Circular RNA circMYL1 Inhibit Proliferation and Promote Differentiation of Myoblasts by Sponging miR-2400.

Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs (ncRNAs) involved in regulating skeletal muscle development by sponging miRNAs. In this study, we found that the circMYL1 expression was down-regulated during myoblast proliferation, while gradually up-regulated in myoblast differentiation. The potential role of circMYL1 was identified in the proliferation of bovine myoblast through mRNA and protein expression of proliferation marker genes (PCNA, CyclinD1, and CDK2), cell counting kit-8 assay, flow cytometry analysis, and 5-ethynyl 2'-deoxyuridine (EdU) assay. Analysis of the expression of differentiation marker genes (MyoD, MyoG, and MYH2) and immunofluorescence of Myosin heavy chain (MyHC) was used to assess cell differentiation. The proliferation analysis revealed that circMYL1 inhibited the proliferation of bovine primary myoblast. Furthermore, the differentiation analysis demonstrated that circMYL1 promoted the differentiation of bovine primary myoblast. The luciferase screening and RNA immunoprecipitation (RIP) assays found that circMYL1 could have interaction with miR-2400. Additionally, we demonstrated that miR-2400 promoted proliferation and inhibited differentiation of bovine primary myoblast, while circMYL1 may eliminate the effects of miR-2400, as showed by rescue experiments. Together, our results revealed that a novel circular RNA of circMYL1 could inhibit proliferation and promote differentiation of myoblast by sponging miR-2400.

A novel 23 bp indel mutation in PRL gene is associated with growth traits in Luxi Blackhead sheep.

Prolactin is a highly versatile pituitary hormone with multiple biological functions. PRL expression is regulated by POU1F1 and the prophet of POU1F1 (PROP1). The aim of this study was to investigate the indel variations in ovine PRL and the directly related (PROP1 and POU1F1) genes, and their associations with growth traits in Luxi Blackhead (LXBH) sheep. A monomorphism in PROP1 and POU1F1 genes, and one novel 23-bp insertion mutation in the PRL gene, were identified in LXBH sheep. The 23 bp insertion mutation within PRL gene was significantly associated with several body measurements (e.g., body weight, body height) in sheep of different ages (p < 0.05). Ram lambs (p = 0.036) of genotype insertion/insertion (II) had significantly higher body weights. Weaners (p = 0.018) of genotypes insertion/insertion (II) and insertion/deletion (ID) also had significantly higher body weights compared with male sheep of deletion/deletion (DD) genotype. Moreover, among ewe lambs, individuals of genotype insertion/insertion (II) had a higher paunch girth compared to those with other genotypes (p = 0.044). These findings indicate that a 23 bp indel variant of the ovine PRL gene is correlated with body measurements in LXBH sheep. The findings have potential utility for sheep breeding programs based on marker-assisted selection.

Transcriptional regulation of the bovine FGFR1 gene facilitates myoblast proliferation under hypomethylation of the promoter.

DNA methylation, which can affect the expression level of genes, is one of the most vital epigenetic modifications in mammals. Fibroblast growth factor receptor 1 (FGFR1) plays an important role in muscle development; however, DNA methylation of the FGFR1 promoter has not been studied to date in cattle. Our study focused on methylation of the FGFR1 promoter and its effect on bovine myoblast proliferation and differentiation. We identified the FGFR1 core promoter by using luciferase reporter assays; we then studied FGFR1 expression by reverse transcription quantitative polymerase chain reaction, and the methylation pattern in the FGFR1 core promoter by bisulfite sequencing polymerase chain reaction in bovine muscle tissue at three different developmental stages. We used RNAi strategy to investigate the function of FGFR1 in myoblast proliferation and differentiation. Results showed that the FGFR1 core promoters were located at the R2 (-509 to ~-202 bp) and R4 (-1295 to ~-794 bp) regions upstream of the FGFR1 gene. FGFR1 expression level was negatively associated with the degree of methylation of the FGFR1 core promoter during the developmental process. In addition, we found that FGFR1 can promote myoblast proliferation, but had no effect on myoblast differentiation. In conclusion, our results suggest that FGFR1 can promote myoblast proliferation and its transcription can be regulated by the methylation level of the core promoter. Our findings provide a mechanistic basis for the improvement of animal breeding.

Associations of ORMDL1 gene copy number variations with growth traits in four Chinese sheep breeds.

Copy number variations (CNVs) are gains and losses of genomic sequence of more than 50 bp between two individuals of a species. Also, CNV is considered to be one of the main elements affecting the phenotypic diversity and evolutionary adaptation of animals. ORMDL sphingolipid biosynthesis regulator 1 (ORMDL1) is a protein-coding gene associated with diseases and development. In our study, the polymorphism of ORMDL1 gene copy numbers in four Chinese sheep breeds (abbreviated CK, HU, STH, and LTH) was detected. In addition, we analyzed the transcriptional expression level of ORMDL1 gene in different tissues of sheep and examined the association of ORMDL1 CNV with growth traits. The statistical analysis revealed that ORMDL1 CNV was remarkably correlated with body height, heart girth, and circumference of cannon bone in HU sheep ( P < 0.05 ), and there are significant effects on body weight, body height, body length, chest depth, and height of hip cross in STH sheep ( P < 0.05 ). In conclusion, our results provide a basis for the relationship between CNV of ORMDL1 gene and sheep growth traits, suggesting that ORMDL1 CNV may be considered a promising marker for the molecular breeding of Chinese sheep.

Circular RNA TTN Acts As a miR-432 Sponge to Facilitate Proliferation and Differentiation of Myoblasts via the IGF2/PI3K/AKT Signaling Pathway.

Circular RNAs (circRNAs) are ubiquitous endogenous RNA found in various organisms that can regulate gene expression in eukaryotes. However, little is known about potential roles for circRNAs in muscle development. We analyzed circRNA sequencing data of bovine skeletal muscle tissue and found differential expression of circTitin (circTTN) in fetal and adult bovine muscle tissue. We then further studied the role of circTTN in bovine muscle development. Overexpression and inhibition of circTTN together elicited its promoting roles in proliferation and differentiation of bovine primary myoblasts. Mechanistically, circTTN showed interaction with miR-432 by luciferase screening and RNA immunoprecipitation (RIP) assays. Additionally, miR-432 is a regulator of insulin-like growth factor 2 (IGF2), as indicated by luciferase activity, quantitative real-time PCR, and western blotting assays. Increased miR-432 expression inhibited the expression of IGF2, but this effect was remitted by circTTN. Conclusively, our results showed that circTTN promoted proliferation and differentiation of bovine primary myoblasts via competitively combining with miR-432 to activate the IGF2/phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway.

Function and Transcriptional Regulation of Bovine TORC2 Gene in Adipocytes: Roles of C/EBP, XBP1, INSM1 and ZNF263.

The TORC2 gene is a member of the transducer of the regulated cyclic adenosine monophosphate (cAMP) response element binding protein gene family, which plays a key role in metabolism and adipogenesis. In the present study, we confirmed the role of TORC2 in bovine preadipocyte proliferation through cell cycle staining flow cytometry, cell counting assay, 5-ethynyl-2'-deoxyuridine staining (EdU), and mRNA and protein expression analysis of proliferation-related marker genes. In addition, Oil red O staining analysis, immunofluorescence of adiponectin, mRNA and protein level expression of lipid related marker genes confirmed the role of TORC2 in the regulation of bovine adipocyte differentiation. Furthermore, the transcription start site and sub-cellular localization of the TORC2 gene was identified in bovine adipocytes. To investigate the underlying regulatory mechanism of the bovine TORC2, we cloned a 1990 bp of the 5' untranslated region (5'UTR) promoter region into a luciferase reporter vector and seven vector fragments were constructed through serial deletion of the 5'UTR flanking region. The core promoter region of the TORC2 gene was identified at location -314 to -69 bp upstream of the transcription start site. Based on the results of the transcriptional activities of the promoter vector fragments, luciferase activities of mutated fragments and siRNAs interference, four transcription factors (CCAAT/enhancer-binding protein C/BEP, X-box binding protein 1 XBP1, Insulinoma-associated 1 INSM1, and Zinc finger protein 263 ZNF263) were identified as the transcriptional regulators of TORC2 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. Furthermore, we also identified that C/EBP, XBP1, INSM1 and ZNF263 regulate TORC2 gene as activators in the promoter region. We can conclude that TORC2 gene is potentially a positive regulator of adipogenesis. These findings will not only provide an insight for the improvement of intramuscular fat in cattle, but will enhance our understanding regarding therapeutic intervention of metabolic syndrome and obesity in public health as well.