Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (20): 4287-4296.doi: 10.3864/j.issn.0578-1752.2020.20.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Effects of PSMB5 on the Proliferation and Myogenic Differentiation of Skeletal Muscle Satellite Cells

LAI YuTing(),ZHU FeiFei,WANG YiMin,GUO Hong,ZHANG LinLin,LI Xin,GUO YiWen,DING XiangBin()   

  1. Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry/College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384
  • Received:2019-11-13 Accepted:2020-04-02 Online:2020-10-16 Published:2020-10-26
  • Contact: XiangBin DING E-mail:yutinglai@163.com;xiangbinding@163.com

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Abstract:

【Objective】 The aim of this study was to investigate the effects of proteasome subunit beta type-5(PSMB5) on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells. 【Method】 In this study, the in vitro induced myogenic differentiation model of bovine skeletal muscle satellite cells was used to simulate the growth and development of bovine skeletal muscle. Firstly, the expression of PSMB5 in bovine skeletal muscle satellite cells before and after the differentiation was detected. The mRNA expression level of PSMB5 was detected by qRT-PCR, and the protein expression level of PSMB5 was detected by western blotting. Then, the small interfering RNA si-RNA-PSMB5 (si-PSMB5) was designed and synthesized, and PSMB5 overexpression plasmid pcDNA3.1-PSMB5 (pcDNA-PSMB5) was constructed, which were transfected into bovine skeletal muscle satellite cells by lipofectamine 3000, and the transfection effect was detected by qRT-PCR and Western blotting. Finally, EdU staining was used to detect the effects of PSMB5 interference and overexpression on the proliferation of bovine skeletal muscle satellite cells. Further, in vitro myogenic differentiation of bovine skeletal muscle satellite cells was induced, and the myotube formation of bovine skeletal muscle satellite cells was observed under light microscope. The expression of myosin heavy chain (MyHC) protein, a marker of differentiation, was detected by western blotting. The effects of PSMB5 on proliferation and differentiation of bovine skeletal muscle satellite cells were analyzed. 【Result】There was a significant difference in the expression level of PSMB5 before and after the differentiation of bovine skeletal muscle satellite cells. After the differentiation of bovine skeletal muscle satellite cells, the expression level of PSMB5 mRNA and protein was significantly higher than that in the proliferation period (P<0.05). When PSMB5 was interfered or overexpressed, there was no significant difference in EdU positive cell rate between the two groups (P>0.05). After interfering with the expression of PSMB5, the number of myotubes formed by cell differentiation was significantly less than that of the control group, and the protein expression level of MyHC was significantly lower than that of the control group (P<0.05); however, after overexpression of PSMB5, the number of myotubes formed by cell differentiation was significantly higher than that of the control group, and the protein expression level of MyHC was significantly higher than that of the control group (P<0.05). 【Conclusion】The results showed that the PSMB5 had no significant effect on the proliferation of bovine skeletal muscle satellite cells, but it had a significant regulatory effect on the myogenic differentiation process of bovine skeletal muscle satellite cells. Interference with PSMB5 expression could inhibit the myogenic differentiation process, while overexpression of PSMB5 could promote the myogenic differentiation process of bovine skeletal muscle satellite cells in vitro. In this study, the specific regulatory effect of PSMB5 on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells was explored, which laid a foundation for further study on the regulatory mechanism of PSMB5 in bovine muscle myogenic differentiation.

Key words: bovine, PSMB5, skeletal muscle satellite cells, cell proliferation, myogenic differentiation

Table 1

The primer information of qRT-PCR"

引物名称
Primer name		 引物序列
Primer sequence		 片段长度
Fragment length
PSMB5-F 5′-TCAAGATGTCAGCCACACCC -3′ 112
PSMB5-R 5′-ACCACGCCTCCATACACAAG -3′
MyHC-F 5′- CTGGAATCCGGAGGCAGAA-3′ 105
MyHC-R 5′- TTTTCGAAGGTAGGGAGCGG-3′
GAPDH-F 5′- TGTTGTGGATCTGACCTGCC-3′ 135
GAPDH-R 5′- AAGTCGCAGGAGACAACCTG-3′

Fig. 1

Expression of PSMB5 in proliferation and differentiation of bovine skeletal muscle satellite cells Comment: GM indicates the period of proliferation of bovine skeletal muscle satellite cells; DM indicates the differentiation of bovine skeletal muscle satellite cells for 72 hours"

Fig. 2

Detection of interference effects of si-PSMB5"

Fig. 3

Detection of pcDNA-PSMB5 overexpression effect"

Fig. 4

EdU detects proliferating cells of bovine skeletal muscle satellite cells after knockdown and overexpression of PSMB5"

Fig. 5

Effects of interference and overexpression of PSMB5 on the differentiation of bovine skeletal muscle satellite cells Comment: (A-B)Effect of transfection of siRNA-PSMB5 and pcDNA-PSMB5 and control group bovine skeletal muscle satellite cells on myogenic differentiation (100×)(C-D)Western blotting to detect the effect of interfering with PSMB5 on the expression levels of PSMB5 and differentiation markers MyHC(E-F)Western blotting to detect the effect of over-expressing PSMB5 on the protein expression levels of PSMB5 and differentiation markers MyHC"

[1] 李兴爽, 王洋, 金一. 泛素-蛋白酶体系统研究进展. 畜牧与兽医, 2016,48(3):137-141.
LI X S, WANG Y, JIN Y. Research progress on ubiquitin-proteasome system. Animal Husbandry & Veterinary Medicine, 2016,48(3):137-141. (in Chinese)
[2] 杨立坤, 傅力, 牛燕媚. 泛素-蛋白酶体在运动调节骨骼肌代谢中的作用研究进展. 中国运动医学杂志, 2018,37(10):59-62.
YANG L K, FU L, NIU Y M. Research progress of ubiquitin proteasome system in motor regulation of skeletal muscle metabolism. Chinese Journal of Sports Medicine, 2018,37(10):59-62. (in Chinese)
[3] 兰秋艳, 高媛, 李衍常, 洪学传, 徐平. 泛素、泛素链和蛋白质泛素化研究进展. 生物工程学报, 2016(1):14-30.
LAN Q Y, GAO Y, LI Y C, HONG X C, XU P. Progress in ubiquitin, ubiquitin chain and protein ubiquitination. Chinese Journal of Biotechnology, 2016,32(1):14-30. (in Chinese)
[4] NATALIA S, DALE S H. Ub on the move. Journal of Cellular Biochemistry, 2004,93(1):11-19.
doi: 10.1002/jcb.20130 pmid: 15352157
[5] KAANG B K, CHOI J H. Synaptic protein degradation in memory reorganization. Advances in Experimental Medicine & Biology, 2012,970(970):221-240.
[6] 程思源, 张苑, 陈磊磊. 泛素-蛋白酶体系统在心血管疾病中的研究进展. 医学综述 2018,24(15):2954-2960.
CHENG S Y, ZHANG Y, CHEN L L. Research progress of ubiquitin-proteasome system in cardiovascular diseases. Medical Recapitulate, 2018,24(15):2954-2960. (in Chinese)
[7] HARSHBARGER W, MILLER C, DIEDRICH C, Crystal structure of the human 20S proteasome in complex with carfilzomib. Structure, 2015,23(2):418-424.
pmid: 25599644
[8] 刘香娥, 季保启, 鲁彬, 李晓涛. 生物体内蛋白质降解系统的研究进展. 生命的化学, 2017, ( 5):699-706.
LIU X E, JI B Q, LU B, LI X T. Protein degradation pathways in eukaryotes.Chemistry of Life, 2017(5):699-706. (in Chinese)
[9] LUO H, WONG J, WONG B. Protein degradation systems in viral myocarditis leading to dilatedcardiomyopathy. Cardiovascular Research, 2010,85(2):347-356.
doi: 10.1093/cvr/cvp225 pmid: 19578074
[10] GROLL M, DITZEL L, LOWE J. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature, 1997,386(6624):463-471.
doi: 10.1038/386463a0 pmid: 9087403
[11] YOSHIMITSU S, TARO K, OSAMU T, NEAL G, COPELAND, DEBRA J, GILBERT, NANCY A, JENKINS, TAKEDA K, AKIRA S. Mouse proteasomal ATPases Psmc3 and Psmc4: Genomic organization and gene targeting. Genomics, 2000,67(1) : 1-7.
doi: 10.1006/geno.2000.6231 pmid: 10945464
[12] 吕莹, 胡勇. 泛素-蛋白酶体途径与肝脏疾病的研究进展. 医学分子生物学志, 2018,15(2):120-124.
LÜ Y, HU Y. Advances in the ubiquitin-proteasome pathway and liver diseases. Journal of Medical Molecular Biology, 2018,15(2):120-124. (in Chinese)
[13] 鄢雯, 李囡, 刘立新. 泛素-蛋白酶体系统在心肌肥厚中的研究进展. 医学综述, 2016,22(17):3329-3332. DOI: 10.3969/j.issn.10062084.201617.001.
YAN W, LI N, LIU L X. Research advance of ubiquitin-proteasome system in myocardial hypertrophy. Medical Recapitulate, 2016,22(17):3329-3332. DOI: 10.3969/j.issn.10062084.201617.001. (in Chinese)
[14] BILODEAU P A, COYNE E S, WING S S. The ubiquitin proteasome system in atrophying skeletalmuscle: roles and regulation. American Journal of Physiology-Cell Physiology, 2016,311(3):392-403.
[15] HASSELGREN P O, WRAY C, MAMMEN J. Molecular regulation of muscle cachexia: it may be more than the proteasome. Biochemical and Biophysical Research Communications, 2002,290(1):1-10.
doi: 10.1006/bbrc.2001.5849 pmid: 11779124
[16] 李程浩. 过表达PSMB5抑制人骨髓间充质干细胞衰老进程[D]. 太原:山西医科大学, 2015. DOI: 10.7666/d.D649761.
LI C H. Overexpression of PSMB5 gene promotes human mesenchymal stem cells differentiation into neuron-likecells[D]. Taiyuan: Shanxi Medical University, 2015. DOI: 10.7666/d.D649761. (in Chinese)
[17] 李程浩, 赵晨, 贺婵婷, 陆利, 杨桂姣, PSMB5 基因过表达促人骨髓间充质干细胞向神经元样细胞分化. 神经解剖学杂志, 2015,31(4):428-432.
LI C H, ZHAO C, HE C T, LU L, YANG G J. Overexpression of PSMB5 gene promotes human mesenchymal stem cells differentiation into neuron-like cells. Chinese Journal of Neuroanatomy, 2015,31(4):428-432. (in Chinese)
[18] 刘立新, 鄢雯, 李囡, 田翠, 王红霞, 李汇华. 免疫蛋白酶体β 5i亚基在DOCA盐敏感高血压性心肌肥厚中的作用及机制. 中国分子心脏病学杂志, 2015,5:1469-1471.
LIU L X, YAN W, LI N, TIAN C, WANG H X, LI H H. The role of immunoproteasome β5i Subunit in DOCA Salt-induced cardiac hypertrophy and the underlying mechanism. Molecular Cardiology of China, 2015,5:1469-1471. (in Chinese)
[19] 李睿, 黄菲菲, 陆利, 赵云鹤. 靶向PSMB5基因的shRNA慢病毒载体对神经干细胞增殖和分化潜能的影响. 神经解剖学杂志, 2017,33(5):599-604.
LI R, HUANG F F, LU L, ZHAO Y H. Effects of shRNA lentiviral vectors targeting PSMB5 on the proliferation and differentiation of neural stem cells. Chinese Journal of Neuroanatomy, 2017,33(5):599-604. (in Chinese)
[20] LI N, WANG H X, HAN Q Y, LI W J, ZHANG Y L, DU J, XIA Y L, LI H H. Activation of the cardiac proteasome promotes angiotensionII- induced hypertrophy by down-regulation of ATRAP. Journal of Molecular and Cellular Cardiology, 2015,79:303-314.
doi: 10.1016/j.yjmcc.2014.12.007 pmid: 25526681
[21] 王轶敏, 代阳, 刘新峰, 刘中伟, 李吉霞, 郭宏, 丁向彬. 牛骨骼肌卫星细胞的分离鉴定和诱导分化. 中国畜牧兽医, 2014,41(7):142-147.
WANG Y M, DAI Y, LIU X F, LIU Z W, LI J X, GUO H, DING X B. Isolation identification and differentiation of bovine skeletal muscle satellite cells. Chinese Animal Husbandry and Veterinary Medicine, 2014,41(7):142-147. (in Chinese)
[22] DAI Y, WANG Y M, ZHANG W R, LIU X F, LI X, DING X B, GUO H. The role of microRNA-1 and microRNA-206 in the proliferation and differentiation of bovine skeletal muscle satellite cells. In Vitro Cellular & Developmental Biology-Animal, 2016,52(1):27-34.
doi: 10.1007/s11626-015-9953-4 pmid: 26424132
[23] COLLINS C A, OLSEN I, ZAMMIT P S, HESLOP L, PETRIE A, PARTRIDGE T A, MORGAN J E. Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell, 2005,122(2):289-301.
doi: 10.1016/j.cell.2005.05.010 pmid: 16051152
[24] MONTARRAS D, MORGAN J, COLLINS C, RELAIX F, ZAFFRAN S, CUMANO, PARTRIDGE T, BUCKINGHAM M. Direct isolation of satellite cells for skeletal muscle regeneration. Science, 2005,309(5743):2064-2067.
doi: 10.1126/science.1114758 pmid: 16141372
[25] 郭卉, 张雪竹. 泛素-蛋白酶体途径及其生物学作用的研究进展. 现代医学生物进展, 2008,8(9):1786-1788.
GUO H, ZHANG X Z. Studies of ubiquitin/proteasome system and its function in biology. Progress in Modern Biomedicine, 2008,8(9):1786-1788. (in Chinese)
[26] 肖玲, 孙瑞红. 泛素-蛋白酶体系统在动脉粥样硬化形成中的作用. 卒中与神经疾病, 2008,15(3):180-181.
XIAO L, SUN R H. The role of ubiquitin-proteasome system in the formation of atherosclerosis. Stroke and Nervous Diseases, 2008,15(3):180-181. (in Chinese)
[27] SOHNS W, VAN VEEN TA, VAN DER HEYDEN MA. Regulatory roles of the ubiquitin-proteasome system in cardiomyocyte apoptosis. Current Molecular Medicine, 2010,10(1):1-13.
doi: 10.2174/156652410791065426 pmid: 20205676
[28] KITAJIMA Y, SUZUKI N, NUNOMIYA A. The ubiquitin-proteasome system is indispensable for the maintenance of muscle stem cells. Stem Cell Reports,2018, 11(6):1523-1538. DOI: 10.1016/j.stemcr.
doi: 10.1016/j.stemcr.2018.10.009 pmid: 30416048
[29] 李强. 蛋白酶体抑制剂MG-132对大鼠神经损伤-肌肉萎缩防治作用的实验研究[D]. 陕西: 第四军医大学, 2007. DOI: 10.7666/d.d036427.
LI Q. Preventive and therapeutic effects of proteasome inhibitor MG-132 on nerve injured muscle atrophy of rats[D]. Shaanxi: The Fourth Military Medical University, 2007. DOI: 10.7666/d.d036427. (in Chinese)
[30] KITAJIMA Y, TASHIRO Y, SUZUKIL N, WARITAL H, KATO M, TATEYAMA M, ANDO R, IZUMI R, YAMAZAKI M, ABE M, SAKIMURA K, ITO H, URUSHITANI M, NAGATOMI R, TAKAHASHI R, AOKIL M. Proteasome dysfunction induces muscle growth defects and protein aggregation. Journal of Cell Science, 2014,127(24):5204-5217. DOI: 10.1242/jcs
doi: 10.1242/jcs.150961
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