牛LncRNA-133a对骨骼肌卫星细胞增殖分化的影响

doi:10.3864/j.issn.0578-1752.2019.01.013

Abstract

Abstract:

【Objective】The objective of this paper was to investigate the effects of long non-coding RNA LncRNA-133a on the proliferation and differentiation of bovine skeletal muscle satellite cells. 【Method】 This study used qRT-PCR to detect the expression level of LncRNA-133a in the skeletal muscle tissues of 3, 6 and 9 months old fetal cattle and 24 months old adult bovine skeletal muscle, and obtained the tissue temporal expression profile of LncRNA-133a. The in vitro induced myoblast differentiation model of bovine skeletal muscle satellite cells was constructed to simulate the growth and development of bovine skeletal muscle. The qRT-PCR was used to detect the cells temporal expression profiles of LncRNA-133a and myocyte differentiation markers MyoG and MHC. The bovine skeletal muscle satellite cells were transfected with LncRNA-133a overexpression vector (pCDNA3.1-EGFP- LncRNA-133a) or LncRNA-133a inhibitor (si-LncRNA 133a), and the transfection efficiency and the mRNA expression levels of LncRNA-133a, MyoD, MyoG and MHC were detected by qRT-PCR in each transfection treatment group, then the protein expression level of MHC gene was detected by western blotting. In addition, the cell proliferation of the bovine skeletal muscle satellite cells and the extent of myotube fusion at the differentiation stage were detected by EdU cell proliferation assay and immunofluorescence protein staining, respectively. 【Result】 Tissue expression profiling revealed that LncRNA-133a had the highest expression in the muscle tissue of 3 months old fetal bovine, followed by the 6-month-old fetal bovine muscle tissue, and the lowest expression in the 9-month-old fetus and adult bovine muscle tissue, which demonstrated that the time expression showed a downward trend. Cell-time expression profiles of LncRNA-133a, MyoG, and MHC were analyzed by a successfully constructed bovine skeletal muscle satellite cell differentiation model in vitro, and the results showed that the expression levels of myogenic differentiation markers MyoG and MHC gradually increased during the differentiation of bovine skeletal muscle satellite cells (D0-D3). The expression of LncRNA- 133a increased in the differentiation stage, and the expression level reached the highest at 48 h of differentiation (D2). The bovine skeletal muscle satellite cell model of overexpressing LncRNA-133a or inhibiting LncRNA-133a was constructed successfully, and in the proliferative phase (D0): the number of EdU positive cells in the overexpressed LncRNA-133a-treated group was significantly increased (P<0.01), and the number of EdU positive cells in the LncRNA-133a inhibition treatment group was significantly decreased (P<0.01), compared with the control group. At 48 h of differentiation (D2): compared with the control group, the results of LncRNA-133a overexpression treatment showed that mRNA expression levels of myocyte differentiation markers MyoD, MyoG and MHC were significantly increased (P<0.05). Western blotting showed that the expression of MHC protein was also significantly increased (P<0.01), and the immunofluorescence protein staining of MHC protein showed that the volume of fusion myotubes was larger. On the contrary, in the LncRNA-133a inhibition treatment group, the mRNA expression levels of MyoD, MyoG and MHC were decreased, and MyoG was significantly decreased (P<0.05). Meanwhile, the expression of MHC protein was significantly decreased (P<0.01), and the volume fraction of MHC protein fusion myotubes was also decreased. 【Conclusion】Thus, this study confirmed that LncRNA-133a promoted the proliferation and differentiation of bovine skeletal muscle satellite cells, which laid a foundation for further research on the regulatory network mechanism of LncRNA-133a regulating the proliferation and differentiation of bovine skeletal muscle satellite cells.

Key words: LncRNA-133a, bovine, skeletal muscle satellite cells, proliferation, differentiation

LI Yan,CHEN MingMing,ZHANG JunXing,ZHANG LinLin,LI Xin,GUO Hong,DING XiangBin,LIU XinFeng. Effects of Bovine LncRNA-133a on the Proliferation and Differentiation of Skeletal Muscle Satellite Cells[J].Scientia Agricultura Sinica, 2019, 52(1): 143-153.

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References 36

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基因 ID Gene ID	引物序列(5′-3′) Primers sequence (5′-3′)		产物长度 Product length (bp)
LncRNA-133a	Forward	GCATAGCCGGTGTCTGAGAG	108
LncRNA-133a	Reverse	CGGCCGCTTGTATATTGTCC	108
MHC	Forward	GTGGAATCCGGAGGCAGAA	105
MHC	Reverse	TTTTCGAAGGTAGGGAGCGG	105
MyoG	Forward	GGCTGACAAATGCCAGACTATCC	140
MyoG	Reverse	TGGTCCCTTGCTTTATCTCCCT	140
MyoD	Forward	GACGGCTCTCTCTGCAACTT	101
MyoD	Reverse	CGGCGCGGATCCAGGT	101
GAPDH-1	Forward	ACAGTCAAGGCAGAGAACGG	98
GAPDH-1	Reverse	CCAGCATCACCCCACTTGAT	98
GAPDH-2	Forward	CCTGCCCGTTCGACAGATAG	153
GAPDH-2	Reverse	ATGGCGACGATGTCCACTTT	153

Effects of Bovine LncRNA-133a on the Proliferation and Differentiation of Skeletal Muscle Satellite Cells

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