Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (24): 4944-4955.doi: 10.3864/j.issn.0578-1752.2023.24.012

• ANIMAL SCIENCE・VETERINARY SCIENCE • Previous Articles     Next Articles

LNC721 Targeted Regulation MMP9 Affects Bovineskeletal Muscle Satellite Cell Proliferation and Differentiation

GUO YunPeng(), TAN HaoYun, GUO Hong, FU MengYun, LI Xin, HU DeBao, ZHANG LinLin, DING XiangBin, GUO YiWen()   

  1. School of Animal Science and Animal Medicine, Tianjin Agricultural College/Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, Tianjin 300384
  • Received:2023-05-06 Accepted:2023-07-04 Online:2023-12-16 Published:2023-12-21
  • Contact: GUO YiWen

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Abstract:

【Background】The muscular system is an important basis for maintaining the survival and growth of the animal body. In the mammalian muscle, nearly half of the muscle is skeletal muscle, skeletal muscle through cell multiplication to migration fusion, and gradually formed a mature muscle bundle attached to the bone with contractile ability. In the animal body, skeletal muscle is not only involved in animal growth, but also in physiological activities, such as respiration and metabolism. At present, many studies have shown that lncRNA had the effect of regulating muscle growth and development, and was a key factor affecting skeletal muscle function and diseases. However, due to the complex mechanism of action of lncRNA, the variety of methods, and the very low conservation type between species, the relationship between lncRNAs of different species is not large, and most of the studies exist in organisms such as humans and mice, and there are few studies on the effect on bovine muscle growth. In recent years, it has been discovered that lncRNAs interact with certain target genes to regulate the process of muscle cell genesis. In the early stage of this experiment, the high-throughput sequencing was performed by collecting bovine muscles of different tissues of different months, the lncRNA with high expression difference was obtained, and the mechanism of its regulation of the myogenic process was studied. 【Objective】This study aimed to explore the interaction between long non-coding RNA lnc721 and its target gene MMP9, and the effects of MMP9 on the growth and development of bovine skeletal muscle cells, in order to provide a reference for the study of the regulatory mechanism of bovine skeletal muscle development.【Method】 The previous experiments showed that interference with lnc721 had a positive regulatory effect on the proliferation of bovine skeletal muscle satellite cells and negatively regulated its differentiation. Three groups of interfering lnc721 bovine skeletal muscle satellite cells and three control groups were set up to sequence transcriptome using NGS technology in the differentiation stage of bovine skeletal muscle satellite cells, in order to obtain the lnc721 differential target gene and to further study the regulatory pathway of lnc721 on bovine skeletal muscle development. According to the screening results and the verification results of qRT-PCR, MMP9 was selected as the target gene of lnc721, and the binding ability of lnc721 and MMP9 was predicted through the CatRAPID website. The interference sequences of lnc721 and MMP9 were designed and synthesized, transfected into bovine skeletal muscle satellite cells, and the effect of lnc721 on MMP9 expression was down-regulated by qRT-PCR and Western blot technology at the mRNA level and protein level. After down-regulation of MMP9, qRT-PCR, Western blot and EdU were used to detect the expression of proliferation marker factors Ki67 and Pax7 and differentiation marker factors MyHC and MyOG, so as to reflect the effect of down-regulation of MMP9 on the growth and development of bovine skeletal muscle satellite cells. 【Result】 MMP9 was identified as a target gene for lnc721 to regulate the interaction of bovine skeletal muscle satellite cells. They were found to interact and bind to each other by RIP. After interfering with lnc721, qRT-PCR analysis showed that down-regulation of lnc721 significantly inhibited MMP9 expression (P<0.01) during the proliferative phase, while it significantly promoted MMP9 expression (P<0.01) during the differentiation phase. Downregulation of MMP9 resulted in a highly significant upregulation of Ki67 mRNA level expression in proliferating cells (P<0.01) and the Pax7 protein expression (P ˂0.05). As also, it could significant increase the positive cell rate of EdU labled cells. At the stage of cell differentiation, the downregulation of MMP9 could inhibit muscle myotube formation. On the other hand, the mRNA and protein expressions of MyHC were significantly decreased (P<0.01); MyoG protein expression was significantly down-regulated (P<0.05). 【Conclusion】 lnc721 could bind to MMP9. Interfering with lnc721 was significantly inhibited MMP9 expression during the proliferative phase of cells, while promoting MMP9 expression during the differentiation phase. MMP9 inhibition could promoted cell proliferation and inhibited differentiation. This study demonstrated that lnc721 targeting MMP9 regulated the development of bovine skeletal muscle satellite cells.

Key words: lnc721, MMP9, bovine skeletal muscle satellite cells, proliferation, differentiation

Table 1

Primers of qRT-PCR"

基因
Gene		 引物序列
Primer sequences
(5′-3′)		 片段大小
Product length (bp)
Lnc721 F GGGATCACAGCCTGCCAACAC 119
R AGACAGCGACATCCTCAGTGACTC
MyoG F GGCTGACAAATGCCAGACTATCC 140
R TGGTCCCTTGCTTTATCTCCCT
MyHC F CTGGAATCCGGAGGCAGAA 105
R TTTTCGAAGGTAGGGAGCGG
GAPDH F TGTTGTGGATCTGACCTGCC 135
R AAGTCGCAGGAGACAACCTG
Pax7 F AGCCAGAGTTTCAACGGGAG 93
R GTCGCCAACAGACAACACAC
Ki-67 F GAGGTGGCTCAGGTTCGTC 97
R AAAGGGTTGGTGGTAAGTGGC
MMP9 F TACCTCCACCCTGTCTACCAG 112
R CCTGAGGACCAATGAGAGTGG

Fig. 1

Analysis of lnc721 subcellular localization and coding potential A: Prediction of CPC website protein coding ability; B: Subcellular localization detection of lnc721"

Fig. 2

Tissue series expression profile of lnc721 A: 3-month-old fetus; B: 6-month-old fetus; C: 9-month-old fetus; D: Adult cattle"

Fig. 3

Volcano map of gene expression differential analysis"

Fig. 4

Real-time quantitative PCR results of differential gene A: qRT PCR detects the expression of the up-regulated gene; B: qRT-PCR fetects the expression of the down-regulated gene"

Fig. 5

catRAPID predicts the binding site of lnc721 and MMP9"

Fig. 6

Enrichment of lnc721 with MMP9 protein by RIP"

Fig. 7

Downregulation of lnc721 on MMP9 A: Changes in mRNA levels of MMP9 during proliferation and differentiation after down-regulation of lnc721; B: Detection of lnc721 and MMP9 protein expression in proliferation stage; C: Detection of lnc721 and MMP9 protein expression in differentiation stage"

Fig. 8

The effect of interfering MMP9 on proliferation of bovine skeletal muscle satellite cells A: Quantitative PCR screening of MMP9 interfering siRNA in bovine skeletal muscle satellite cells; B: qRT-PCR was used to detect the mRNA expression level of increment marker factors; C: EdU staining, nuclei were counterstained with Hoechst 33342 (400×; scale bars 50 μm), EdU labeling index, with EdU. The number of positive cells/total number of cells represents; D: Proliferation markers were detected by Western blotting"

Fig. 9

The effect of interfering MMP9 on differentiation of bovine skeletal muscle satellite cells A: After transfection of ASO-MMP9, the growth status of differentiated cells was observed by inverted microscope (200×); B: mRNA expression levels of cell differentiation markers; C: Proliferation markers were detected by Western blotting"

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