Yun Ma

CircFUT10 reduces proliferation and facilitates differentiation of myoblasts by sponging miR-133a.

Circular RNAs (circRNAs) have been identified in various tissues and cell types from human, monkey, porcine, and mouse. However, knowledge on circRNAs in bovine muscle development is limited. We downloaded and analyzed the circRNAs sequencing data of bovine skeletal muscle tissue, and further characterized the role of a candidate circRNA (circFUT10) in muscle development. Quantitative real‐time PCR (qPCR) and Western blot assays were used to confirm the expression of genes involved in myoblasts differentiation and proliferation. Flow cytometry was performed to assess cell cycle distribution and cell apoptosis. EdU incorporation and CCK‐8 assay were performed to demonstrate cell proliferation. We demonstrated that circFUT10 was highly (but differentially) expressed in embryonic and adult skeletal muscle tissue. circFUT10 induced bovine primary myoblasts differentiation and increased the expression of MyoD, MyoG, and MyhC in mRNA and protein levels. circFUT10 increased the number of myoblasts in the G0/G1 phase of the cell cycle, and decreased the proportion of cells in the S‐phase. circFUT10 inhibited the proliferation of myoblasts and promoted them apoptosis. Via a luciferase screening assay, circFUT10 is observed to sponge to miR‐133a with three potential binding sites. Specifically, we show that circFUT10 regulated myoblasts differentiation and cell survival by directly binding to miR‐133a and inhibiting miR‐133a activity. Modulation of circFUT10 expression in muscle tissue may emerge as a potential target in breeding strategies attempting to control muscle development in cattle.

circFGFR4 Promotes Differentiation of Myoblasts via Binding miR-107 to Relieve Its Inhibition of Wnt3a

Muscle development is regulated under a series of complicate processes, and non-coding RNAs, such as microRNAs (miRNAs) and circular RNAs (circRNAs), have been reported to play important roles in regulating myoblast proliferation and differentiation. We found that miR-107 expression was high in skeletal muscle of Qinchuan cattle. Overexpression of miR-107 inhibited bovine myoblasts differentiation and protected cells from apoptosis. Wnt3a was identified as a target of miR-107 by luciferase activity, real-time qPCR, and western blotting assays. Knockdown of Wnt3a inhibited bovine myoblasts differentiation and apoptosis, and this effect was similar to miR-107 overexpression. We also found circFGFR4 to promote myoblasts differentiation and to induce cell apoptosis. Via luciferase screening and RNA pull-down assays, circFGFR4 was observed to sponge miR-107. Overexpression of circFGFR4 increased the expression of Wnt3a, whereas this effect was abolished by miR-107. These results demonstrated that circFGFR4 binding miR-107 promotes cell differentiation via targeting Wnt3a in bovine primary myoblasts.

Genetic variations of ANGPTL6 gene and their associations with growth traits and slaughter traits in Qinchuan cattle

Angiopoietin-like protein 6 (ANGPTL6), which plays an important role in angiogenesis and energy metabolism as a circulating orphan peptide secreted by liver, could produce a potential effect on growth and development of animals. The objective of this study was to detect genetic variations of ANGPTL6 gene in 732 Qinchuan cattle, as well as to analyze their associations with growth traits and carcass weight. Herein, three novel mutations (T2359C, C2403A and G3258T) were identified in cattle for the first time. Chi-square test showed T2359C and C2403A loci were in Hardy–Weinberg equilibrium except G3258T locus. Haplotype with TCG (wild type) was dominant with frequency of 40.0xa0% among eight different haplotypes. Statistical analysis showed body height (BH) and hucklebone width (HW) of individuals with genotype CC were significant higher than other genotypes in T2359C locus (Pxa0<xa00.05). In the C2403A locus, chest girth (CG), chest depth (CD), and chest breadth (CB) of individuals with genotype CA were all extremely significant higher (Pxa0<xa00.01) while BH was significant higher (Pxa0<xa00.05) than genotype CC. The association analysis of combined sites showed BH, CG, CB and HW of individuals with combined genotype TC–CA were significant higher than other combined genotypes (Pxa0<xa00.05) in ScaI–VspI site. BH, CG and CB of individuals with genotype CA–GG were significant higher (Pxa0<xa00.05) as well as CD was extremely significant higher (Pxa0<xa00.01) than other genotypes in VspI–RsaI site. For ScaI–VspI–RsaI site, CD and HW of individuals with genotype TC–CA–GG were significant higher (Pxa0<xa00.05) than those of other combined genotypes. Slaughter test also revealed that the effect of the combined genotypes was extremely significant on the carcass weight. Carcass weight of individuals with genotype TT–CA and TT–CA–GT were extremely significant heavier than other combined genotypes (Pxa0<xa00.01) in the ScaI–VspI and ScaI–VspI–RsaI sites. And those with genotype TT–GT of ScaI–RsaI were significantly heavy (Pxa0<xa00.05) as well as the individuals with genotype CA–GT of VspI–RsaI. In conclusion, the present results provided evidence that polymorphisms of ANGPTL6 gene were associated with growth and slaughter traits, and may apply to Qinchuan cattle breeding program as a possible candidate for marker-assisted selection (MAS).

Novel SNPs in the bovine Transmembrane protein 18 gene, their linkage and their associations with growth traits in Nanyang cattle

Transmembrane protein 18 (TMEM18) gene is expressed in the central nervous system that has recently been linked to human obesity and body mass index (BMI) in genome wide association studies (GWAS). In this study, two novel single nucleotide polymorphisms (SNPs), g.3835 G > A (aa. Gly > Ser) and g.3865 A > G were detected in cattle TMEM18 gene by DNA sequencing. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and forced PCR-RFLP methods were used to genotype the two SNPs in 1218 Chinese native cattle individuals. The association between novel SNPs and growth traits (especially on body weight and body height) in Nanyang cattle showed that individuals with GG genotype was higher than those with AA genotype at g.3835G>A locus (P < 0.05) in Nanyang cattle. Significant association also revealed that individuals of AA genotype at g. 3865 A>G locus were higher in terms of body weight, average daily gain, chest circunmference, body length (P < 0.05), body height and hucklebone width (P < 0.01) than GG genotypes in Nanyang cattle. Moreover, the effect of XspI-MluI combinations with growth traits in Nanyang cattle revealed that AAGG type had higher body weight, body length, chest circumference and average daily gain (P < 0.05) than GGAG in Nanyang cattle aged at 6 months.

Population structure and ancestry of Qinchuan cattle

The aim of this study was to estimate population structure and ancestry of Qinchuan cattle by genotyping 27 individuals using the GeneSeek HD 77k BeadChip, and another 1355 cattle representing breeds distributed worldwide, which had been genotyped using the Illumina Bovine 50k BeadChip. Qinchuan cattle were characterized by a dominant Bos taurus ancestry, accompanied by a considerable proportion of Bos indicus ancestry based on principal components analysis and supervised admixture analysis. A small proportion of Bos javanicus ancestry was detected as well. A similar admixture pattern in both Qinchuan and Turkish cattle breeds reflects their similar degrees of zebu introgression. Our study presents a relatively clear view of the population structure and ancestry of Qinchuan cattle, serving to benefit our understanding of this breed and leading to better targeted conservation approaches moving forward.

Long Non-coding RNA Profiling Reveals an Abundant MDNCR that Promotes Differentiation of Myoblasts by Sponging miR-133a

Muscle development is regulated by a series of complicate processes, and non-coding RNAs (ncRNAs) such as lncRNA have been reported to play important roles in regulating skeletal myogenesis and diseases. Here we profile the expression of lncRNA in cattle skeletal muscle tissue from fetus and adult developmental stages and detect 13,580 lncRNA candidates. Many of these lncRNAs are differentially expressed between two developmental stages. We further characterize one abundant lncRNA with the highest expression level of all downregulated lncRNAs, which we named muscle differentiation-associated lncRNA (MDNCR). Via luciferase screening, RNA binding protein immunoprecipitation (RIP), and RNA pull-down assays, MDNCR was observed to directly bind to miR-133a with 32 potential binding sites. GosB was identified as a target of miR-133a by luciferase activity, quantitative real-time qPCR, and western blotting assays. Overexpression of MDNCR increased the expression of GosB, whereas this effect was abolished by miR-133a. We found that MDNCR promotes myoblast differentiation and inhibits cell proliferation by sponging miR-133a. These results demonstrate that MDNCR binding miR-133a promotes cell differentiation by targeting GosB in cattle primary myoblasts.

MiR-208b regulates cell cycle and promotes skeletal muscle cell proliferation by targeting CDKN1A: WANG et al.

Skeletal muscle is the most abundant tissue in the body. The development of skeletal muscle cell is complex and affected by many factors. A sea of microRNAs (miRNAs) have been identified as critical regulators of myogenesis. MiR-208b, a muscle-specific miRNA, was reported to have a connection with fiber type determination. However, whether miR-208b has effect on proliferation of muscle cell was under ascertained. In our study, cyclin-dependent kinase inhibitor 1A (CDKN1A), which participates in cell cycle regulation, was predicted and then validated as one target gene of miR-208b. We found that overexpression of miR-208b increased the expression of cyclin D1, cyclin E1, and cyclin-dependent kinase 2 at the levels of messenger RNA and protein in cattle primary myoblasts in vivo and in vitro. Flow cytometry showed that forced expression of miR-208b increased the percentage of cells at the S phase and decreased the percentage of cells at the G0/G1 phase. These results indicated that miR-208b participates in the cell cycle regulation of cattle primary myoblast cells. 5-Ethynyl-20-deoxyuridine and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidexa0assays showed that overexpression of miR-208b promoted the proliferation of cattle primary myoblasts. Therefore, we conclude that miR-208b participates in the cell cycle and proliferation regulation of cattle primary skeletal muscle cell through the posttranscriptional downregulation of CDKN1A.

MiR-499 regulates myoblast proliferation and differentiation by targeting transforming growth factor β receptor 1: WU et al.

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 β receptor 1 (TGFβR1), a known regulator of skeletal myoblast development. Additionally, our RNA interference analysis, in which TGFβR1 was downregulated, showed that TGFβR1 significantly promoted the differentiation of C2C12 cells and inhibited their proliferation.

Over-expression of DEC1 inhibits myogenic differentiation by modulating MyoG activity in bovine satellite cell

Differentiated embryo chondrocyte 1 (DEC1), a member of basic‐helix‐loop‐helix transcription factor Bhlhe40, also called stimulated by retinoic acid 13, STRA13, plays an important role in the regulation of adipogenesis, tumorigenesis, peripheral circadian output, response to hypoxia, and development of metabolic syndrome. Previous studies suggested that DEC1 was involved in skeletal muscle development; however, its precise role in myoblast differentiation has not been determined. In the present study, we showed that DEC1 expressed ubiquitously in different bovine tissues and was down‐regulated in differentiated bovine satellite cells. Expression of muscle specific transcription factors (Myf5, MyoD, MyoG, and MHC) was significantly down‐regulated when DEC1 was over‐expressed by both CoCl2‐simulated hypoxia and Adenovirus‐mediated transduction in bovine satellite cells. Consistent with that, promoter analyses via luciferase reporter assay also revealed that overexpression of bovine DEC1 could inhibit MyoG promoter activity. In conclusion, overexpression of DEC1 blocked myogenesis by inhibiting MyoG promoter activity in bovine. Our results provided a new mechanism for the muscle growth, which would contribute to increase cattle meat productivity.

Integrating CNVs into meta-QTL identified GBP4 as positional candidate for adult cattle stature

Copy number variation (CNV) of DNA sequences, functionally significant but yet fully ascertained, is believed to confer considerable increments in unexplained heritability of quantitative traits. Identification of phenotype-associated CNVs (paCNVs) therefore is a pressing need in CNV studies to speed up their exploitation in cattle breeding programs. Here, we provided a new avenue to achieve this goal that is to project the published CNV data onto meta-quantitative trait loci (meta-QTL) map which connects causal genes with phenotypes. Any CNVs overlapping meta-QTL therefore will be potential paCNVs. This study reported potential paCNVs in Bos taurus autosome 3 (BTA3). Notably, overview indexes and CNVs both highlighted a narrower region (BTA3 54,500,000–55,000,000xa0bp, named BTA3_INQTL_6) within one constructed meta-QTL. Then, we ascertained guanylate-binding protein 4 (GBP4) among the nine positional candidate genes was significantly associated with adult cattle stature, including body weight (BW, Pu2009<u20090.05) and withers height (WHT, Pu2009<u20090.05), fitting GBP4 CNV either with three levels or with six levels in the model. Although higher copy number downregulated the mRNA levels of GBP2 (Pu2009<u20090.05) and GBP4 (Pu2009<u20090.05) in 1-Mb window (54.0–55.0xa0Mb) in muscle and adipose, additional analyses will be needed to clarify the causality behind the ascertained association.