N6-methyladenosine (m6A) is the most prevalent methylation modification of eukaryotic mRNA, and it plays an important role in regulating gene expression. Previous studies have found that m6A methylation plays a role in mammalian skeletal muscle development. However, the effect of m6A on bovine skeletal myogenesis are still unclear. Here, we selected proliferating myoblasts (GM) and differentiated myotubes (on the 4th day of differentiation, DM) for m6A-seq and RNA-seq to explore the m6A methylation modification pattern during bovine skeletal myogenesis. m6A-seq analysis revealed that m6A methylation was an abundant modification of the mRNA in bovine myoblasts and myotubes. We scanned 5,691–8,094 m6A-modified transcripts, including 1,437 differentially methylated genes (DMGs). GO and KEGG analyses revealed that DMGs were primarily involved in transcriptional regulation and RNA metabolism, as well as insulin resistance and metabolic pathways related to muscle development. The combined analysis further identified 268 genes that had significant changes at both m6A and mRNA levels, suggesting that m6A modification may regulate myoblast differentiation by mediating the expression of these genes. Furthermore, we experimentally confirmed four genes related to myogenesis, including MYOZ2, TWIST1, KLF5 and MYOD1, with differential changes in both m6A and mRNA levels during bovine myoblast differentiation, indicating that they can be potential candidate targets for m6A regulation of skeletal myogenesis. Our results may provide new insight into molecular genetics and breeding of beef cattle, and provide a reference for investigating the mechanism of m6A regulating skeletal muscle development.
Intramuscular fat (IMF) is a quality index associated with the taste and juiciness of meat. The deposition of IMF is affected by genetic and non-genetic factors, such as age, slaughter location, gender of the animal, and diet. Micro-ribonucleic acids (miRNA) are transcriptional regulators involved in adipogenesis, but the specific role of miR-376a in regulation of bovine adipocytes remains unknown. Our findings indicated that miR-376a was a potential negative regulator of bovine adipocyte differentiation. A bta-miR-376a mimic inhibited mRNA and protein expression of the marker genes, CDK1, CDK2, PCNA, C/EBPα, FAS, and PPAR γ, and significantly reduced ratios (%) of S-phase cells, the number of cells stained with 5-ethynyl-2′-deoxyuridine, and adipocyte proliferation. Oil red O staining and triglyceride content analysis also confirmed that bta-miR-376a was involved in adipocyte differentiation. Luciferase activities confirmed that Krüppel-like transcription factor 15 (KLF15) was a direct target gene of bta-miR-376a, and that KLF15 was a key transcription factor in adipogenesis. Therefore, bta-miR-376a might be a target for increasing beef IMF.
Background Intramuscular preadipocyte differentiation plays a critical role in bovine intramuscular fat (IMF) deposition. However, the roles of different RNAs, including mRNAs, circRNAs, lncRNAs and miRNAs, in regulating the adipogenic differentiation of intramuscular preadipocytes remain largely unclear. Results In the present study, a whole transcriptome sequencing and analysis, including the analysis of mRNAs, circRNAs, lncRNAs and miRNAs, during different differentiation stages (0, 3, 6, and 9 d) of intramuscular preadipocytes from Qinchuan cattle was performed. All samples were prepared with 3 biological replicates. Here, a total of 27,153 mRNAs, 14,070 circRNAs, 7035 lncRNAs, and 427 miRNAs were annotated. Among them, we identified 4848 differentially expressed mRNAs (DEMs), 181 DE circRNAs (DECs), 501 DE lncRNAs (DELs) and 77 DE miRNAs (DEmiRs) between 0 d and other differentiation days (3, 6, and 9 d). GO and KEGG functional enrichment analyses showed that these differentially expressed genes were mainly enriched in cell differentiation, fat metabolism and adipogenesis-related pathways. Furthermore, weighted gene coexpression network analysis (WGCNA) and co-expression network analysis screened out multiple important mRNAs, circRNAs and lncRNAs related to intramuscular adipogenesis. Based on the competing endogenous RNA (ceRNA) regulatory mechanism, we finally identified 24 potential ceRNA networks and 31 potential key genes, including FOXO1/miR-330/circRNA2018/MSTRG.20301, GPAM/miR-27b/ciRNA489 and SESN3/miR-433/circRNA2627MSTRG.20342. Conclusions This study provides new insights into the differential expression patterns of different transcript types (i.e., mRNAs, circRNAs, lncRNAs and miRNAs) in intramuscular preadipocyte differentiation. Our findings provide data support for studying the molecular mechanism of key mRNAs and noncoding RNAs in IMF deposition, and provide new candidate markers for the molecular breeding of beef cattle.
Micro RNA (miR) are recognized for their important roles in biological processes, particularly in regulatory componentization. Among the miR, miR-150 has been the focus of intense scrutiny, mostly due to its role in malignant tumors. A comparison between steer and bull adipose tissues identified bta-miR-150 as one of the nine downregulated miRNAs, although its function remains unknown (GEO:GSE75063). The present study aimed to further characterize the role of bta-miR-150 in cattle. bta-miR-150 has a negative regulatory effect on the differentiation of bovine adipocytes and promotes proliferation. Overexpression of bta-miR-150 can promote mRNA and protein expression of the marker genes CDK1, CDK2, and PCNA, increase the number of EdU-stained cells, promote adipocyte proliferation, inhibit adipocyte differentiation, and reduce lipid droplet formation. Results of RNA-seq and WGCNA analyses showed that the mammalian target of the rapamycin signaling pathway, which plays a major regulatory role, is dysregulated by the overexpression and inhibition of miR-150. We found that the target gene of bta-miR-150 is AKT1 and that bta-miR-150 affects AKT1 phosphorylation levels. These results showed that bta-miR-150 plays a role in adipogenic differentiation and might therefore have applications in the beef industry.
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