| Some studies indicated mi RNAs plays important role in muscle growth and development. However, fewer researches were focused on bovine muscle mi RNAs and its functions in muscle satellite cell proliferation and differentiation. In this study, bovine skeletal muscle satellite cells were isolated, purified and identified, and myogenic differentiation model was established in vitro. The expression changes of mi RNAs in the process of myogenic differentiation were detected by mi RNA microarray tests, followed by real-time PCR analysis, and candidate mi RNAs related to bovine muscle satellite cell differentiation were selected. The target genes of candidate mi RNAs were predicted by bioinformatics software, and Gene Ontology and KEGG pathway analysis were conducted to view the network between mi RNAs and target genes. When the differentiation related mi RNAs were confirmed, mi RNAs mimics and mi RNAs inhibitors were used to change mi RNAs expression levels in bovine muscle satellite cells, and its functions were evaluated by cell proliferation and differentiation detection. Through these studies, the relationship between mi RNAs and bovine muscle satellite cell will be clarified, which can provide theoretical support for further research on mechanism of mi RNAs regulating muscle proliferation and differentiation, and offer new view for beef cattle breeding.1. In this study, bovine skeletal muscle satellite cells were isolated and identified, muscle tissues were digested by collagenase and trypsinase, then the satellite cells were purified and induced differentiate into myotubes. The muscle satellite cells and myotubes were observed, and detected by RT-PCR and immunofluorescent staining technology. The results showed that the muscle satellite cells are in healthy morphology, and more than 93 percent of the cells expressed the marker genes paired box protein(Pax7) and myogenic determination factor(Myo D). After myogenesis induction, satellite cells began to convergence, and finally formed multinucleated myotubes expression of myogenin(Myo G) and myosin heavy chain(MHC). Our results indicated that high purity skeletal muscle satellite cells were successfully isolated and, which can provide cell source for muscle development and bovine muscle quality improvement study.2. In this study, bovine skeletal muscle satellite cells were isolated and cultured. The growth and differentiation of muscle satellite cells were observed, and the curve of cell growth and labeling with Ed U was used to study the effect of basic fibroblast growth factor(b FGF) on the proliferation of bovine skeletal muscle satellite cells. The results showed that b FGF could promote proliferation of bovine skeletal muscle satellite cells efficiently in growth medium and differential medium. Our study established an efficient method to culture bovine skeletal muscle satellite cells, which can provide reference for studying and using of skeletal muscle satellite cells.3. To investigate the mechanisms of mi RNAs-mediated regulation during the myogenic differentiation process, we performed a mi RNAs microarray to detect 783 bovine mi RNAs in bovine skeletal muscle satellite cell myogenic differentiation, there are 17 mi RNAs were significantly different between bovine skeletal muscle satellite cells and differentiated myotubes. The results were further confirmed by a quantitative real-time RT-PCR assay and 15 mi RNAs were confirmed, in which twelve were significantly up-regulated and three were down-regulated in myotubes, the up-regulated mi RNAs including mi R-1, mi R-206, mi R-128, mi R-133 a, mi R-133 b, mi R-143, mi R-145, mi R-193 b, mi R-139, mi R-378, mi R-378 b and mi R-378 c, the down-regulated mi RNAs including mi R-222, mi R-335 and mi R-376 e.4. Furthermore, using bioinformatics methods, the targets of differentially expressed mi RNAs were predicted, and were further subjected to Gene Ontology and KEGG analysis. A total of 3077 potential target genes were produced, and the highly enriched GOs and KEGG pathways showed that these genes together formed a regulatory network that involved in cell proliferation, cell differentiation, and multiple biological molecular signaling processes. Taken together, the results of the current study suggested the potential regulating roles of these differentially expressed mi RNAs in bovine myogenic differentiation.5. mi R-1 and mi R-206 contain the same “seed” sequences and they maybe regulate the same or similar target genes. Therefore, mi R-1 and mi R-206 were selected for further investigation in this study. The target genes for the mi R-1 and mi R-206 were predicted and Pax7 and HDAC4 were selected for further study. mi R-1 and mi R-206 were shown to target to the 3’ UTR of the HDAC4 and Pax7 in bovine skeletal muscle satellite cells by dual-luciferase assays and target genes regulation detection, which including over-expression or down-regulation of mi R-1 and mi R-206. The results of RT-PCR and western blotting detection demonstrated that mi R-1 and mi R-206 post-transcriptionally inhibited Pax7 and HDAC4 translation. Furthermore, we also found that mi R-1 and mi R-206 were involved in the regulation of the cell cycle progression, the over-expression of mi R-1 and mi R-206 suppress the proliferative potential of bovine satellite cells and promote their myogenic differentiation, however, when mi R-1 and mi R-206 were inhibited, bovine skeletal muscle proliferation was improved. In our work, we identified mi R-1 and mi R-206 as novel mi RNAs that played active role in bovine skeletal muscle development.6. Since Mi R-1 and mi R-206 are muscle-specific mi RNAs, we want to know whether the other selected bovine myogenic related mi RNAs can regulate the bovine muscle development process. According to our microarray analysis results and preliminary research works, mi R-139 was selected for further investigation. The target genes of mi R-139 were predicted mainly focused on TGFβ, Wnt, Rho and MAPK/PI3 K pathways. N-ras, TGFβ3 and IGF1 R were then selected as potential targets of mi R-139 for further analysis. Our results showed that the mi R-139 direct target N-ras and IGF1 R. Over-expression of mi R-139 inhibited the proliferation and myogenic differentiation of satellite cells, while down-regulation of mi R-139 promoted the myogenic differentiation process, however, the proliferation of satellite cells was still inhibited. In this study, we think the proliferation regulation ability of mi R-139 is mainly through MAPK signaling pathway, and the myogenic differentiation regulation ability of mi R-139 is mainly through PI3K/Akt signaling pathway. IGF1 R and N-ras expression level decreased when mi R-139 over-expressed, and followed by p-Akt and p-ERK1/2 down regulation. The down regulation of p-ERK1/2 inhibited the cell proliferation, while the down regulation of p-Akt inhibited the myogenic differentiation process. The inhibition of IGF1 R and N-ras expression were released when mi R-139 were inhibited, and followed by p-Akt and p-ERK1/2 up regulation. The increase of p-Akt induced Myo D expression, and thus promoted the myogenic differentiation process. The increase of p-ERK1/2 does not promote the cell proliferation, probably because the high Myo D expression level is more effective in cell cycle regulation. Our results revealed that the mi R-139 could mediate the bovine skeletal muscle satellite cells proliferation and myogenic differentiation through MAPK and PI3K/Akt signaling pathways. Moreover, we found mi R-139 could improve the muscle fiber diameter of denervated gastrocnemius. Such information would be helpful for understanding the mechanisms of muscle development, muscle damage repair and regeneration, and helpful for beef cattle breeding. |
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