Genetic variant of the sheep E2F8 gene and its associations with litter size.

The economic efficiency of sheep breeding, aiming to enhance productivity, is a focal point for improvement of sheep breeding. Recent studies highlight the involvement of the Early Region 2 Binding Factor transcription factor 8 (E2F8) gene in female reproduction. Our group's recent genome-wide association study (GWAS) emphasizes the potential impact of the E2F8 gene on prolificacy traits in Australian White sheep (AUW). Herein, the purpose of this study was to assess the correlation of the E2F8 gene with litter size in AUW sheep breed. This work encompassed 659 AUW sheep, subject to genotyping through PCR-based genotyping technology. Furthermore, the results of PCR-based genotyping showed significant associations between the P1-del-32bp bp InDel and the fourth and fifth parities litter size in AUW sheep; the litter size of those with genotype ID were superior compared to those with DD and II genotypes. Thus, these results indicate that the P1-del-32bp InDel within the E2F8 gene can be useful in marker-assisted selection (MAS) in sheep.

Regulation of myogenic cell proliferation and differentiation during mammalian skeletal myogenesis.

Mammalian skeletal myogenesis is a complex process that allows precise control of myogenic cells' proliferation, differentiation, and fusion to form multinucleated, contractile, and functional muscle fibers. Typically, myogenic progenitors continue growth and division until acquiring a differentiated state, which then permanently leaves the cell cycle and enters terminal differentiation. These processes have been intensively studied using the skeletal muscle developing models in vitro and in vivo, uncovering a complex cellular intrinsic network during mammalian skeletal myogenesis containing transcription factors, translation factors, extracellular matrix, metabolites, and mechano-sensors. Examining the events and how they are knitted together will better understand skeletal myogenesis's molecular basis. This review describes various regulatory mechanisms and recent advances in myogenic cell proliferation and differentiation during mammalian skeletal myogenesis. We focus on significant cell cycle regulators, myogenic factors, and chromatin regulators impacting the coordination of the cell proliferation versus differentiation decision, which will better clarify the complex signaling underlying skeletal myogenesis.

CircRNA Profiling Reveals CircPPARγ Modulates Adipogenic Differentiation via Sponging miR-92a-3p.

Adipogenesis describes the proliferation, differentiation, and apoptosis of mature adipocytes from primary adipocytes and is regulated by post-transcriptional modifications. Circular RNAs (circRNAs) play critical roles in mammalian development and physiology. However, the circRNA-mediated regulation of adipogenesis remains poorly understood. We profiled circRNA expression during bovine primary adipogenesis, detecting 16 circRNA candidates, including circPPARγ, which was abundant in the adipose tissue. Overexpression (overexpression plasmids) and interference (small interfering RNAs) with circPPARγ in bovine primary adipocytes, and proliferation, differentiation, and apoptosis were analyzed using EdU (5-ethynyl-2'-deoxyuridine) cell proliferation, cell counting kit-8, flow cytometry, TdT-mediated dUTP nick-end labeling apoptosis assay, Oil Red O staining, quantitative real-time PCR, and western blotting assays, which showed that circPPARγ facilitates adipocyte differentiation and inhibits proliferation and apoptosis. Dual-luciferase reporter assay and RNA immunoprecipitation assays indicated that circPPARγ binds miR-92a-3p and YinYang 1 (YY1). A novel regulatory pathway regulating adipogenesis and adipose deposition was revealed.

circSVIL regulates bovine myoblast development by inhibiting STAT1 phosphorylation.

Circular RNAs (circRNAs), a novel class of non-coding RNAs with a loop structure, have recently been shown to participate in various pathophysiological processes. However, the precise role of circRNAs in myoblasts remains unclear. In this report, circSVIL was screened and identified from our previous sequencing analysis; we then performed gain- and loss-of-function experiments on bovine myoblasts by CCK8, EdU, flow cytometry, qRT-PCR, and Western blotting. The results indicate that circSVIL facilitates bovine myoblast proliferation and inhibits cell apoptosis. Using mechanism assays such as bioinformatics prediction, RNA immunoprecipitation (RIP), and cytoplasmic separation, we demonstrate that circSVIL could interact with STAT1 and inhibit STAT1 phosphorylation, thereby restraining STAT1's nuclear translocation and affecting its downstream signal cascade. Our results may elucidate a new regulatory pathway for bovine skeletal muscle development.

Characterization and Transcriptome Analysis of Exosomal and Nonexosomal RNAs in Bovine Adipocytes.

Exosomes are endosome-derived extracellular vesicles that allow intercellular communication. However, the biological significance of adipocyte exosomal RNAs remains unclear. To determine the role of RNAs from bovine adipocytes and exosomes in bovine adipogenesis, exosomal and nonexosomal RNAs were extracted from three bovine primary white adipocyte samples and then profiles were generated using DNBSEQ/BGISEQ-500 technology. The RNAome of adipocytes consisted of 12,082 mRNAs, 8589 lncRNAs, and 378 miRNAs for a higher complexity that that detected in exosomes, with 1083 mRNAs, 105 lncRNAs, and 48 miRNAs. Exosomal miRNA-mRNA and lncRNA-miRNA-mRNA networks were constructed and enrichment analysis was performed to predict functional roles and regulatory mechanisms. Our study provides the first characterization of RNAs from bovine adipocyte and exosomes. The findings reveal that some RNAs are specifically packaged in adipocyte-derived exosomes, potentially enabling crosstalk between adipocytes and/or other cells that is mediated by exosomes. Our results greatly expand our understanding of exosomal RNAs from bovine adipocytes, and provide a reference for future functional investigations of adipocyte exosomal RNAs under normal physiological conditions.

Exosome biogenesis, secretion and function of exosomal miRNAs in skeletal muscle myogenesis.

Exosomes are membrane-bound extracellular vesicles that are produced in the endosomal compartment of most mammalian cell types and then released. Exosomes are effective carriers for the intercellular material transfer of material that can influence a series of physiological and pathological processes in recipient cells. Among loaded cargoes, non-coding RNAs (ncRNAs) vary for the exosome-producing cell and its homeostatic state, and characterization of the biogenesis and secretion of exosomal ncRNAs and the functions of these ncRNAs in skeletal muscle myogenesis remain preliminary. In this review, we will describe what is currently known of exosome biogenesis, release and uptake of exosomal ncRNAs, as well as the varied functions of exosomal miRNAs in skeletal muscle myogenesis.

The Circular RNA circHUWE1 Sponges the miR-29b-AKT3 Axis to Regulate Myoblast Development.

Myogenesis is controlled by a well-established transcriptional hierarchy that coordinates the activities of a set of muscle genes. Recently, roles in myogenesis have been described for non-coding RNAs, including a role of circular RNA (circRNA) to regulate muscle gene expression. However, the functions of circRNA and the underlying mechanism by which circRNAs affect myogenesis remain poorly understood. In this study, we analyzed circRNA high-throughput sequencing results of bovine skeletal muscle samples and constructed a circRNA-miRNA-mRNA network according to the competitive endogenous RNA (ceRNA) theory. The putative circHUWE1-miR-29b-AKT3 network was analyzed and its involvement in myogenesis was confirmed through a series of assays. To assess the potential function of this regulation, bovine myoblasts were infected with overexpression plasmids and small interfering RNAs (siRNAs) that target circHUWE1. Next, cell proliferation, apoptosis, and differentiation were analyzed using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, western blotting, and qRT-PCR assays. The results suggest that circHUWE1 facilitates bovine myoblast proliferation and inhibits cell apoptosis and differentiation. Next, bioinformatics, dual-luciferase reporter assay, and AGO2 RNA immunoprecipitation (RIP) approaches were used to verify the interaction between circHUWE1, miR-29b, and AKT3. Subsequently, we identified that circHUWE1 could directly interfere with the ability of miR-29b to relieve AKT3 suppression, which ultimately activates the AKT signaling pathway. These findings suggested a new regulatory pathway for bovine skeletal muscle development, and they also expand our understanding of circRNA functions in mammals.

Polymorphism in PLIN2 gene and its association with growth traits in Chinese native cattle.

Perilipin 2 (PLIN2) is a cytosolic protein that regulates intracellular lipid storage and mobilization. However, research reports of the relationship between PLIN2 gene and growth traits in cattle are rare. Here, five novel single nucleotide polymorphisms (SNPs)（g.3036G > C, g.3964C > T, g.6458G > T, g.6555C > T and g.8231G > A）were identified within the bovine PLIN2 gene using DNA sequencing and PCR-SSCP methods in 820 individuals from four Chinese indigenous bovine breeds. Overall, five common haplotypes were identified based on the 5 SNPs, with the most common haplotypes (GCGCG) occurring at a frequency of 69.0%. In addition, The 5 novel SNPs were associated with growth traits at 6, 12, 18 and 24 months in Nanyang population, and significant associations were found in body weight and heart girth. These results suggest that PLIN2 possibly is a strong candidate gene marker for body weight in cattle breeding program.

Biogenesis and ceRNA role of circular RNAs in skeletal muscle myogenesis.

Circular RNAs (circRNAs) are novel endogenous non-coding RNAs that are generated by reverse-splicing of precursor mRNA derived from various genes in mammals. Despite low expression, recent studies have shown that circRNA plays an important role in skeletal muscle myogenesis with competing endogenous RNA (ceRNA) functions. However, the potential regulatory role of circRNAs and interactions with miRNAs remain largely unexplored, and the function of circRNAs as miRNA sponges is not yet generally accepted. In this review, we outline the biogenesis and ceRNA mechanisms of circRNAs as well as their involvement in skeletal muscle myogenesis and discuss the conflicting conclusions of recent circRNA-ceRNA studies.

Intron retention as an alternative splice variant of the cattle ANGPTL6 gene.

Angiopoietin-like protein 6, which is encoded by ANGPTL6 gene (also known as angiopoietin growth factor, AGF), has been extensively characterized with regard to its proposed functions as angiogenesis and energy metabolism. The present results showed the occurrence of alternative splicing by intron retention (IR) event in the bovine ANGPTL6 gene (bANGPTL6). By means of RT-PCR, TA clone and sequencing, we have shown that the bANGPTL6 gene has a splice variant generated by the retention of its partial intron 3. The computational analysis of the bANGPTL6 genomic sequence showed that its intron 3 has a high percentage of GC (62.31%) and a length of 199 nt, characteristics that have been associated with an IR event. The IR event does not interfere with the coding region as the bANGPTL6 prepropeptide is entirely coded in the third exon. Additionally, both the intronless (namely, bANGPTL6α) and intron-retaining (namely, bANGPTL6ß) ANGPTL6 transcripts are constitutively co-expressed in the bovine liver. Further, the relative expression level of different variants in liver was tested by both semi-RT-PCR and RT-qPCR methods. The results suggested bANGPTL6ß are significantly higher than bANGPTL6α. Overall, our findings will be helpful for studies on the molecular mechanism of IR events and the functions of ANGPTL6 gene. Specially, bANGPTL6ß gene probably contributes to a new target for treatment of obesity and obesity-related diseases.

Characterization of lncRNA-miRNA-mRNA Network to Reveal Potential Functional ceRNAs in Bovine Skeletal Muscle.

There is growing evidence that non-coding RNAs are emerging as critical regulators of skeletal muscle development. In order to reveal their functional roles and regulatory mechanisms, we constructed a lncRNA-miRNA-mRNA network according to the ceRNA (competitive endogenous RNA) theory, using our high-throughput sequencing data. Subsequently, the network analysis, GO (Gene Ontology) analysis, and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis were performed for functional annotation and exploration of lncRNA ceRNAs. The results uncovered a scale-free characteristics network which exhibited high functional specificity for bovine skeletal muscle development: co-expression lncRNAs were significantly enriched in muscle development related biological processes and the Wnt signaling pathway. Furthermore, GSEA (Gene Set Enrichment Analysis) indicated that the risk score has a tendency to associate with myogenesis, and differentially expressed RNAs were validated by qPCR, further confirming the credibility of our network. In summary, this study provides insights into lncRNA-mediated ceRNA function and mechanisms in bovine skeletal muscle development and will expand our understanding of lncRNA biology in mammals.

Research on association between variants and haplotypes of TRPV1 and TRPA1 genes with growth traits in three cattle breeds.

The objective of this study was to examine the association of transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) genes polymorphisms with growth traits in three Chinese cattle breeds (Jiaxian red cattle, Qinchuan cattle and Luxi cattle). Through experiments we identified three single nucleotide polymorphisms (SNPs) in these three cattle breeds TRPV1 and TRPA1 genes using PCR-SSCP, (forced) PCR-RFLP methods. Three of these polymorphisms are all synonymous mutation which includes (NW_003104493.1: 30327 C > T), (NW_003104493.1: 33394 A > G) and (NW_003104493.1: 38471 G > A) are in exons. The other three polymorphisms are located at 3'UTR. Furthermore, we evaluated the haplotype frequency and the statistical analyses indicated that these SNPs of TRPV1 and TRPA1 genes were associated with bovine body height, body length, waist angle width, hucklebone width, cross ministry height, chest qingwidth (p < 0.05) and recommendation height, cannon circumference (p < 0.01) of Qingchuan cattle; body length, waist angle width (p < 0.05) of Jiaxian red cattle; body weight, Body length, cannon circumference, chest circumference (p < 0.05) and body height (p < 0.01) of Luxi cattle. Our result confirms the polymorphisms in the TRPV1 and TRPA1 genes are associated with growth traits that may be used for marker-assisted selection (MAS) in three beef breeds programs.

MiR-499 regulates myoblast proliferation and differentiation by targeting transforming growth factor ß receptor 1.

MicroRNAs (miRNAs or miRs) are small noncoding RNAs that play critical roles in muscle cell proliferation and differentiation via post-transcriptional regulation of gene expression. Here, based on our previous high-throughput sequencing results, we evaluated miRNA-499 (miR-499) functions during myoblast proliferation and differentiation. In addition, we analyzed miR-499 expression profiles and characterized the associated functional roles. MiR-499 is known to be a skeletal muscle fiber-type-associated miRNA. However, its roles in skeletal myoblast proliferation and differentiation are poorly understood. MiR-499 overexpression promoted C2C12 cell proliferation and significantly attenuated C2C12 cell myogenic differentiation. Furthermore, miR-499 inhibition enhanced C2C12 cell proliferation and suppressed C2C12 cell differentiation. Using dual-luciferase reporter assays and western blot analysis, we confirmed that miR-499 targeted transforming growth factor ß receptor 1 (TGFßR1), a known regulator of skeletal myoblast development. Additionally, our RNA interference analysis, in which TGFßR1 was downregulated, showed that TGFßR1 significantly promoted the differentiation of C2C12 cells and inhibited their proliferation.

miR-101-1 expression pattern in Qinchuan cattle and its role in the regulation of cell differentiation.

MiRNAs have emerged as key regulators of skeletal muscle development, but the knowledge of miRNAs in the molecular network of muscle development remains poorly understood. In this study, we designed to examine the biological function of bovine-miR-101-1. The bovine miR-101-1 was detected in the skeletal muscle of fetal, calf and adult cattle. Its abundance was significantly higher in the skeletal muscle of calf cattle than that in fetal and adult cattle. In the course of C2C12 myoblast differentiation, the expression of miR-101-1 gradually increased. Transfected the exogenous miR-101-1 into the C2C12 myoblast could decrease myotube formation, and the mRNA expression levels of the myogenic marker genes MyOD, MyOG and MyHC were up-regulated. The protein level of MyOD, MyOG and MyHC were also up-regulated. Through TargetScan to predict the target gene of bovine miR-101-1, and the dual luciferase system was used for target gene verification. The results show that amyloid precursor protein (APP) is the target gene of miR-101-1. Therefore, our results shed light on miR-101-1 in the regulation of the skeletal muscle development.

Combined Haplotypes of CaSR Gene Sequence Variants and Their Associations with Growth Traits in Cattle.

The calcium-sensing receptor (CaSR) is a Class C G-protein coupled receptor that regulates food intake and assimilation. However, studies on the relationship between CaSR gene and growth traits in cattle are deficient. The aim of this study was to examine the association of the CaSR polymorphism with growth traits in cattle breeds. Four novel single nucleotide polymorphisms (SNPs) and one previously reported SNP (NC_007299.5: g.67630865T>C, 67638409G>C, 67660395G>C, 67661546C>G, and 67661892A>C) were identified in the bovine CaSR gene using DNA sequencing and PCR-SSCP methods in 520 individuals from three representative breeds. The three SNP P4_2, P7_1, and P7_4 in LX, QC, and JX cattle populations belonged to intermediate genetic diversity (0.25 < PIC < 0.5). In addition, we evaluated the haplotype frequency and linkage disequilibrium coefficient of five sequence variants in the three cattle breeds. LD and haplotype structure of CaSR were different between breeds. LD analysis showed that the P4_2 and P7_4 loci were in complete LD in JX cattle population (r2 = 0.99 and D' = 1). Only 11 haplotypes were listed except for those with a frequency of <0.03. Hap1 (-TGGGC-) had the highest haplotype frequencies in LX (27.30%), Hap6(-TGGCC-) had the highest haplotype frequencies in QC (21.70%) and JX (32.30%). Association analysis indicated that P2, P4_2, and P7_4 loci were all significantly associated with growth traits and combined genotype TTGCGC was highly significantly associated with Chest circumference and body weight than the other genotype in JX cattle population. The results of this study suggest that the CaSR gene possibly is a strong candidate gene that affects growth traits in the Chinese cattle breeding program.

Expression Profiles Analysis and Functional Characterization of MicroRNA-660 in Skeletal Muscle Differentiation.

MicroRNA are a series of small non-coding RNAs that have emerged as critical regulators of skeletal muscle development. Here, we concentrated on the function of miR-660 during bovine skeletal myogenesis from our previous high-throughput sequencing results, then analyzed its expression profiles and characterized related functional roles. Overexpression of miR-660 significantly attenuated myogenic differentiation of C2C12 cells, whereas miR-660 inhibition enhanced C2C12 differentiation. Dual-Luciferase Reporter Assay went for demonstrating that miR-660 directly targeted the 3'-UTR of Rho guanine nucleotide exchange factor 12 (ARHGEF-12). Furthermore, we found an inverse relationship between the expression of miR-660 and ARHGEF12 in both gain- and loss-of-function studies: overexpression of miR-660 declined the mRNA and protein expressions of ARHGEF12 in C2C12 cells differentiation; however, knockdown of miR-660 had completely opposite results. Taken together, these results offered a new perspective for miR-660 in skeletal muscle differentiation. J. Cell. Biochem. 118: 2387-2394, 2017. © 2017 Wiley Periodicals, Inc.

The absorption and excretion of fluoride and arsenic in humans.

The absorption and excretion of fluoride and arsenic were measured in a group of healthy volunteers given drinking water with naturally high concentration of fluoride (F 2.3 mg/l)(,) or high concentration of arsenic (As 0.15 mg/l), or high concentrations of both fluoride and arsenic (F 2.25 mg/l, As 0.23 mg/l and F 4.05 mg/l, As 0.58 mg/l), respectively. The results indicated that, for arsenic, the absorption rate, the proportion of urinary excretion and the biological-half-life did not show statistically significant differences between drinking water containing high arsenic alone and drinking water containing different levels of high arsenic and fluoride. Excretion and retention of arsenic were positively correlated to the total arsenic intake. Similar results were observed for fluoride. This suggests that there are different metabolic processes for arsenic and fluoride in respect to absorption and excretion; and no joint action can be attributed by these two elements.