Lnc721靶向调控MMP9对牛骨骼肌卫星细胞增殖分化的影响

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

【背景】肌肉系统是维持动物体生存生长的重要基础,在哺乳动物肌肉中,将近一半的肌肉为骨骼肌,骨骼肌通过细胞繁育增殖到迁移融合,逐步形成了具有收缩能力的附着在骨骼上的成熟肌束。在机体内,骨骼肌不仅参与动物生长,也参与呼吸、代谢等生理活动。目前许多研究表明,lncRNA具有调控肌肉生长发育的作用,是影响骨骼肌功能、疾病的关键因子。但由于lncRNA作用机制复杂,方式多样,在物种间保守型很低,故不同种属动物lncRNA之间作用关系不大,且大多数研究存在于人和小鼠等生物体内,而对牛肌肉生长影响的研究内容极少。近年来,人们发现lncRNA会与某些靶基因相互作用,进而调控肌肉细胞的发生过程。【目的】在前期采集不同月龄不同组织部位的牛肌肉,进行高通量测序,获得高表达差异的lncRNA,在对其调控成肌过程进行机制研究的基础上,探究长链非编码RNA lnc721与其靶基因MMP9的互作关系,以及MMP9对牛骨骼肌细胞生长发育的影响,以期为牛骨骼肌发育调控机制的研究提供参考。【方法】前期试验发现干扰lnc721对牛骨骼肌卫星细胞增殖具有正调控作用,且对其分化具有负调控作用。为了进一步探究lnc721对牛肌肉发育的调控通路。分别在牛骨骼肌卫星细胞的分化期设置了3个干扰lnc721牛骨骼肌卫星细胞组,与3个对照组采用NGS技术进行转录组测序,以期获得lnc721差异靶基因,进一步研究lnc721对牛骨骼肌发育的调控通路。根据筛选结果以及qRT-PCR的验证结果,试验选取MMP9作为lnc721的靶基因,并通过CatRAPID网站对lnc721与MMP9结合能力进行预测。设计并合成lnc721及MMP9干扰序列,转染至牛骨骼肌卫星细胞中,通过qRT-PCR、Western blot技术在mRNA水平与蛋白质水平分析下调lnc721对MMP9表达情况的影响。并且通过下调MMP9,采用qRT-PCR、Western blot与EdU技术检测增殖标志因子Ki67、Pax7以及分化标志因子MyHC与MyOG的表达情况,以反映下调MMP9对牛骨骼肌卫星细胞生长发育的影响。【结果】CatRAPID结果显示,lnc721与MMP9结合倾向为0.75,共有9个结合位点,二者存在互作结合。干扰lnc721之后,采用qRT-PCR分析,结果表明在细胞增殖期下调lnc721可以极显著抑制MMP9表达(P<0.01),而在分化期则极显著促进MMP9的表达(P<0.01),证实了MMP9为lnc721调控牛骨骼肌卫星细胞互作的靶基因。进而下调MMP9,检测对增殖期细胞的影响,发现Ki67的mRNA水平表达极显著上调(P<0.01),Pax7蛋白表达量显著上调(P<0.05),EdU观察下调MMP9后的阳性细胞率也明显升高。同样检测下调MMP9之后对细胞分化期的影响,发现下调MMP9后镜下观察发现下调MMP9会抑制肌管形成,同时MyHC的mRNA和蛋白表达量极显著下降(P<0.01),MyoG蛋白表达量显著下调(P<0.05)。【结论】lnc721与MMP9相互结合。在细胞增殖期干扰lnc721极显著抑制MMP9表达,而在分化期促进MMP9的表达,且干扰MMP9可促进细胞增殖并抑制分化。证明lnc721靶向MMP9调控牛骨骼肌卫星细胞的发育。

【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(P0.05).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.