MMP14调控骨骼肌卫星细胞分化的分子机制研究

doi:10.11843/j.issn.0366-6964.2024.04.022

摘要/Abstract

摘要： 旨在分析基质金属蛋白酶14(MMP14)调控骨骼肌卫星细胞分化的分子机制。本试验取10只4周龄C57/BL6雌性小鼠，利用胶原酶消化法分离骨骼肌卫星细胞。首先，对骨骼肌卫星细胞进行诱导分化，利用qRT-PCR和Western blot试验分析MMP14在骨骼肌卫星细胞增殖期和分化期表达量的变化。应用siRNA抑制MMP14蛋白表达，分为试验组(si-MMP14)和对照组(si-NC)，每组3个重复：首先诱导细胞分化，应用免疫荧光和qRT-PCR分析试验组和对照组细胞分化水平的差异；随后取增殖期细胞进行蛋白组学测序，结合生物信息学分析鉴定差异蛋白，并筛选MMP14调控的关键差异蛋白和通路。本研究结果表明：1)MMP14在卫星细胞增殖期表达上调，在分化期表达下调；抑制MMP14蛋白会抑制肌管生成，表现为肌管融合指数下降。2)通过蛋白组学分析筛选到549个差异蛋白，其中有66个上调蛋白和483个下调蛋白，差异蛋白主要富集在细胞黏附、脂肪酸代谢以及AMPK通路等，参与调控细胞命运决定、组蛋白甲基化和染色质结构等生物学过程。3)通过蛋白互作关系网络分析发现MMP14与肌源性分化相关蛋白、脂肪生成相关蛋白以及异染色质结构调控蛋白直接互作，抑制MMP14可导致肌分化转录因子(PAX7、MYOD)和H3-K9甲基转移酶(SETDB1、SUV39H1)下调，而成脂分化转录因子(JUN、C/EBPβ)上调。本研究初步分析了MMP14调控骨骼肌卫星细胞分化的机制，MMP14可能通过H3-K9组蛋白甲基化参与卫星细胞命运决定以及成肌与成脂分化的转换，且这种调控作用发生在卫星细胞细胞分化启动前。本研究结果为骨骼肌发育研究提供理论依据。

关键词: 骨骼肌卫星细胞, 卫星细胞的分化, MMP14

Abstract: The aim of this study was to analyze the molecular mechanism by which matrix metalloproteinase 14 (MMP14) regulated the differentiation of skeletal muscle satellite cells. Skeletal muscle satellite cells were isolated from 10 4-week-old C57/BL6 female mice by collagenase digestion. First, skeletal muscle satellite cells were induced to differentiate, and then qRT-PCR and Western blot were used to identify the expression changes of MMP14 during the proliferation and differentiation stages of satellite cells. siRNA was applied to inhibit the expression of MMP14 protein, which was divided into experimental group (si-MMP14) and control group (si-NC), with 3 replicates per group: firstly, the cells were induced to differentiate, and the level of cell differentiation between the experimental group and the control group was analyzed by immunofluorescence and qRT-PCR; furthermore, proteomic analysis was performed, combined with bioinformatics analysis to identify differentially expressed proteins and screen for key differentially expressed proteins and pathways regulated by MMP14. The results of this study show that: 1) The expression level of MMP14 was up-regulated in satellite cell proliferation stage and down-regulated in differentiation stage. Inhibition of MMP14 protein led to the obstruction of myogenesis, which was manifested by the decrease of myotube fusion index. 2) A total of 549 differentially expressed proteins were identified through proteomic analysis, including 66 up-regulated proteins and 483 down-regulated proteins. Differentially expressed proteins were mainly enriched in cell adhesion, fatty acid metabolism, and the AMPK pathway, and were involved in regulating biological processes such as cell fate determination, histone methylation, and chromatin structure. 3) MMP14 was found to directly interact with myogenic differentiation related proteins, lipogenesis related proteins, and heterochromatin structure regulation proteins, through protein interaction network analysis. Furthermore, the inhibition of MMP14 resulted in the down-regulation of myogenic differentiation transcription factors (PAX7 and MYOD) as well as H3-K9 methyltransferases (SETDB1 and SUV39H1), while leading to the up-regulation of lipogenic differentiation transcription factors (JUN and C/EBP-β). This study preliminarily analyzed the mechanism by which MMP14 regulates the differentiation of skeletal muscle satellite cells. MMP14 may be involved in the fate determination of satellite cells and the conversion of myogenic and lipogenic differentiation through H3-K9 histone methylation, which occurs before the initiation of satellite cell differentiation. The results of this study provide a theoretical basis for the study of skeletal muscle development.

Key words: skeletal muscle satellite cells, differentiation of satellite cells, MMP14

中图分类号:

S813.1

刘媛, 李溪月, 张维娅. MMP14调控骨骼肌卫星细胞分化的分子机制研究[J]. 畜牧兽医学报, 2024, 55(4): 1592-1604.

LIU Yuan, LI Xiyue, ZHANG Weiya. Molecular Mechanism of MMP14 Regulating Skeletal Muscle Satellite Cell Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1592-1604.

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