大鼠运动性肌损伤特征microRNA表达及其膜损伤调控靶点的预测
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摘要
本文旨在筛选大鼠运动性肌损伤(exercise-induced muscle damage,EIMD)的特征microRNA(miRNA),并预测分析特征miRNA调控膜损伤的靶点。将24只健康雄性Sprague-Dawley(SD)大鼠平均分为安静对照(C)组、运动后24 h(E24)组、运动后48 h(E48)组,采取一次间歇性离心运动(-16°下坡跑台跑)复制EIMD模型,用伊文氏蓝荧光染色法鉴定EIMD特征,用基因芯片进行差异性miRNA筛选,用RT-q PCR验证芯片检测结果后获得EIMD差异性miRNA表达谱,RT-qPCR检测EIMD膜损伤相关特征蛋白和相关通路核心分子的m RNA表达水平并预测特征miRNA的靶基因。本研究筛选出并验证了2条EIMD特征miRNA:miR-206-3p和miR-139-3p。骨架蛋白dystrophin(r=-0.68)、utrophin(r=-0.64)和MAPK信号通路核心分子JNK(r=-0.62)、ERK1(r=-0.68)均与miR206-3p呈中度负相关(P<0.001),而均与miR-139-3p无显著相关。以上研究结果提示,EIMD可诱导成年大鼠腓肠肌miRNA表达谱明显改变,miR-206-3p和miR-139-3p是EIMD特征miRNA,miR-206-3p可能通过调控骨架蛋白dystrophin、utrophin和MAPK信号通路分子JNK、ERK1调控膜损伤。
In the present study, we were to screen the specific microRNA(miRNA) of exercise-induced muscle damage(EIMD) and assess the EIMD-specific miRNAs-regulated target of sarcolemmal damage in rats. Twenty-four male Sprague-Dawley(SD) rats were randomly divided into 3 groups, which included sedentary(C), 24 h post-exercise(E24) and 48 h post-exercise(E48) groups. Rat EIMD model was established by an acute eccentric exercise, i.e., a downhill running treatment at-16o gradient. EIMD characteristics were verified by Evans blue dye staining, differentially expressed miRNAs were detected by microarray assay, EIMD-specific miRNAs expressions were further validated by real-time quantitative RT-PCR(RT-qPCR), and targets of the miRNAs were predicted based on m RNA expressions of associated proteins and related pathway core molecules of sarcolemmal damage. Two EIMD-specific expressed miRNAs, including miR-206-3p and miR-139-3p, were found in the study. There was a significantly negative correlation(P< 0.05) between miR-206-3p expression and dystrophin(r=-0.68), utrophin(r=-0.64), JNK(r=-0.62) or ERK1(r=-0.68) respectively, but no correlation was found between miR-139-3p and these biomolecules. The results suggest that: i) the expression profile of miRNAs in rat is significantly affected by EIMD, ii) miR-206-3p and miR-139-3p are the EIMD-specific miRNAs, and iii)miR-206-3p may control sarcolemmal damage by regulating dystrophin, utrophin, JNK and ERK1.
In the present study, we were to screen the specific microRNA(miRNA) of exercise-induced muscle damage(EIMD) and assess the EIMD-specific miRNAs-regulated target of sarcolemmal damage in rats. Twenty-four male Sprague-Dawley(SD) rats were randomly divided into 3 groups, which included sedentary(C), 24 h post-exercise(E24) and 48 h post-exercise(E48) groups. Rat EIMD model was established by an acute eccentric exercise, i.e., a downhill running treatment at-16o gradient. EIMD characteristics were verified by Evans blue dye staining, differentially expressed miRNAs were detected by microarray assay, EIMD-specific miRNAs expressions were further validated by real-time quantitative RT-PCR(RT-qPCR), and targets of the miRNAs were predicted based on m RNA expressions of associated proteins and related pathway core molecules of sarcolemmal damage. Two EIMD-specific expressed miRNAs, including miR-206-3p and miR-139-3p, were found in the study. There was a significantly negative correlation(P< 0.05) between miR-206-3p expression and dystrophin(r=-0.68), utrophin(r=-0.64), JNK(r=-0.62) or ERK1(r=-0.68) respectively, but no correlation was found between miR-139-3p and these biomolecules. The results suggest that: i) the expression profile of miRNAs in rat is significantly affected by EIMD, ii) miR-206-3p and miR-139-3p are the EIMD-specific miRNAs, and iii)miR-206-3p may control sarcolemmal damage by regulating dystrophin, utrophin, JNK and ERK1.
引文
1 Brooks SV,Zerba E,Faulkner JA.Injury to muscle fibres after single stretches of passive and maximally stimulated muscles in mice.J Physiol 1995;488:459-469.
2 Lieber RL,Thornell LE,Fridén J.Muscle cytoskeletal disruption occurs within the first 15 min of cyclic eccentric contractions.J Appl Physiol 1996;80(1):278-284.
3 Nosaka K,Clarkson PM.Muscle damage following repeated bouts of high force eccentric exercise.Med Sci Sports Exerc1995;27:1263-1269.
4 Stauber WT,Knack KK,Miller GR,Grimmett JG.Fibrosis and intercellular collagen connections from four weeks of muscle strains.Muscle Nerve 1996;19:423-430.
5 Belcastro AN,Shewchuk LD,Raj DA.Exercise-induced muscle injury:a calpain hypothesis.Mol Cell Biochem1998;179(1):135-145.
6 Lovering RM,De Deyne PG.Contractile function,sarcolemma integrity,and the loss of dystrophin after skeletal muscle eccentric contraction-induced injury.Am J Physiol Cell Physiol 2004;286(2):C230-C238.
7 Xu YM,Lee JP,Fang WB,Wang RY.The effects of hypoxic uphill exercise on the changes of sarcolemma integrity and dystrophin content.J Exerc Sci Fit 2008;6(2):97-105.
8 Bartel DP.Micro RNAs:genomics,biogenesis,mechanism,and function.Cell 2004;116(2):281-297.
9 Griffiths-Jones S,Grocock RJ,van Dongen S,Bateman A,Enright AJ.mi RBase:micro RNA sequences,targets and gene nomenclature.Nucleic Acids Res 2006;34(1):D140-D144.
10 Naguibneva I,Ameyar-Zazoua M,Polesskaya A,Ait-Si-Ali S,Groisman R,Souidi M,Cuvellier S,Harel-Bellan A.The micro RNA mi R-181 targets the homeobox protein Hox-A1during mammalian myoblast differentiation.Nat Cell Biol2006;8(3):278-284.
11 Chen Y,Melton DW,Gelfond JA,Mc Manus LM,Shireman PK.Mi R-351 transiently increases during muscle regeneration and promotes progenitor cell proliferation and survival upon differentiation.Physiol Genomics 2012;44(21):1042-1051.
12 Liu N,Williams AH,Maxeiner JM,Bezprozvannaya S,Shelton JM,Richardson JA,Bassel-Duby R,Olson EN.micro RNA-206 promotes skeletal muscle regeneration and delays progression of Duchenne muscular dystrophy in mice.J Clin Invest 2012;122(6):2054-2065.
13 Dey BK,Gagan J,Yan Z,Dutta A.mi R-26a is required for skeletal muscle differentiation and regeneration in mice.Genes Dev 2012;26(19):2180-2191.
14 Yin H,Pasut A,Soleimani VD,Bentzinger CF,Antoun G,Thorn S,Seale P,Fernando P,van Ijcken W,Grosveld F,Dekemp RA,Boushel R,Harper ME,Rudnicki MA.Micro RNA-133 controls brown adipose determination in skeletal muscle satellite cells by targeting Prdm16.Cell Metab 2013;17(2):210-224.
15 Armstrong RB,Ogilvie RW,Schwane JA.Eccentric exercise-induced injury to rat skeletal muscle.J Appl Physiol1983;54:80-93.
16 Pica AJ,Brooks GA.Effects of training and age on VO2max in laboratory rats.Med Sci Sports Exerc 1982;14(3):249-252.
17 Bedford TG,Tipton CM,Wilson NC,Oppliger RA,Gisolfi CV.Maximum oxygen consumption of rats and its changes with various experimental procedures in identically trained young and old rats.J Appl Physiol Respir Environ Exerc Physiol 1979;17:1278-1283.
18 Xu YM(徐玉明),Li JP,Wang RY.Changes of dystrophin and desmin in rat gastrocnemius under micro-damage induced by hypoxia.Acta Physiol Sin(生理学报)2010;62(4):339-348(in Chinese with English abstract).
19 Xu YM(徐玉明),Cao JM,Chen YP,Wang P,Han TT,Huang QT.The specific micro RNA expression influenced by hypocia of exercise-induced muscle damage rats.Chin JSports Med(中国运动医学杂志)2016;35(10):914-921(in Chinese with English abstract).
20 Black MA,Doerge RW.Calculation of the minimum number of replicate spots required for detection of significant gene expression fold change in microarray experiments.Bioinformatics 2002;18(12):1609-1616.
21 Baldi P,Long AD.A Bayesian framework for the analysis of microarray expression data:regularized t-test and statistical inferences of gene changes.Bioinformatics 2001;17(6):509-519.
22 Heidi JP,Gary JL.Normalization of micro RNA expression levels in quantitative RT-PCR assays:Identification of suitable reference RNA targets in normal and cancerous human solid tissues.RNA 2008;14(5):844-852.
23 Zhou Y(周越),Li Y,Wang R,Li S,Long G,Yang Z.Comparation of evaluating indicators for exercise-induced muscle damage.Chin J Sports Med(中国运动医学杂志)2008;27(2):206-208(in Chinese with English abstract).
24 Guo J,Miao Y,Xiao B,Huan R,Jiang Z,Meng D,Wang Y.Differential expression of micro RNA species in human gastric cancer versus nontumorous tissues.J Gastroenterol Hepatol 2009;24(4):652-657.
25 Chavali S,Bruhn S,S?trom P,Barren?s F,Saito T,Kanduri K,Wang H,Benson M.Micro RNAs act complementarily to regulate disease-related m RNA modules in human diseases.RNA 2013;19(11):1552-1562.
26 Schmitz KJ,Helwig J,Bertram S,Sheu SY,Suttorp AC,Seggewiss J,Willscher E,Walz MK,Worm K,Schmid KW.Differential expression of micro RNA-675,micro RNA-139-3p and micro RNA-335 in benign and malignant adrenocortical tumours.J Clin Pathol 2011;64(6):529-535.
27 Rao PK,Kumar RM,Farkhondeh M,Baskerville S,Lodish HF.Myogenic factors that regulate expression of musclespecific micro RNAs.Proc Natl Acad Sci U S A 2006;103(23):8721-8726.
28 Mc Carthy JJ,Esser KA,Andrade FH.Micro RNA-206 is overexpressed in the diaphragm but not the hindlimb muscle of mdx mouse.Am J Physiol Cell Physiol 2007;293(1):C451-C457.
29 Greco S,Simone MD,Colussi C,Zaccagnini G,Fasanaro P,Pescatori M,Cardani R,Perbellini R,Isaia E,Sale P,Meola G,Capogrossi MC,Gaetano C,Martelli F.Common micro-RNA signature in skeletal muscle damage and regeneration induced by Duchenne muscular dystrophy and acute ischemia.FASEB J 2009;23(10):3335-3346.
30 Yuasa K,Hagiwara Y,Ando M,Nakamura A,Takeda S,Hijikata T.Micro RNA-206 is highly expressed in newly formed muscle fibers:implications regarding potential for muscle regeneration and maturation in muscular dystrophy.Cell Struct Funct 2008;33(2):163-169.
31 Rybakova IN,Humston JL,Sonnemann KJ,Ervasti JM.Dystrophin and utrophin bind actin through distinct modes of contact.J Biol Chem 2006;281(15):9996-10001.
32 Rosenberg MI,Georges SA,Asawachaicharn A,Analau E,Tapscott SJ.Myo D inhibits Fstl1 and Utrn expression by inducing transcription of mi R-206.J Cell Biol 2006;175(1):77-85.
33 Williamson DL,Kubica N,Kimball SR,Jefferson LS.Exercise-induced alterations in extracellular signal-regulated kinase 1/2 and mammalian target of rapamycin(m TOR)signalling to regulatory mechanisms of m RNA translation in mouse muscle.J Physiol 2006;573(2):497-510.
34 Spence HJ,Dhillon AS,James M,Winder SJ.Dystroglycan,a scaffold for the ERK-MAP kinase cascade.EMBO Rep2004;5(5):484-489.
35 Campanelli JT,Roberds SL,Campbell KP,Scheller RH.Arole for dystrophin-associated glycoproteins and utrophin in agrin-induced ACh R clustering.Cell 1994;77(5):663-674.
2 Lieber RL,Thornell LE,Fridén J.Muscle cytoskeletal disruption occurs within the first 15 min of cyclic eccentric contractions.J Appl Physiol 1996;80(1):278-284.
3 Nosaka K,Clarkson PM.Muscle damage following repeated bouts of high force eccentric exercise.Med Sci Sports Exerc1995;27:1263-1269.
4 Stauber WT,Knack KK,Miller GR,Grimmett JG.Fibrosis and intercellular collagen connections from four weeks of muscle strains.Muscle Nerve 1996;19:423-430.
5 Belcastro AN,Shewchuk LD,Raj DA.Exercise-induced muscle injury:a calpain hypothesis.Mol Cell Biochem1998;179(1):135-145.
6 Lovering RM,De Deyne PG.Contractile function,sarcolemma integrity,and the loss of dystrophin after skeletal muscle eccentric contraction-induced injury.Am J Physiol Cell Physiol 2004;286(2):C230-C238.
7 Xu YM,Lee JP,Fang WB,Wang RY.The effects of hypoxic uphill exercise on the changes of sarcolemma integrity and dystrophin content.J Exerc Sci Fit 2008;6(2):97-105.
8 Bartel DP.Micro RNAs:genomics,biogenesis,mechanism,and function.Cell 2004;116(2):281-297.
9 Griffiths-Jones S,Grocock RJ,van Dongen S,Bateman A,Enright AJ.mi RBase:micro RNA sequences,targets and gene nomenclature.Nucleic Acids Res 2006;34(1):D140-D144.
10 Naguibneva I,Ameyar-Zazoua M,Polesskaya A,Ait-Si-Ali S,Groisman R,Souidi M,Cuvellier S,Harel-Bellan A.The micro RNA mi R-181 targets the homeobox protein Hox-A1during mammalian myoblast differentiation.Nat Cell Biol2006;8(3):278-284.
11 Chen Y,Melton DW,Gelfond JA,Mc Manus LM,Shireman PK.Mi R-351 transiently increases during muscle regeneration and promotes progenitor cell proliferation and survival upon differentiation.Physiol Genomics 2012;44(21):1042-1051.
12 Liu N,Williams AH,Maxeiner JM,Bezprozvannaya S,Shelton JM,Richardson JA,Bassel-Duby R,Olson EN.micro RNA-206 promotes skeletal muscle regeneration and delays progression of Duchenne muscular dystrophy in mice.J Clin Invest 2012;122(6):2054-2065.
13 Dey BK,Gagan J,Yan Z,Dutta A.mi R-26a is required for skeletal muscle differentiation and regeneration in mice.Genes Dev 2012;26(19):2180-2191.
14 Yin H,Pasut A,Soleimani VD,Bentzinger CF,Antoun G,Thorn S,Seale P,Fernando P,van Ijcken W,Grosveld F,Dekemp RA,Boushel R,Harper ME,Rudnicki MA.Micro RNA-133 controls brown adipose determination in skeletal muscle satellite cells by targeting Prdm16.Cell Metab 2013;17(2):210-224.
15 Armstrong RB,Ogilvie RW,Schwane JA.Eccentric exercise-induced injury to rat skeletal muscle.J Appl Physiol1983;54:80-93.
16 Pica AJ,Brooks GA.Effects of training and age on VO2max in laboratory rats.Med Sci Sports Exerc 1982;14(3):249-252.
17 Bedford TG,Tipton CM,Wilson NC,Oppliger RA,Gisolfi CV.Maximum oxygen consumption of rats and its changes with various experimental procedures in identically trained young and old rats.J Appl Physiol Respir Environ Exerc Physiol 1979;17:1278-1283.
18 Xu YM(徐玉明),Li JP,Wang RY.Changes of dystrophin and desmin in rat gastrocnemius under micro-damage induced by hypoxia.Acta Physiol Sin(生理学报)2010;62(4):339-348(in Chinese with English abstract).
19 Xu YM(徐玉明),Cao JM,Chen YP,Wang P,Han TT,Huang QT.The specific micro RNA expression influenced by hypocia of exercise-induced muscle damage rats.Chin JSports Med(中国运动医学杂志)2016;35(10):914-921(in Chinese with English abstract).
20 Black MA,Doerge RW.Calculation of the minimum number of replicate spots required for detection of significant gene expression fold change in microarray experiments.Bioinformatics 2002;18(12):1609-1616.
21 Baldi P,Long AD.A Bayesian framework for the analysis of microarray expression data:regularized t-test and statistical inferences of gene changes.Bioinformatics 2001;17(6):509-519.
22 Heidi JP,Gary JL.Normalization of micro RNA expression levels in quantitative RT-PCR assays:Identification of suitable reference RNA targets in normal and cancerous human solid tissues.RNA 2008;14(5):844-852.
23 Zhou Y(周越),Li Y,Wang R,Li S,Long G,Yang Z.Comparation of evaluating indicators for exercise-induced muscle damage.Chin J Sports Med(中国运动医学杂志)2008;27(2):206-208(in Chinese with English abstract).
24 Guo J,Miao Y,Xiao B,Huan R,Jiang Z,Meng D,Wang Y.Differential expression of micro RNA species in human gastric cancer versus nontumorous tissues.J Gastroenterol Hepatol 2009;24(4):652-657.
25 Chavali S,Bruhn S,S?trom P,Barren?s F,Saito T,Kanduri K,Wang H,Benson M.Micro RNAs act complementarily to regulate disease-related m RNA modules in human diseases.RNA 2013;19(11):1552-1562.
26 Schmitz KJ,Helwig J,Bertram S,Sheu SY,Suttorp AC,Seggewiss J,Willscher E,Walz MK,Worm K,Schmid KW.Differential expression of micro RNA-675,micro RNA-139-3p and micro RNA-335 in benign and malignant adrenocortical tumours.J Clin Pathol 2011;64(6):529-535.
27 Rao PK,Kumar RM,Farkhondeh M,Baskerville S,Lodish HF.Myogenic factors that regulate expression of musclespecific micro RNAs.Proc Natl Acad Sci U S A 2006;103(23):8721-8726.
28 Mc Carthy JJ,Esser KA,Andrade FH.Micro RNA-206 is overexpressed in the diaphragm but not the hindlimb muscle of mdx mouse.Am J Physiol Cell Physiol 2007;293(1):C451-C457.
29 Greco S,Simone MD,Colussi C,Zaccagnini G,Fasanaro P,Pescatori M,Cardani R,Perbellini R,Isaia E,Sale P,Meola G,Capogrossi MC,Gaetano C,Martelli F.Common micro-RNA signature in skeletal muscle damage and regeneration induced by Duchenne muscular dystrophy and acute ischemia.FASEB J 2009;23(10):3335-3346.
30 Yuasa K,Hagiwara Y,Ando M,Nakamura A,Takeda S,Hijikata T.Micro RNA-206 is highly expressed in newly formed muscle fibers:implications regarding potential for muscle regeneration and maturation in muscular dystrophy.Cell Struct Funct 2008;33(2):163-169.
31 Rybakova IN,Humston JL,Sonnemann KJ,Ervasti JM.Dystrophin and utrophin bind actin through distinct modes of contact.J Biol Chem 2006;281(15):9996-10001.
32 Rosenberg MI,Georges SA,Asawachaicharn A,Analau E,Tapscott SJ.Myo D inhibits Fstl1 and Utrn expression by inducing transcription of mi R-206.J Cell Biol 2006;175(1):77-85.
33 Williamson DL,Kubica N,Kimball SR,Jefferson LS.Exercise-induced alterations in extracellular signal-regulated kinase 1/2 and mammalian target of rapamycin(m TOR)signalling to regulatory mechanisms of m RNA translation in mouse muscle.J Physiol 2006;573(2):497-510.
34 Spence HJ,Dhillon AS,James M,Winder SJ.Dystroglycan,a scaffold for the ERK-MAP kinase cascade.EMBO Rep2004;5(5):484-489.
35 Campanelli JT,Roberds SL,Campbell KP,Scheller RH.Arole for dystrophin-associated glycoproteins and utrophin in agrin-induced ACh R clustering.Cell 1994;77(5):663-674.