋白（fat mass and obesity-associated protein，FTO）、甲基转移酶样蛋白3（methyltransferase like 3，METTL3）、甲基转移酶样蛋白14（methyltransferase like 14，METTL14）和成肾细胞瘤1-结合蛋白（Wilms’tumor 1-associating protein，WTAP）在鸡骨骼肌发育过程中的表达，分析其与骨骼肌m6A甲基化水平的相关性。首先，利用实时荧光定量PCR技术检测m6A甲基化相关基因在金茅花鸡12（E12）、14（E14）、16（E16）、18（E18）胚龄和1日龄腿肌和胸肌组织中mRNA表达水平，以及其在鸡成肌细胞50%、100%增殖期和1、2、3、4、5 d分化期的mRNA表达水平；随后，利用m6A甲基化试剂盒检测金茅花鸡E12和1日龄腿肌和胸肌组织中m6A甲基化修饰水平，与m6A甲基化相关基因表达水平进行相关性分析。结果显示，m6A去甲基化基因ALKBH5和FTO mRNA表达水平在骨骼肌发育过程中显著上调（P<0.05），即在E12、E14低表达，E16、E18逐渐上调，1日龄达到最高。m6A甲基化写入基因METTL14、METTL3和WTAP mRNA表达水平在E12、E14、E16逐渐上升，E18下降，随后至1日龄表达量回升。在细胞增殖过程中，ALKBH5、FTO、METTL14、METTL3和WTAP基因表达均上调；在细胞分化过程中ALKBH5和FTO基因表达水平显著上调（P<0.05），在分化第5天达到最高。METTL14、METTL3和WTAP基因mRNA表达水平在细胞诱导分化的1、2、3、4 d表达量呈下降趋势，而在诱导分化的第5天有所回升。甲基化水平检测结果显示，腿肌和胸肌m6A甲基化水平变化趋势一致，均在胚胎发育过程中显著下降（P<0.05），至1日龄达到最低。相关性分析结果显示，鸡骨骼肌RNA m6A甲基化水平与m6A去甲基化修饰基因ALKBH5、FTO mRNA表达水平呈显著负相关（P<0.05）。综合以上试验结果，推测m6A甲基化修饰与鸡骨骼肌发育相关，而去甲基化基因ALKBH5、FTO可能通过调控RNA m6A甲基化水平，影响鸡骨骼肌发育。本研究结果为进一步研究m6A甲基化修饰调控鸡骨骼肌生长发育的功能和分子机制提供理论依据。

关键词: m6A甲基化; 鸡; 骨骼肌发育; 基因表达

Abstract: The purpose of this experiment was to study the expression of RNA m6A modification related genes ALK B homologue 5 (ALKBH5),fat mass and obesity associated protein (FTO),methyltransferase like 3 (METTLl3),methyltransferase like 14 (METTL14) and Wilms' tumor 1-associated protein (WTAP) in chicken skeletal muscle development,and analyzed its correlation with the level of skeletal muscle m6A methylation.Firstly,Real-time quantitative PCR was used to detect the mRNA expression levels of m6A methylation related genes in 12 (E12),14 (E14),16 (E16),18 (E18) embryonic and 1-day-old leg and chest muscles of Jinmao Hua chicken.And the mRNA expression levels in chicken myoblasts at 50%,100% proliferation and 1,2,3,4,5 d differentiation phases.Subsequently,the level of m6A methylation modification in the E12 and 1-day-old leg muscles and chest muscle tissues of Jinmao Hua chicken were detected by m6A methylation reagent test kit,and the correlation between the methylation level and the expression level of genes related to m6A methylation was analyzed.The results showed that the mRNA expression levels of m6A demethylation genes ALKBH5 and FTO were significantly up-regulated during skeletal muscle development (P<0.05),that wass,low expression at E12 and E14,and gradually up-regulated at E16 and E18,reaching the highest level at 1-day-old.The mRNA expression levels of the m6A methylation write genes METL14,METTL3 and WTAP gradually increased at E12,E14 and E16,and decreased at E18,and then the expression level rose back to 1-day-old.In the process of cell proliferation,the expression of ALKBH5, FTO,METL14,METL3 and WTAP genes were all up-regulated,during the process of cell differentiation,the expression levels of ALKBH5 and FTO genes were significantly up-regulated (P<0.05),reaching the highest on the fifth day of differentiation.The mRNA expression levels of METTL14,METTL3 and WTAP genes showed a downward trend on the 1st,2nd,3rd,and 4th day after cell differentiation,but increased on the 5th day after differentiation.The methylation level detection results showed that the change trend of the m6A methylation level of leg muscles and chest muscles was the same,and both decreased significantly during embryonic development (P<0.05),and reached the lowest at 1-day-old.The results of correlation analysis showed that the methylation level of chicken skeletal muscle RNA m6A was significantly negatively correlated with the mRNA expression levels of m6A demethylation modifier genes ALKBH5 and FTO (P<0.05).Based on the above experimental results,that m6A methylation modification was related to chicken skeletal muscle development,and the demethylation genes ALKBH5 and FTO might affect chicken skeletal muscle development by regulating the level of RNA m6A methylation.This study provided theoretical basis for further research on the function and molecular mechanism of m6A methylation in regulating chicken skeletal muscle growth and development.

Key words: m6A methylation; chicken; skeletal muscle development; gene expression

中图分类号:

S831.2

丁浩, 林月月, 张涛, 张闪闪, 吴玉麟, 段严军, 巩用双, 谢恺舟, 王金玉, 戴国俊, 张跟喜. m6A甲基化在鸡肌肉生长发育中的表达研究[J]. 中国畜牧兽医, 2021, 48(5): 1525-1534.

DING Hao, LIN Yueyue, ZHANG Tao, ZHANG Shanshan, WU Yulin, DUAN Yanjun, GONG Yongshuang, XIE Kaizhou, WANG Jinyu, DAI Guojun, ZHANG Genxi. Study on the Expression of m6A Methylation in Chicken Muscle Growth and Development[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(5): 1525-1534.

参考文献

[1] FORNTERA W R,OCHALA J.Skeletal muscle:A brief review of structure and function[J].Calcified Tissue International,2015,96(3):83-95.
[2] 丁丽艳,殷元虎,韩永胜,等.分子育种技术在肉牛育种中的应用研究进展[J].现代畜牧科技,2018,10:21-22. DING L Y,YIN Y H,HAN Y S,et al.Application research progress of molecular breeding technology in beef cattle breeding[J].Modern Animal Husbandry Science & Technology,2018,10:21-22.(in Chinese)
[3] TIZARD M L,JENKINS K A,COOPER C A,et al.Potential benefits of gene editing for the future of poultry farming[J].Transgenic Research,2019,28(S2):87-92.
[4] KIM G D,LEE J H,SONG S,et al.Generation of myostatin-knockout chickens mediated by D10A-Cas9 nickase[J].FASEB Journal,2020,34(4):5688-5696.
[5] WILLIAM A C,PAMELA F.C,JEF R,et al.The RNA modification database,RNAMDB:2011 update[J].Nucleic Acids Research,2011,39,195-201.
[6] MEYER K D,SALETORE Y,ZUMBO P,et al.Comprehensive analysis of mRNA methylation reveals enrichment in 3'UTRs and near stop codons[J].Cell,2012,149(7):1635-1646.
[7] LIU J,YUE Y,HAN D,et al.A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation[J].Nature Chemical Biology,2014,10(2):93-95.
[8] SORCI M,IANNIELLO Z,CRUCIANI S,et al.METTL3 regulates WTAP protein homeostasis[J].Cell Death Disease,2018,9(8):796.
[9] HAUSSMANN I U,BODI Z,SANCHEZ-MORAN E,et al.m⁶A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination[J].Nature,2016,540(7632):301-304.
[10] FUSTIN J M,DOI M,YAMAGUCHI Y,et al.RNA-methylation-dependent RNA processing controls the speed of the circadian clock[J].Cell,2013,155(4):793-806.
[11] GEULA S,MOSHITCH-MOSHKOVITZ S,DOMI-NISSINI D,et al.Stem cells.m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation[J].Science,2015,347(6225):1002-1006.
[12] COOTS R A,LIU X M,MAO Y,et al.m(6) A Facilitates eIF4F-Independent mRNA translation[J].Molecular Cell,2017,68(3):504-514.
[13] HAN B,CHU C,SU X,et al.N⁶-methyladenosine-dependent primary microRNA-126 processing activated PI3K-AKT-mTOR pathway drove the development of pulmonary fibrosis induced by nanoscale carbon black particles in rats[J].Nanotoxicology,2020,14(1):1-20.
[14] WANG Q,CHEN C,DING Q,et al.METTL3-mediated m⁶A modification of HDGF mRNA promotes gastric cancer progression and has prognostic significance[J].Gut,2020,69(7):1193-1205.
[15] HAN J,WANG J Z,YANG X,et al.METTL3 promote tumor proliferation of bladder cancer by accelerating pri-miR221/222 maturation in m6A-depen-dent manner[J].Molecular Cancer,2019,18(1):110.
[16] MENDEL M,CHEN K M,HOMOLKA D,et al.Methylation of structured RNA by the m⁶A Writer METTL16 is essential for mouse embryonic develop-ment[J].Molecular Cell,2018,71(6):986-1000.
[17] ZHANG S,ZHAO B S,ZHOU A,LIN K,et al.m(6) A demethylase ALKBH5 maintains tumorigenicity of glioblastoma stem-like cells by sustaining FOXM1 expression and cell proliferation program[J].Cancer Cell,2017,31(4):591-606.
[18] LEE H,BAO S,QIAN Y,et al.Stage-specific requirement for Mettl3-dependent m⁶A mRNA methylation during haematopoietic stem cell differentiation[J].Nature Cell Biology,2019,21(6):700-709.
[19] CHEN J N,CHEN Y,WEI Y Y,et al.Regulation of m^(6) A RNA methylation and its effect on myogenic differentiation in murine myoblasts[J].Molekuliarraia Biolgogiia,2019,53(3):436-445.
[20] KUDOU K,KOMATSU T,NOGAMI J,et al.The requirement of Mettl3-promoted MyoD mRNA maintenance in proliferative myoblasts for skeletal muscle differentiation[J].Open Biology,2017,7(9):170119.
[21] WANG X,HUANG N,YANG M,et al.FTO is required for myogenesis by positively regulating mTOR-PGC-1alpha pathway-mediated mitochondria biogenesis[J].Cell Death Disease,2017,8(3):e2702.
[22] LIU J,JIA G.Methylation modifications in eukaryotic messenger RNA[J].Journal of Genetics and Genomics,2014,41(1):21-33.
[23] ALDERMAN M H 3RD,XIAO A Z.N(6)-Methyla-denine in eukaryotes[J].Cellular and Molecular Life Sciences,2019,76(15):2957-2966.
[24] 黄芳芳,周诺.METTL3在RNA m⁶A甲基化修饰的研究进展[J].生理科学进展,2019,50(6):458-463. HUANG F F,ZHOU N.Advances in METTL3-mediated m⁶A RNA methylation[J].Progress in Physiological Sciences,2019,50(6):458-463.(in Chinese)
[25] JIAN D D,WANG Y,JIAN L G,et al.METTL14 aggravates endothelial inflammation and atheroscle-rosis by increasing FOXO1 N6-methyladeosine modi-fications[J].Theranostics,2020,10(20):8939-8956.
[26] 汪毅,张杰,皮静楠,等.WTAP基因在胃癌组织中低表达[J].基础医学与临床,2016,36(11):1548-1553. WANG Y,ZHANG J,PI J N,et al.Low expression of WTAP gene in gastric cancer[J].Basic & Clinical Medicine,2016,36(11):1548-1553.(in Chinese)
[27] 冯冲.RNA m⁶A去甲基化酶Alkbh5的结构与功能研究[D].北京:中国农业大学,2014. FENG C.structure and function of RNA m⁶A demethylase Alkbh5[D].Beijing:China Agricultural University,2014.(in Chinese)
[28] FU Y,JIA G,PANG X,et al.FTO mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA[J].Nature Communications, 2013,4:1798.
[29] 梁忠,黄华云,宋迟,等.FTO基因在不同品种鸡胚胎与生长期骨骼肌中的表达[J].安徽农业大学学报,2016,43(5):694-697. LIANG Z,HUANG H Y,SONG C,et al.Expression of FTO in skeletal muscles of different breeds between embryonic period and growth period[J].Journal of Anhui Agricultural University,2016,43(5):694-697.(in Chinese)
[30] 林惠然,贾新正,聂庆华. FTO基因在粤黄鸡胚胎期肌肉和肝脏组织中的表达规律[A].广东省遗传学会第九届代表大会暨学术研讨会论文及摘要汇编[C].2014. LIN H R,JIA X Z,NIE Q H.Expression of FTO gene in embryonic muscle and liver tissues of Yuehuang chicken[A].The 9th Congress of Guangdong Genetics Society and its abstracts Compilation[C].2014.(in Chinese)
[31] ZHAO X,YANG Y,SUN B F,et al.FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis[J].Cell Research,2014,24(12):1403-1419.
[32] 黄海涛,李灵,陈善,等.去甲基化酶ALKBH5在肺腺癌组织中的表达及其与细胞增殖的关系[J].肿瘤,2018,38(6):572-580. HUANG H T,LI L,CHEN S,et al.Expression of demethylase ALKBH5 in lung adenocarcinoma and its relationship with cell proliferation[J].Tumor,2018,38(6):572-580.(in Chinese)
[33] ZHENG G,DAHL J A,NIU Y,et al.ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility[J].Molecular Cell,2013,49(1):18-29.