MiR-460b-5p promotes proliferation and differentiation of chicken myoblasts and targets RBM19 gene
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ABSTRACT:
SUBMITTER: Zhou K
PROVIDER: S-EPMC10685028 | biostudies-literature | 2023 Oct
REPOSITORIES: biostudies-literature
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Project description:The proliferation and differentiation of myoblasts is an important process of skeletal muscle development. In this process, microRNAs (miRNAs) play an important role in the proliferation and differentiation of chicken primary myoblasts (CPMs). Our previous study found that miR-214 and the tRNA methyltransferase 61A (TRMT61A) gene were differentially expressed in different stages of proliferation and differentiation. Therefore, this study aimed to explore the effect of miR-214 on the proliferation and differentiation of CPMs and the functional relationship between miR-214 and TRMT61A. In this study, we detected the effect of miR-214 on the proliferation of CPMs by qPCR, flow cytometry, CCK-8, and EdU after the overexpression and interference of miR-214. qPCR, Western blotting, and indirect immunofluorescence were used to detect the effect of miR-214 on the differentiation of the CPMs. The expression patterns of miR-214 and TRMT61A were observed at different time points of differentiation induced by the CPMs. The results show that miR-214 inhibited the proliferation of the CPMs and promoted the differentiation of the CPMs. The Dual-Luciferase Reporter assay and the expression pattern of miR-214 and TRMT61A suggested that they had a negative regulatory target relationship. This study revealed the function and regulatory mechanism of miR-214 in the proliferation and differentiation of CPMs.
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Project description:Several recent studies investigated the role of the miR-29 family in muscle development. However, only a few studies focused on chicken skeletal muscle. In the present study, cell cycle, 5-ethynyl-2'-deoxyuridine (EdU), cell counting kit-8 (CCK-8), and other assays indicated that miR-29b-1-5p can inhibit the proliferation of chicken primary myoblasts (CPMs); the western blot assay and immunofluorescence detection of MYHC demonstrated that miR-29b-1-5p can promote the differentiation of myoblasts. The functional enrichment analysis revealed that the target genes of miR-29b-1-5p may be involved in muscle tissue development, muscle organ development, and striated muscle tissue development, which are biological processes related to muscle development. The correlation analysis showed that these 6 genes, that is, ankyrin repeat domain 9 (ANKRD9), lactate dehydrogenase A (LDHA), transcription factor 12 (TCF12), FAT atypical cadherin 1 (FAT1), lin-9 homolog (LIN9), and integrin beta 3 binding protein (ITGB3BP), can be used as effective candidate target genes of miR-29b-1-5p. Moreover, miR-29b-1-5p inhibits the expression of ANKRD9 by directly binding the 3'UTR of ANKRD9. Overall, these data indicate that miR-29b-1-5p inhibits the proliferation of primary chicken myoblasts, stimulates their differentiation, and is involved in the process of muscle development and that its effective target gene is ANKRD9. This study identified the molecular mechanism of miR-29b-1-5p in chicken muscle development.
| S-EPMC8639469 | biostudies-literature
Project description:Proliferation, differentiation, and apoptosis are three essential stages in cell development, and miRNAs can achieve extensive regulation of cellular developmental processes by repressing the expression of target genes. According to our previous RNA-seq results, miRNA-10a-5p was differentially expressed at different periods in chicken myoblasts, revealing a possible association with muscle development. In this study, we concluded that miRNA-10a-5p inhibited chicken myoblasts' proliferation and differentiation and promoted chicken myoblasts' apoptosis by directly targeting BCL6, a critical transcription factor involved in muscle development and regeneration. Overexpression of BCL6 significantly facilitated myoblasts' proliferation and differentiation and suppressed myoblasts' apoptosis. On the contrary, knockdown of BCL6 significantly repressed myoblasts' proliferation and differentiation and induced myoblasts' apoptosis. The results above suggest that miRNA-10a-5p plays a potential role in skeletal muscle growth, development and autophagy by targeting the BCL6 gene. We first revealed the functions of miRNA-10a-5p and BCL6 in the proliferation, differentiation, and apoptosis of chicken myoblasts.
| S-EPMC9455618 | biostudies-literature
Project description:The proliferation and differentiation of myoblasts are considered the key biological processes in muscle development and muscle-related diseases, in which the miRNAs involved remain incompletely understood. Previous research reported that miR-424(322)-5p is highly expressed in mouse skeletal muscle. Therefore, C2C12 cells are used as a model to clarify the effect of miR-424(322)-5p on the proliferation and differentiation of myoblasts. The data show that miR-424(322)-5p exhibits a decreasing trend upon myogenic differentiation. Overexpression of miR-424(322)-5p inhibits the proliferation of myoblasts, manifested by downregulation of proliferation marker genes ( CCNB1, CCND2, and CDK4), decreased percentage of EdU + cells, and reduced cell viability. In contrast, these phenotypes are promoted in myoblasts treated with an inhibitor of miR-424(322)-5p. Interestingly, its gain of function inhibits the expression of myogenic regulators, including MyoD, MyoG, MyHC, and Myf5. Additionally, immunofluorescence staining of MyHC and MyoD shows that overexpression of miR-424(322)-5p reduces the number of myotubes and decreases the myotube fusion index. Consistently, inhibition of its function mediated by an inhibitor promotes the expressions of myogenic markers and myotube fusion. Mechanistically, gene prediction and dual-luciferase reporter experiments confirm that enhancer of zeste homolog 1 ( Ezh1) is one of the targets of miR-424(322)-5p. Furthermore, knockdown of Ezh1 inhibits the proliferation and differentiation of myoblasts. Compared with NC and inhibitor treatment, inhibitor+si- EZH1 treatment rescues the phenotypes of proliferation and differentiation mediated by the miR-424(322)-5p inhibitor. Taken together, these data indicate that miR-424(322)-5p targets Ezh1 to negatively regulate the proliferation and differentiation of myoblasts.
| S-EPMC10160230 | biostudies-literature
Project description:The growth performance of chickens contributes to the sustainable development of the broiler industry, and skeletal muscle development is particularly crucial for chicken production. MicroRNAs (miRNAs) are noncoding RNAs that can participate in the regulation of many biological processes. MiR-34a-5p is a miRNA closely related to chicken skeletal muscle growth and development. However, the specific role of miR-34a-5p in chicken skeletal muscle development remains unknown. Characteristic analysis revealed that the structures and functions of miR-34a-5p from 14 species were similar. Transmembrane domain, hydropathicity/hydrophobicity, and signal peptide analyses of the chicken NOTCH1 protein demonstrated that NOTCH1 may participate in cellular life processes. Motif identification and gene structure analysis revealed that the NOTCH genes of H. sapiens, M. musculus, and G. gallus had similar gene structures, indicating that they may have similar biological functions. In this study, miR-34a-5p significantly inhibited chicken primary myoblast proliferation and differentiation (P < 0.05). Through miRDB and TargetScan database prediction, NOTCH1 was speculated to be one of the target genes of miR-34a-5p. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses predicted that NOTCH1 was related to skeletal muscle development. Dual-luciferase reporter, RT-qPCR, and rescue experiments confirmed the target relationship between miR-34a-5p and NOTCH1. mRNA quantitative analysis and western blot, CCK-8, and EdU findings revealed that NOTCH1 significantly promoted chicken myoblast proliferation (P < 0.05). mRNA quantitative analysis, Western blotting, and indirect immunofluorescence of myotubes revealed that NOTCH1 significantly promoted chicken myoblast differentiation (P < 0.05). In summary, miRNA-34a-5p inhibited chicken myoblast proliferation and differentiation by inhibiting NOTCH1 expression.
| S-EPMC11880695 | biostudies-literature
Project description:The proliferation and differentiation of chicken primary myoblasts (CPMs) play an important role in the development of skeletal muscle. In our previous research, RNA-seq analysis showed that microRNA-7 (miR-7) was relatively highly expressed in the proliferation phase of CPMs, but its expression level decreased significantly after CPMS-induced differentiation. Meanwhile, the mechanism by which the miR-7 regulates the proliferation and differentiation of CPMs is still unknown. In this study, we found that the expression levels of miR-7 and the Krüppel-like factor 4 (KLF4) gene were negatively correlated during the embryonic phase, and in vitro induced differentiation. A dual-luciferase assay and a rescue experiment show that there is a target relationship between miR-7 and the KLF4 gene. Meanwhile, the results show that overexpression of miR-7 inhibited the proliferation and differentiation of CPMs, while inhibition of miR-7 had the opposite effects. Furthermore, overexpression of the KLF4 gene was found to significantly promote the proliferation and differentiation of CPMs. Conversely, inhibition of the KLF4 gene was able to significantly decrease the proliferation and differentiation of CPMs. Our results demonstrate, for the first time, that miR-7 inhibits the proliferation and differentiation of myoblasts by targeting the KLF4 gene in chicken primary myoblasts.
| S-EPMC7530283 | biostudies-literature
Project description:BackgroundIn the poultry and livestock industries, precise genetic information is crucial for improving economic traits. Thus, functional genomic studies help to generate faster, healthier, and more efficient animal production. Chicken myoblast cells, which are required for muscle development and regeneration, are particularly important because chicken growth is closely related to muscle mass.ResultsIn this study, we induced expression of microRNA-146b-5p mediated by the piggyBac transposon system in primary chicken myoblast (pCM) cells. Subsequently, we analyzed and compared the proliferation and differentiation capacity and also examined the expression of related genes in regular pCM (rpCM) cells and pCM cells overexpressing miRNA-146b-5p (pCM-146b OE cells). pCM-146b OE cells showed increased proliferation and upregulated gene expression related to cell proliferation. In addition, next-generation sequencing analyses were performed to compare global gene expression patterns between rpCM cells and pCM-146b OE cells. We found that the higher proliferation in pCM-146b OE cells was the result of upregulation of gene sets related to the cell cycle. Moreover, miRNA-146b-5p overexpression had inhibitory effects on myotube differentiation in pCM cells.ConclusionsCollectively these results demonstrate that miR-146b-5p is closely related to the proliferation and differentiation of chicken myogenic cells as a modulator of post-transcription.
| S-EPMC7260857 | biostudies-literature
Project description:The quality and quantity of animal meat are closely related to the development of skeletal muscle, which, in turn, is determined by myogenic cells, including myoblasts and skeletal muscle satellite cells (SMSCs). Circular RNA, an endogenous RNA derivative formed through specific reverse splicing in mRNA precursors, has the potential to influence muscle development by binding to miRNAs or regulating gene expression involved in muscular growth at the transcriptional level. Previous high-throughput sequencing of circRNA in chicken liver tissue revealed a circular transcript, circIGF2BP3, derived from the gene encoding insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3). In this study, we confirmed the presence of the natural circular molecule of circIGF2BP3 through an RNase R enzyme tolerance assay. RT-qPCR results showed high circIGF2BP3 expression in the pectoral and thigh muscles of Yuexi frizzled feather chickens at embryonic ages 14 and 18, as well as at 7 weeks post-hatch. Notably, its expression increased during embryonic development, followed by a rapid decrease after birth. As well as using RT-qPCR, Edu, CCK-8, immunofluorescence, and Western blot techniques, we demonstrated that overexpressing circIGF2BP3 could promote the proliferation and differentiation of chicken primary myoblasts through upregulating genes such as proliferating cell nuclear antigen (PCNA), cyclin D1 (CCND1), cyclin E1 (CCNE1), cyclin dependent kinase 2 (CDK2), myosin heavy chain (MyHC), myoblast-determining 1 (MyoD1), myogenin (MyoG), and Myomaker. In conclusion, circIGF2BP3 promotes the proliferation and differentiation of myoblasts in chickens. This study establishes a foundation for further investigation into the biological functions and mechanisms of circIGF2BP3 in myoblasts proliferation and differentiation.
| S-EPMC10650573 | biostudies-literature
Project description:As key post-transcriptional regulators, microRNAs (miRNAs) play an indispensable role in skeletal muscle development. Our previous study suggested that miR-34b-5p and IGFBP2 could have a potential role in skeletal muscle growth. Our goal in this study is to explore the function and regulatory mechanism of miR-34b-5p and IGFBP2 in myogenesis. In this study, the dual-luciferase reporter assay and Western blot analysis showed that IGFBP2 is a direct target of miR-34b-5p. Flow cytometric analysis and EdU assay showed that miR-34b-5p could repress the cell cycle progression of myoblasts, and miR-34b-5p could promote the formation of myotubes by promoting the expression of MyHC. On the contrary, the overexpression of IGFBP2 significantly facilitated the proliferation of myoblasts and hampered the formation of myotubes. Together, our results indicate that miR-34b-5p could mediate the proliferation and differentiation of myoblasts by targeting IGFBP2.
| S-EPMC6523632 | biostudies-literature
Project description:During myogenesis and regeneration, the proliferation and differentiation of myoblasts play key regulatory roles and may be regulated by many genes. In this study, we analyzed the transcriptomic data of chicken primary myoblasts at different periods of proliferation and differentiation with protein‒protein interaction network, and the results indicated that there was an interaction between cyclin-dependent kinase 1 (CDK1) and ribonucleotide reductase regulatory subunit M2 (RRM2). Previous studies in mammals have a role for RRM2 in skeletal muscle development as well as cell growth, but the role of RRM2 in chicken is unclear. In this study, we investigated the effects of RRM2 on skeletal muscle development and regeneration in chickens in vitro and in vivo. The interaction between RRM2 and CDK1 was initially identified by co-immunoprecipitation and mass spectrometry. Through a dual luciferase reporter assay and quantitative real-time PCR, we identified the core promoter region of RRM2, which is regulated by the SP1 transcription factor. In this study, through cell counting kit-8 assays, 5-ethynyl-2'-deoxyuridine incorporation assays, flow cytometry, immunofluorescence staining, and Western blot analysis, we demonstrated that RRM2 promoted the proliferation and inhibited the differentiation of myoblasts. In vivo studies showed that RRM2 reduced the diameter of muscle fibers and slowed skeletal muscle regeneration. In conclusion, these data provide preliminary insights into the biological functions of RRM2 in chicken muscle development and skeletal muscle regeneration.
| S-EPMC10825555 | biostudies-literature