MSTN基因对牛肌动蛋白细胞骨架调节通路的影响

doi:10.16431/j.cnki.1671-7236.2021.10.006

摘要/Abstract

摘要： 为探究肌肉生长抑制素（myostatin，MSTN）对牛骨骼肌生长发育的作用机制，本研究前期利用定量蛋白质组学与磷酸化蛋白质组学分析野生型蒙古牛（MG.WT）和MSTN^+/-蒙古牛（MG.MSTN^+/-）腿臀肌肌肉组织中蛋白质水平和磷酸化修饰水平的差异变化，使用已建立的牛骨骼肌卫星细胞体外诱导成肌分化模型，检测设计合成的MSTN siRNA （si-MSTN）干扰效果；采用实时荧光定量PCR和Western blotting方法检测转染si-MSTN的增殖期（GM）和分化第3天（DM3）牛骨骼肌卫星细胞中肌动蛋白细胞骨架调节通路相关基因的mRNA和蛋白水平的表达变化，研究敲低MSTN表达对肌动蛋白细胞骨架调节通路的影响。结果显示，在MSTN^+/-蒙古牛肌肉组织中共鉴定到16个肌动蛋白细胞骨架调节通路相关基因表达丰度上调；转染si-MSTN细胞中的MSTN表达水平极显著降低（P<0.01）；在转染si-MSTN的GM期牛骨骼肌卫星细胞中，肌动蛋白细胞骨架调节通路相关基因ENAH、ACTN4和Cdc42的mRNA水平均显著升高（P<0.05），PFN1、RhoA和ACTN4的蛋白水平均显著或极显著升高（P<0.05；P<0.01）；在转染si-MSTN的DM3牛骨骼肌卫星细胞中，ENAH、CFL1、SCIN和Cdc42基因mRNA水平均显著升高（P<0.05），RhoA基因mRNA水平极显著升高（P<0.01），PFN1和ACTN4的蛋白水平均显著升高（P<0.05）。结果表明，干扰MSTN可以促进肌动蛋白细胞骨架调节通路相关基因的表达，探明了MSTN可能通过介导肌动蛋白细胞骨架调节通路影响牛骨骼肌卫星细胞增殖和成肌分化的分子机制，为进一步研究MSTN对牛成肌分化的调控机制提供参考。

关键词: 牛; 肌肉生长抑制素; 骨骼肌卫星细胞; 肌动蛋白细胞骨架调控通路; 增殖; 成肌分化

Abstract: In order to explore the mechanism of myostatin (MSTN) on the growth and development of bovine skeletal muscle, quantitative proteomics and phosphorylated proteomics were used to analyze the differences of protein and phosphorylated modification levels in leg gluteal muscle of wild type Mongolian cattle (MG. WT) and MSTN^+/- Mongolian cattle (MG. MSTN^+/-). The established model of muscle differentiation of bovine skeletal muscle satellite cells in vitro was used to detect the interference effect of the designed and synthesized MSTN siRNA (si-MSTN). At the same time, Real-time quantitative PCR and Western blotting were used to detect the expression of mRNA and protein levels of genes related to actin cytoskeleton regulatory pathway in proliferative (GM) and day 3 of differentiation (DM3) bovine skeletal muscle satellite cells transfected with si-MSTN, and study the effect of knockout MSTN expression on actin cytoskeleton regulatory pathway. The results showed that 16 genes related to actin cytoskeleton regulatory pathway were up-regulated in muscle tissue of MSTN^+/- Mongolian cattle, the expression level of MSTN in si-MSTN transfected cells was extremely significantly decreased (P<0.01). In si-MSTN transfected GM phase bovine skeletal muscle satellite cells, the mRNA levels of ENAH, ACTN4 and Cdc42 genes related to actin cytoskeleton regulatory pathway were significantly increased (P<0.05), and the protein levels of PFN1, RhoA and ACTN4 were significantly or extremely significantly increased (P<0.05;P<0.01). In si-MSTN transfected DM3 bovine skeletal muscle satellite cells, the mRNA levels of ENAH, CFL1, SCIN and Cdc42 genes were significantly increased (P<0.05), the mRNA level of RhoA gene was extremely significantly increased (P<0.01), and the protein levels of PFN1 and ACTN4 were significantly increased (P<0.05). The results of this study indicated that interfering with MSTN could promote the expression of genes related to actin cytoskeleton regulatory pathway, and explore the molecular mechanism that MSTN might mediate actin cytoskeleton regulatory pathway to affect the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells, which provided reference for further study of the regulatory mechanism of MSTN on bovine myogenic differentiation.

Key words: bovine; MSTN; skeletal muscle satellite cells; actin cytoskeleton regulatory pathway; proliferation; myogenic differentiation

中图分类号:

盛辉, 郭益文, 张林林, 谭皓云, 胡德宝, 李新, 丁向彬, 郭宏. MSTN基因对牛肌动蛋白细胞骨架调节通路的影响[J]. 中国畜牧兽医, 2021, 48(10): 3554-3564.

SHENG Hui, GUO Yiwen, ZHANG Linlin, TAN Haoyun, HU Debao, LI Xin, DING Xiangbin, GUO Hong. Effect of MSTN Gene on Actin Cytoskeleton Regulatory Pathway[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3554-3564.

参考文献

[1] GULLER I, RUSSELL A P.microRNAs in skeletal muscle:Their role and regulation in development, disease and function[J]. The Journal of Physiology, 2010, 588(Pt21):4075-4087.
[2] JWNSEN P B, PEDERSEN L, KRISHNA S, et al.A Wnt oscillator model for somitogenesis[J]. Biophysical Journal, 2010, 98(6):943-950.
[3] BLAKE J A, ZIMAN M R.Pax genes:Regulators of lineage specification and progenitor cell maintenance[J]. Development, 2014, 141(4):737-751.
[4] CHANG N C, RUDNICKI M A.Satellite cells:The architects of skeletal muscle[J]. Current Topics in Developmental Biology, 2014, 107:161-181.
[5] 张萍, 马月辉, 焦淑清, 等.骨骼肌卫星细胞的研究进展[J]. 黑龙江畜牧兽医, 2015, 13:58-62. ZHANG P, MA Y H, JIAO S Q, et al.Research progress of skeletal muscle satellite cells[J]. Heilongjiang Animal Science and Veterinary Medicine, 2015, 13:58-62.(in Chinese)
[6] DING S J, WANG F, LIU Y, et al.Characterization and isolation of highly purified porcine satellite cells[J]. Cell Death Discovery, 2017, 3:17003.
[7] LEE S J, MCPHERRON A C.Myostatin and the control of skeletal muscle mass[J]. Current Opinion in Genetics & Development, 1999, 9(5):604-607.
[8] SHARMA M, LANGLEY B, BASS J, et al.Myostatin in muscle growth and repair[J]. Exercise and Sport Sciences Reviews, 2001, 29(4):155-158.
[9] LEE S J, MCPHERRON A C.Regulation of myostatin activity and muscle growth[J]. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(16):9306-9311.
[10] MANFREDI L H, PAULA-GOMES S, ZANON N M, et al.Myostatin promotes distinct responses on protein metabolism of skeletal and cardiac muscle fibers of rodents[J]. Brazilian Journal of Medical and Biological Research, 2017, 50(12):e6733.
[11] MOSHER D S, QUIGNON P, BUSTAMANTE C D, et al.A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs[J]. PLoS Genetics, 2007, 3(5):e79.
[12] WANG K, OUYANG H, XIE Z, et al.Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system[J]. Scientific Reports, 2015, 5:16623.
[13] ELKASRAWY M N, HAMRICK M W.Myostatin (GDF-8) as a key factor linking muscle mass and bone structure[J]. Journal of Musculoskeletal & Neuronal Interactions, 2010, 10(1):56-63.
[14] KOLLIAS H D, MCDERMOTT J C.Transforming growth factor-beta and myostatin signaling in skeletal muscle[J]. Journal of Applied Physiology, 2008, 104(3):579-587.
[15] STEELMAN C A, RECKNOR J C, NETTLETON D, et al.Transcriptional profiling of myostatin-knockout mice implicates Wnt signaling in postnatal skeletal muscle growth and hypertrophy[J]. FASEB Journal, 2006, 20(3):580-582.
[16] MORISSETTE M R, COOK S A, BURANASOMBATI C, et al.Myostatin inhibits IGF-Ⅰ-induced myotube hypertrophy through Akt[J]. American Journal of Physiology-Cell Physiology, 2009, 297(5):C1124-C1132.
[17] ALLENDORPH G P, VALE W W, CHOE S.Structure of the ternary signaling complex of a TGF-beta superfamily member[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(20):7643-7648.
[18] JOULIA-EKAZA D, CABELLO G.The myostatin gene:Physiology and pharmacological relevance[J]. Current Opinion in Pharmacology, 2007, 7(3):310-315.
[19] LIPINA C, KENDALL H, MCPHERRON A C, et al.Mechanisms involved in the enhancement of mammalian target of rapamycin signalling and hypertrophy in skeletal muscle of myostatin-deficient mice[J]. FEBS Letters, 2010, 584(11):2403-2408.
[20] WANG D T, YANG Y J, HUANG R H, et al.Myostatin activates the ubiquitin-proteasome and autophagy-lysosome systems contributing to muscle wasting in chronic kidney disease[J]. Oxidative Medicine and Cellular Longevity, 2015, 2015:684965.
[21] HUANG P, PANG D, WANG K, et al.The possible role of complete loss of myostatin in limiting excessive proliferation of muscle cells (C2C12) via activation of microRNAs[J]. International Journal of Molecular Sciences, 2019, 20(3):643.
[22] XIN X B, YANG S P, LI X, et al.Proteomics insights into the effects of MSTN on muscle glucose and lipid metabolism in genetically edited cattle[J]. General and Comparative Endocrinology, 2020, 291:113237.
[23] AVERSA Z, BONETTO A, PENNA F, et al.Changes in myostatin signaling in non-weight-losing cancer patients[J]. Annals of Surgical Oncology, 2012, 19(4):1350-1356.
[24] LENK K, SCHUR R, LINLE A, et al.Impact of exercise training on myostatin expression in the myocardium and skeletal muscle in a chronic heart failure model[J]. European Journal of Heart Failure, 2009, 11(4):342-348.
[25] MCPHERRON A C, LEE S J.Suppression of body fat accumulation in myostatin-deficient mice[J]. Journal of Clinical Investigation, 2002, 109(5):595-601.
[26] ZHANG C, MCFARLANE C, LOKIREDDY S, et al.Myostatin-deficient mice exhibit reduced insulin resistance through activating the AMP-activated protein kinase signalling pathway[J]. Diabetologia, 2011, 54(6):1491-1501.
[27] PAAVILAINEN V O, BERTLING E, FALCK S, et al.Regulation of cytoskeletal dynamics by actin-monomer-binding proteins[J]. Trends in Cell Biology, 2004, 14(7):386-394.
[28] 王轶敏, 代阳, 刘新峰, 等.牛骨骼肌卫星细胞的分离鉴定和诱导分化[J]. 中国畜牧兽医, 2014, 41(7):142-147. WANG Y M, DAI Y, LIU X F, et al.Isolation identification and induction differentiation of bovine skeletal muscle satellite cells[J]. China Animal Husbandry & Veterinary Medicine, 2014, 41(7):142-147.(in Chinese)
[29] 代阳, 王轶敏, 刘新峰, 等.兔骨骼肌卫星细胞成肌诱导分化及鉴定[J]. 黑龙江畜牧兽医, 2014, 21:176-179. DAI Y, WANG Y M, LIU X F, et al.Myogenic differentiation and identification of rabbit skeletal muscle satellite cells[J]. Heilongjiang Animal Science and Veterinary Medicine, 2014, 21:176-179.(in Chinese)
[30] 易健明, 屈武斌, 张成岗.实时荧光定量PCR的数据分析方法[J]. 生物技术通讯, 2015, 26(1):140-145. YI J M, QU W B, ZHANG C G.Data analysis methods of Real-time fluorescent quantitative PCR[J]. Letters in Biotechnology, 2015, 26(1):140-145.(in Chinese)
[31] FENG Y, CAO J H, LI X Y, et al.Inhibition of miR-214 expression represses proliferation and differentiation of C2C12 myoblasts[J]. Cell Biochemistry and Function, 2011, 29(5):378-383.
[32] SCHIAFFINO S, SANDRI M, MURGUA M.Activity-dependent signaling pathways controlling muscle diversity and plasticity[J]. Physiology (Bethesda, Md), 2007, 22:269-278.
[33] RAFTOPOULOU M, HALL A.Cell migration:Rho GTPases lead the way[J]. Developmental Biology, 2004, 265(1):23-32.
[34] WEN J, QIAN C, PAN M, et al.Lentivirus-mediated RNA interference targeting RhoA slacks the migration, proliferation, and myelin formation of Schwann cells[J]. Molecular Neurobiology, 2017, 54(2):1229-1239.
[35] ZHAN Y, LIANG X, LI L, et al.microRNA-548j functions as a metastasis promoter in human breast cancer by targeting Tensin1[J]. Molecular Oncology, 2016, 10(6):838-849.
[36] GAGAT M, HALASW M, ZIELINSKA W, et al.The effect of piperlongumine on endothelial and lung adenocarcinoma cells with regulated expression of profilin-1[J]. OncoTargets and Therapy, 2018, 11:8275-8292.
[37] WANG L, YANG L, TIAN L, et al.Cannabinoid receptor 1 mediates homing of bone marrow-derived mesenchymalstem cells triggered by chronic liver injury[J]. Journal of Cellular Physiology, 2017, 232(1):110-121.
[38] 朱菲菲, 张俊星, 张林林, 等.干扰MSTN对牛骨骼肌卫星细胞增殖分化的影响[J]. 中国畜牧兽医, 2020, 47(2):479-487. ZHU F F, ZHANG J X, ZHANG L L, et al.Effect of interfering with MSTN on the proliferation and differentiation of bovine skeletal muscle satellite cells[J]. China Animal Husbandry & Veterinary Medicine, 2020, 47(2):479-487.(in Chinese)