关键词: lnc721, MMP9, 牛骨骼肌卫星细胞, 增殖, 分化

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