Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (18): 3670-3681.doi: 10.3864/j.issn.0578-1752.2023.18.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

CircCEP85L Regulates the Proliferation and Myogenic Differentiation of Bovine MuSCs

WEI Yao1(), ZHANG RuiMen1, AN Qiang1, WANG LeYi1, ZHANG YongWang1, ZOU ChaoXia1, ZHANG ErKang1, MO BiYun2, SHI DeShun1, YANG SuFang1, DENG YanFei1(), WEI YingMing3()   

  1. 1 State Key Laborator for Conservation and Untilization of Subtropical Agro-bioresources /College of Animal Science and Technology, Guangxi University, Nanning 530004
    2 Qinzhou Center for Animal Disease Control and Prevention, Qinzhou 535099, Guangxi
    3 Research Institute of Agriculture and Animal Husbandry Industry Development, Guangxi University, Nanning 530004
  • Received:2022-11-01 Accepted:2023-05-16 Online:2023-09-16 Published:2023-09-21
  • Contact: DENG YanFei, WEI YingMing

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Abstract:

【Objective】At present, studies have proved that circRNA plays important roles in the development of bovine muscle, but its molecular regulation mechanism remain elusive. Screening circRNAs related to bovine muscle development can lay a foundation for further elucidating the molecular mechanism of bovine muscle development.【Method】In this study, RNA-seq sequencing results of proliferating (GM) and myogenic differentiation (DM) yellow bovine muscle stem cells (MuSCs) analyzed in the previous stage were used to screen for significantly differentially expressed circRNA, circCEP85L. Tissue samples of heart, liver, spleen, lung, kidney, muscle, intestine and stomach were collected aseptically from fresh yellow fetal calves, and yellow muscle stem cells were isolated and cultured and induced into myogenic differentiation. GM and DM cells cultured in vitro from yellow calves were collected, RNA was extracted and reverse transcribed into cDNA, respectively. Quantitative real time PCR (qRT-PCR) was used to detect the expression of circCEP85L in different tissues and different cell states. Meanwhile, specific primers were designed to amplify the full length of circCEP85L, and the overexpression vector p-circCEP85L was constructed. The plasmid was transfected into MuSCs and overexpressed circCEP85L cell samples were collected. Using overexpression plasmid pCD5-ciR cell samples as control, qRT-PCR, flow cytometry, Western Blot and immunofluorescence were used to detect the effects of overexpression of circCEP85L on proliferation, apoptosis and myogenic differentiation of yellow bovine MuSCs.【Result】The electrophoresis of PCR product proved the existence of circCEP85L. CircCEP85L was expressed in various tissues, and the expression level in DM stage was significantly higher than that in GM stage (P<0.001). To further investigate the effect on circCEP85L scallion MuSCs. The overexpression vector p-circCEP85L was transfected with the control vector pCD5-ciR in vitro cultured yellow bovine MuSCs and the EdU results showed that overexpression of circCEP85L significantly reduced the proportion of EdU positive cells (P<0.001) after continuing the culture for 24 h. Flow cycle analysis showed that overexpression of circCEP85L increased the proportion of cells in G0/G1 phase and significantly decreased the proportion of cells in S phase (P<0.001). Flow cytometry showed that overexpression of circCEP85L significantly inhibited the apoptosis rate of MuSCs (P<0.05). qRT-PCR and western blot were used to detect the expression of proliferation and apoptosis-related genes in MuSCs, respectively. The results showed that overexpression of circCEP85L significantly reduced the mRNA expression levels of proliferation and apoptosis-related genes in bovine MuSCs (P<0.001), and the expression of apoptotic protein BAX was also significantly reduced (P<0.01). In addition, in order to detect the effect of circCEP85L overexpression on myogenic differentiation of cattle MuSCs, the differentiation medium was replaced 24 hours after transfection to induce cell differentiation. Western blot and immunofluorescence results showed that overexpression of circCEP85L significantly promoted the expression level of differentiation marker gene MyH6 (P<0.001), and the number and size of myotubes formed by cell fusion were significantly higher than those of the control group.【Conclusion】The results of this study indicate that circCEP85L affects the growth and development process of skeletal muscle in cattle by inhibiting the proliferation and apoptosis of MuSCs and promoting myogenic differentiation of cells, which is expected to be a key circRNA for subsequent mechanistic studies to regulate the growth and development process of skeletal muscle in cattle.

Key words: circCEP85L, muscle stem cells, cell proliferation, myogenic differentiation

Table 1

qRT-PCR gene primer synthesis sequence"

基因名
Gene		 序列号
Accession		 引物序列（5′-3′）
Primer sequence		 产物大小
Product length（bp）
PCNA NM_001034494 F: TCCAGAACAAGAGTATAGC
R: TACAACAGCATCTCCAAT		 94
CDK2 NM_001014934 F: TCTTTGCTGAGATGGTGACCC
R: CATCTTCATCCAGGGGAGGC		 201
CyclinD1 NM_001046273 F: AACCTCCTCAACGACCGAGT
R: CGGTCCAGGTAGTTCATGGC		 220
BAX NM_173894 F: TGCAGAGGATGATCGCAGCTGT
R: CCAATGTCCAGCCCATCATGGT		 198
Caspase9 NM_001205504 F: GCTCCAGGCACGACATTCTT
R: CAAAGGAAGGGCCTGACTCC		 191
β-Actin NM_173979 F: GCCCTGGCACCCAGCACAAT
R: GGAGGGGCCGGACTCATCGT		 188
circCEP85L F: GAGACAACAAAATGAAAATGCC
R: CTGGGAATGGGATGCTGACT		 142

Fig. 1

Isolation, culture and identification of bovine MuSCs A: 3-month-old scalper fetus; B: Proliferative phase (GM) growth status of bovine MuSCs, Scale bars = 100; C: Differentiation phase（DM）growth status of bovine MuSCs, Scale bars = 100; D: Immunofluorescence showed that bovine MuSCs expressed PAX7. Scale bars = 100/400 μm"

Fig. 2

Expression profile of circCEP85L A: Tissue expression profile of circCEP85L; B: Expression of circCEP85L in bovine MuSCs before and after differentiation. The chart 'abcd' indicates the significance of the difference, first arranged according to the average from largest to smallest, the largest is marked as 'a', any insignificant difference is marked as 'a', until the significant difference is marked as 'b'（P<0.05）,and so on;*P<0.05,**P<0.01,***P<0.001,None * indicates that the difference is not significant(P<0.05). The same as below"

Fig. 3

The overexpression vector of circCEP85L was constructed M1: Supercoiled DNA ladder marker; Ⅰ: PCR amplification product of cirCEP85L; M2: DL2000 Plus DNA marker; Ⅱ: pCD5-ciR plasmid; Ⅲ: p-circCEP85L plasmid"

Fig. 4

Effect of circCEP85L overexpression on the proliferation of bovine MuSCs A: Overexpression efficiency of circCEP85L; B, C: EdU method was used to detect cell proliferation and count the number of positive cells; D: Cell proliferation related gene mRNA expression detection; E, F: Detection of the expression of proliferation-related genes and proteins; G, H: The effect of circCEP85L overexpression on cell cycle distribution was evaluated by flow cytometry analysis. Scale bars = 400 μm"

Fig. 5

Effect of circCEP85L overexpression on apoptosis of bovine MuSCs A: The mRNA expression of apoptosis related genes was detected; B, C: Detection of apoptosis marker gene BAX protein expression; D, E: Effect of circCEP85L overexpression on cell apoptosis was evaluated by flow cytometry analysis"

Fig. 6

Effect of circCEP85L overexpression on differentiation of bovine MuSCs A: Morphology of cattle MuSCs after 4 days of differentiation; B: Immunofluorescence showed the expression of MyH6 after induced differentiation; C, D: The expression of MyH6 protein was detected by Western Blot, Scale bars =100/ 400 μm"

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