甲基转移酶METTL21C影响鸡骨骼肌细胞成肌的发育
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摘要
本研究旨在探究甲基转移酶METTL21C在家禽骨骼肌发育分化过程中的作用。采用实时荧光定量PCR (quantitative Real-time PCR, qPCR)检测METTL21C基因在鸡不同组织中的表达情况,绘制其组织表达谱;选取3个时间点,检测其在骨骼肌组织中的表达情况。通过酶消化法从鸡骨骼肌组织中分离得到原代细胞;将METTL21C超表达载体转染至原代鸡骨骼肌细胞,通过qPCR和Western blotting检测Pax7、MyoD、Myf5、MyoG等基因的表达水平。结果显示,METTL21C在心肌和骨骼肌组织中的表达量显著高于其他组织,在胚胎期和幼龄期骨骼肌中的表达呈上升趋势;超表达METTL21C后,成肌相关基因Pax7、MyoD、Myf5、MyoG的表达量显著升高。本研究初步发现甲基转移酶METTL21C具有促进家禽骨骼肌发育分化的作用,为骨骼肌发育的分子机理的研究及相关医学研究提供数据支持。
The aim of this study was to inves tigate the role of METTL21 C gene in the development and differentiation of skeletal muscle in poultry. The relative mRNA expression of METTL21 C in different tissues of chicken was detected by real-time fluorescence quantitative PCR to generate the expression profile. The expression level of METTL21 C gene was tested in skeletal muscle tissue at three timing. Primary cells were isolated from chicken skeletal muscle tissue by enzyme digestion. Overexpression vector of METTL21 C gene was transfected into primary skeletal muscle cells. The expression of Pax7, MyoD, Myf5 and MyoG genes were detected by q PCR and Western blotting. Expression levels of METTL21 C gene in cardiac muscle and skeletal muscle were much higher than that of other tissues, and presented an upward trend in skeletal muscle embryonic stage and juvenile stage. The expression of muscle-related genes Pax7, MyoD, Myf5 and MyoG was increased significantly with overexpression of METTL21 C. This study found that METTL21 C can promote the development and differentiation of skeletal muscle in poultry, which could provide data support for the molecular mechanism of skeletal muscle development and corresponding medical research.
The aim of this study was to inves tigate the role of METTL21 C gene in the development and differentiation of skeletal muscle in poultry. The relative mRNA expression of METTL21 C in different tissues of chicken was detected by real-time fluorescence quantitative PCR to generate the expression profile. The expression level of METTL21 C gene was tested in skeletal muscle tissue at three timing. Primary cells were isolated from chicken skeletal muscle tissue by enzyme digestion. Overexpression vector of METTL21 C gene was transfected into primary skeletal muscle cells. The expression of Pax7, MyoD, Myf5 and MyoG genes were detected by q PCR and Western blotting. Expression levels of METTL21 C gene in cardiac muscle and skeletal muscle were much higher than that of other tissues, and presented an upward trend in skeletal muscle embryonic stage and juvenile stage. The expression of muscle-related genes Pax7, MyoD, Myf5 and MyoG was increased significantly with overexpression of METTL21 C. This study found that METTL21 C can promote the development and differentiation of skeletal muscle in poultry, which could provide data support for the molecular mechanism of skeletal muscle development and corresponding medical research.
引文
Choi J.,Jang H.,Kim H.,Lee J.H.,Kim S.T.,Cho E.J.,and Youn H.D.,2014,Modulation of lysine methylation in myocyte enhancer factor 2 during skeletal muscle cell differentiation,Nucleic Acids Res.,42(1):224-234
Cloutier P.,Lavallee-Adam M.,Faubert D.,Blanchette M.,and Coulombe B.,2013,A newly uncovered group of distantly related lysine methyltransferases preferentially interact with molecular chaperones to regulate their activity,PLoS Genet,9(1):e1003210
Dey B.K.,Gagan J.,and Dutta A.,2011,mi R-206 and-486 induce myoblast differentiation by downregulating Pax7,Mol.Cell Biol.,31(1):203-214
Feng Y.Q.,Li Z.,Chu W.L.,Yu Y.H.,Ma L.,and Chai J.K.,2016,Study on culture,identification and differentiation of primary rat skeletal muscle satellite cells,Zhonghua Yi Xue Za Zhi,96(12):971-974
Gao L.,Gu D.H.,Xu Z.Q.,Ge C.R.,Jia J.J.,Rong H.,and Li Q.H.,2015,Research progress on the effect of the myogenic regulatory factors(MRFs)gene family on meat quality,Heilongjiang Xumu Shouyi(Heilongjiang Animal Science and Veterinary Medicine),9(5):65-67(高玲,谷大海,徐志强,葛长荣,贾俊静,荣华,李琦华,2015,MRFs基因家族对肉品质影响的研究进展,黑龙江畜牧兽医,9(5):65-67)
Huang J.,Hsu Y.H.,Mo C.,Abreu E.,Kiel D.P.,Bonewald L.F.,Brotto M.,and Karasik D.,2014,METTL21C is a potential pleiotropic gene for osteoporosis and sarcopenia acting through the modulation of the NF-κB signaling pathway,J.Bone Miner.Res.,29(7):1531-1540
He Y.L.,Wu Y.H.,Quan F.S.,Liu Q.,and Zhang Y.,2012,Isolation and transfection of skeletal muscle satellite cells from Qinchuan fetal bovine,Xibei Nonglin Keji Daxue Xuebao(Zirankexue Ban)(Journal of Northwest A&F University(Natural Science Edition)),40(6):1-6(何玉龙,吴月红,权富生,刘琴,张涌,2012,秦川牛胎儿骨骼肌卫星细胞的分离培养,西北农林科技大学学报(自然科学版),40(6):1-6)
Kernstock S.,Davydova E.,Jakobsson M.,Moen A.,Pettersen S.,Maelandsmo G.M.,Egge-Jacobsen W.,and Falnes P.O.,2012,Lysine methylatio n of VCP by a member of a novel human protein methyltransferase family,Nat.Commun.,3(3):1038
Ling B.M.,Bharathy N.,Chung T.K.,Kok W.K.,Li S.,Tan Y.H.,Rao V.K.,Gopinadhan S.,Sartorelli V.,Walsh M.J.,and Taneja R.,2012,Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation,Proc.Natl.Acad.Sci.USA,109(3):841-846
Ren T.,Guo Y.Y.,Zhang J.,Zhao L.H.,and Jin Y.,2014,Effect of myogenic regulatory factors(MRFs)to meat quality,Shipin Gongye Keji(Science and Technology of Food Industry),35(16):363-366(任霆,郭月英,张静,赵丽华,靳烨,2014,MRFs基因家族对肉品质的影响,食品工业科技,35(16):363-366)
Wang Y.F.T.,Ren T.,Zhu S.H.,Liang S.R.,He K.,and Zhao A.Y.,2017,Expression patterns of MRFs,Pax3,and Pax7 during skeletal muscle development in Zhedong white geese,Zhejiang Nonglin Daxue Xuebao(Journal of Zhejiang A&F University),34(6):1065-1070(王吟方亭,任婷,朱松会,梁世日,何珂,赵阿勇,2017,浙东白鹅骨骼肌发育相关因子MRFs,Pax3和Pax7基因的表达规律,浙江农林大学学报,34(6):1065-1070)
Yang M.J.,and Chen H.,2010,Research progress of MRFs gene family in bovine,Zhongguo Niuye Kexue(China Cattle Science),36(6):51-55(杨明娟,陈宏,2010,牛MRFs基因家族研究进展,中国牛业科学,36(6):51-55)
Zhong X.,Wang Q.Q.,Li J.W.,Zhang Y.M.,An X.R.,and Hou J.,2017,Ten-eleven translocation-2(Tet2)is involved in myogenic differentiation of skeletal myoblast cells in vitro,Sci.Rep.,7:43539
Zhu C.H.,Tao Z.Y.,Ji G.G.,Zhu W.Q.,Song W.T.,Song W.T.,Song C.,and Li H.F.,2014,Development of skeletal muscle and expression of myogenic regulatory factors during embryonic stage in Jinding ducks(Anas platyrhynchos domestica),Zhongguo Jiaqin(China Poultry),36(20):6-11(朱春红,陶志云,姬改革,朱文奇,宋卫涛,宋迟,李慧芳,2014,金定鸭胚胎期骨骼肌发育模式及MRFs家族基因表达变化研究,中国家禽,36(20):6-11)
Cloutier P.,Lavallee-Adam M.,Faubert D.,Blanchette M.,and Coulombe B.,2013,A newly uncovered group of distantly related lysine methyltransferases preferentially interact with molecular chaperones to regulate their activity,PLoS Genet,9(1):e1003210
Dey B.K.,Gagan J.,and Dutta A.,2011,mi R-206 and-486 induce myoblast differentiation by downregulating Pax7,Mol.Cell Biol.,31(1):203-214
Feng Y.Q.,Li Z.,Chu W.L.,Yu Y.H.,Ma L.,and Chai J.K.,2016,Study on culture,identification and differentiation of primary rat skeletal muscle satellite cells,Zhonghua Yi Xue Za Zhi,96(12):971-974
Gao L.,Gu D.H.,Xu Z.Q.,Ge C.R.,Jia J.J.,Rong H.,and Li Q.H.,2015,Research progress on the effect of the myogenic regulatory factors(MRFs)gene family on meat quality,Heilongjiang Xumu Shouyi(Heilongjiang Animal Science and Veterinary Medicine),9(5):65-67(高玲,谷大海,徐志强,葛长荣,贾俊静,荣华,李琦华,2015,MRFs基因家族对肉品质影响的研究进展,黑龙江畜牧兽医,9(5):65-67)
Huang J.,Hsu Y.H.,Mo C.,Abreu E.,Kiel D.P.,Bonewald L.F.,Brotto M.,and Karasik D.,2014,METTL21C is a potential pleiotropic gene for osteoporosis and sarcopenia acting through the modulation of the NF-κB signaling pathway,J.Bone Miner.Res.,29(7):1531-1540
He Y.L.,Wu Y.H.,Quan F.S.,Liu Q.,and Zhang Y.,2012,Isolation and transfection of skeletal muscle satellite cells from Qinchuan fetal bovine,Xibei Nonglin Keji Daxue Xuebao(Zirankexue Ban)(Journal of Northwest A&F University(Natural Science Edition)),40(6):1-6(何玉龙,吴月红,权富生,刘琴,张涌,2012,秦川牛胎儿骨骼肌卫星细胞的分离培养,西北农林科技大学学报(自然科学版),40(6):1-6)
Kernstock S.,Davydova E.,Jakobsson M.,Moen A.,Pettersen S.,Maelandsmo G.M.,Egge-Jacobsen W.,and Falnes P.O.,2012,Lysine methylatio n of VCP by a member of a novel human protein methyltransferase family,Nat.Commun.,3(3):1038
Ling B.M.,Bharathy N.,Chung T.K.,Kok W.K.,Li S.,Tan Y.H.,Rao V.K.,Gopinadhan S.,Sartorelli V.,Walsh M.J.,and Taneja R.,2012,Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation,Proc.Natl.Acad.Sci.USA,109(3):841-846
Ren T.,Guo Y.Y.,Zhang J.,Zhao L.H.,and Jin Y.,2014,Effect of myogenic regulatory factors(MRFs)to meat quality,Shipin Gongye Keji(Science and Technology of Food Industry),35(16):363-366(任霆,郭月英,张静,赵丽华,靳烨,2014,MRFs基因家族对肉品质的影响,食品工业科技,35(16):363-366)
Wang Y.F.T.,Ren T.,Zhu S.H.,Liang S.R.,He K.,and Zhao A.Y.,2017,Expression patterns of MRFs,Pax3,and Pax7 during skeletal muscle development in Zhedong white geese,Zhejiang Nonglin Daxue Xuebao(Journal of Zhejiang A&F University),34(6):1065-1070(王吟方亭,任婷,朱松会,梁世日,何珂,赵阿勇,2017,浙东白鹅骨骼肌发育相关因子MRFs,Pax3和Pax7基因的表达规律,浙江农林大学学报,34(6):1065-1070)
Yang M.J.,and Chen H.,2010,Research progress of MRFs gene family in bovine,Zhongguo Niuye Kexue(China Cattle Science),36(6):51-55(杨明娟,陈宏,2010,牛MRFs基因家族研究进展,中国牛业科学,36(6):51-55)
Zhong X.,Wang Q.Q.,Li J.W.,Zhang Y.M.,An X.R.,and Hou J.,2017,Ten-eleven translocation-2(Tet2)is involved in myogenic differentiation of skeletal myoblast cells in vitro,Sci.Rep.,7:43539
Zhu C.H.,Tao Z.Y.,Ji G.G.,Zhu W.Q.,Song W.T.,Song W.T.,Song C.,and Li H.F.,2014,Development of skeletal muscle and expression of myogenic regulatory factors during embryonic stage in Jinding ducks(Anas platyrhynchos domestica),Zhongguo Jiaqin(China Poultry),36(20):6-11(朱春红,陶志云,姬改革,朱文奇,宋卫涛,宋迟,李慧芳,2014,金定鸭胚胎期骨骼肌发育模式及MRFs家族基因表达变化研究,中国家禽,36(20):6-11)
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