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"

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