A Novel Dnmt3a1 Transcript Inhibits Adipogenesis

DNA (cytosine-5)-methyltransferase 3a (Dnmt3a) is an enzyme that catalyzes the transfer of methyl groups to specific CpG forms in DNA. In mammals, two variant transcripts of Dnmt3a have been successfully identified. To the best of our knowledge, no Dnmt3a transcripts in an avian have been successfully identified. This study was performed to detect different transcripts of Dnmt3a in chickens and to examine whether a novel Dnmt3a transcript named Dnmt3a1 may regulate adipogenesis. In addition to cloning, sequencing, transcript detection, and expression studies, a novel Dnmt3a1 transcript overexpression and knockdown were conducted to explore the potential role of Dnmt3a1 in preadipocyte proliferation and the early stage of adipocyte differentiation. In chicken abdominal fat tissue, we detected a novel Dnmt3a1 transcript that differs from Dnmt3a by lacking 23 amino acids at the exon-1/exon-2 border. Dnmt3a1 mRNA was ubiquitously expressed in a variety of tissues or cells and highly expressed in chicken adipose tissue/cells. The expression of Dnmt3a1 was regulated under different physiological conditions including aging, fasting, and high-fat diet. In addition, overexpression of Dnmt3a1 significantly decreased preadipocyte proliferation and induced cell-cycle arrest while its inhibition increased cell proliferation and S-phase cells. Furthermore, the overexpression of Dnmt3a1 significantly upregulated the mRNA level of cell-cycle-related genes, such as CDKN1A, CDKN1B, CCNB3, CCND2, CCNG2, CDKN2B, and CDK9, or the protein level of CDKN1A, CDKN1B, and CCNG2. Conversely, the knockdown of Dnmt3a1 by siRNA had the opposite effects. Moreover, during early adipocyte differentiation, the overexpression of Dnmt3a1 significantly decreased the mRNA and the protein levels of PPAR-γ, C/EBP-α, ADIPOR1, and STAT3, and the mRNA levels of FAS, LEPR, LPL, PRKAB2, and ATGL. In contrast, their expression was significantly increased after the knockdown of Dnmt3a1. Taken together, we identified a novel transcript of Dnmt3a, and it played a potential role in adipogenesis

Gga-miR-205a Affecting Myoblast Proliferation and Differentiation by Targeting CDH11

Non-coding RNAs especially miRNAs have been found to play important roles during skeletal muscle development. Our previous RNA-Seq performed on breast muscle tissue from 7 weeks old Recessive White Rock and Xinhua Chicken and leg muscle tissue from female Xinghua Chicken at three development time points (11 embryo age, 16 embryo age, and 1 day post hatch) (accession number GSE62971 and GSE89355, respectively) showed that miR-205a and CDH11 were differentially expressed genes. In this study, we found that overexpression of CDH11 significantly facilitated Quail muscle clone (QM7) and chicken primary myoblast (CPM) proliferation and hampered CPM differentiation. MiR-205a can directly binding to the 3′UTR of CDH11 and the overexpression of miR-205a could inhibit both cell lines (QM7) and CPM proliferation, at the meantime promote the differentiation of myoblasts. The Dual-Luciferase Reporter Assay results and qRT-PCR results showed that myogenin (MyoG) could regulate the expression of miR-205a by binding to the active region of miR-205a. Altogether our data suggest that MyoG could stimulate miR-205a expression to suppress CDH11, which promotes myoblasts proliferation while represses the differentiation

Circular RNA circSVIL Promotes Myoblast Proliferation and Differentiation by Sponging miR-203 in Chicken

Circular RNAs (circRNAs), expressed abundantly and universally in various eukaryotes, are involved in growth and development of animals. Our previous study on circRNA sequencing revealed that circSVIL, an exonic circular, expressed differentially among skeletal muscle at 11 embryo age (E11), 16 embryo age (E16), and 1 day post-hatch (P1). In this study, we aim to investigate the effect of circSVIL on the development of skeletal muscle. We detected the expression level of circSVIL in embryonic leg muscle during E10 to P1. As a result, we found that circSVIL had a high expression level during late embryonic development of skeletal muscle. Through dual-luciferase assay, RNA immunoprecipitation and biotin-coupled miRNA pull down, we found chicken circSVIL could functions as miR-203 sponges and upregulated the mRNA level of c-JUN and MEF2C. In chicken, circSVIL could promote the proliferation and differentiation of myoblast, and antagonize the functions of miR-203. Altogether our data suggest that circSVIL promotes the embryonic skeletal muscle development by sequestering miR-203 in chicken

A novel transcript of MEF2D promotes myoblast differentiation and its variations associated with growth traits in chicken

Background Development of skeletal muscle is closely related to broiler production traits. The myocyte-specific enhancer binding factor (MEF) 2D gene (MEF2D) and its variant transcripts play important parts in myogenesis. Methods To identify the transcript variants of chicken MEF2D gene and their function, this study cloned chicken MEF2D gene and identified its transcript variants from different tissue samples. The expression levels of different transcripts of MEF2D gene in different tissues and different periods were measured, and their effects on myoblast proliferation and differentiation were investigated. Variations in MEF2D were identified and association analysis with chicken production traits carried out. Results Four novel transcript variants of MEF2D were obtained, all of which contained highly conserved sequences, including MADS-Box and MEF2-Domain functional regions. Transcript MEF2D-V4 was expressed specifically in muscle, and its expression was increased during embryonic muscle development. The MEF2D-V4 could promote differentiation of chicken myoblasts and its expression was regulated by RBFOX2. The single nucleotide polymorphism g.36186C > T generated a TAG stop codon, caused MEF2D-V4 to terminate translation early, and was associated with several growth traits, especially on early body weight. Conclusion We cloned the muscle-specific transcript of MEF2D and preliminarily revealed its role in embryonic muscle development

A Novel Circular RNA Generated by FGFR2 Gene Promotes Myoblast Proliferation and Differentiation by Sponging miR-133a-5p and miR-29b-1-5p

It is well known that fibroblast growth factor receptor 2 (FGFR2) interacts with its ligand of fibroblast growth factor (FGF) therefore exerting biological functions on cell proliferation and differentiation. In this study, we first reported that the FGFR2 gene could generate a circular RNA of circFGFR2, which regulates skeletal muscle development by sponging miRNA. In our previous study of circular RNA sequencing, we found that circFGFR2, generated by exon 3–6 of FGFR2 gene, differentially expressed during chicken embryo skeletal muscle development. The purpose of this study was to reveal the real mechanism of how circFGFR2 affects skeletal muscle development in chicken. In this study, cell proliferation was analyzed by both flow cytometry analysis of the cell cycle and 5-ethynyl-2′-deoxyuridine (EdU) assays. Cell differentiation was determined by analysis of the expression of the differentiation marker gene and Myosin heavy chain (MyHC) immunofluorescence. The results of flow cytometry analysis of the cell cycle and EdU assays showed that, overexpression of circFGFR2 accelerated the proliferation of myoblast and QM-7 cells, whereas knockdown of circFGFR2 with siRNA reduced the proliferation of both cells. Meanwhile, overexpression of circFGFR2 accelerated the expression of myogenic differentiation 1 (MYOD), myogenin (MYOG) and the formation of myotubes, and knockdown of circFGFR2 showed contrary effects in myoblasts. Results of luciferase reporter assay and biotin-coupled miRNA pull down assay further showed that circFGFR2 could directly target two binding sites of miR-133a-5p and one binding site of miR-29b-1-5p, and further inhibited the expression and activity of these two miRNAs. In addition, we demonstrated that both miR-133a-5p and miR-29b-1-5p inhibited myoblast proliferation and differentiation, while circFGFR2 could eliminate the inhibition effects of the two miRNAs as indicated by rescue experiments. Altogether, our data revealed that a novel circular RNA of circFGFR2 could promote skeletal muscle proliferation and differentiation by sponging miR-133a-5p and miR-29b-1-5p

Associations of IGF2 and DRD2 polymorphisms with laying traits in Muscovy duck

Insulin-like growth factor 2 (IGF2) and dopamine receptor 2 (DRD2) play important roles in ovarian follicular development. In this study, we analyzed tissue-specific expression of the Muscovy duck IGF2 and DRD2 genes and cloned those genes transcripts. Polymorphisms in these genes were tightly linked with egg production traits and both genes were highly expressed in the ovary. Moreover, we identified five single nucleotide polymorphisms (SNPs) for IGF1 and 28 for DRD2. Mutations A-1864G and C-1704G of IGF2 were positively correlated with increased egg laying at 59 weeks (E59W) (P < 0.05). The C+7T and C+364G mutations of DRD2 were highly and significantly associated with first-egg age (FEA) and egg numbers at 300 days (E300D) (P < 0.01). Moreover, C+3301G and C+3545G of DRD2 were highly significantly associated with FEA, E59W and E300D (P < 0.01). Other mutations were positively associated with FEA or E300D or E59W (P < 0.05). These data suggest specific roles for IGF1 and DRD2 polymorphisms in egg production in Muscovy ducks

Deep Sequencing Analysis of miRNA Expression in Breast Muscle of Fast-Growing and Slow-Growing Broilers

Growth performance is an important economic trait in chicken. MicroRNAs (miRNAs) have been shown to play important roles in various biological processes, but their functions in chicken growth are not yet clear. To investigate the function of miRNAs in chicken growth, breast muscle tissues of the two-tail samples (highest and lowest body weight) from Recessive White Rock (WRR) and Xinghua Chickens (XH) were performed on high throughput small RNA deep sequencing. In this study, a total of 921 miRNAs were identified, including 733 known mature miRNAs and 188 novel miRNAs. There were 200, 279, 257 and 297 differentially expressed miRNAs in the comparisons of WRRh vs. WRRl, WRRh vs. XHh, WRRl vs. XHl, and XHh vs. XHl group, respectively. A total of 22 highly differentially expressed miRNAs (fold change &gt; 2 or &lt; 0.5; p-value &lt; 0.05; q-value &lt; 0.01), which also have abundant expression (read counts &gt; 1000) were found in our comparisons. As far as two analyses (WRRh vs. WRRl, and XHh vs. XHl) are concerned, we found 80 common differentially expressed miRNAs, while 110 miRNAs were found in WRRh vs. XHh and WRRl vs. XHl. Furthermore, 26 common miRNAs were identified among all four comparisons. Four differentially expressed miRNAs (miR-223, miR-16, miR-205a and miR-222b-5p) were validated by quantitative real-time RT-PCR (qRT-PCR). Regulatory networks of interactions among miRNAs and their targets were constructed using integrative miRNA target-prediction and network-analysis. Growth hormone receptor (GHR) was confirmed as a target of miR-146b-3p by dual-luciferase assay and qPCR, indicating that miR-34c, miR-223, miR-146b-3p, miR-21 and miR-205a are key growth-related target genes in the network. These miRNAs are proposed as candidate miRNAs for future studies concerning miRNA-target function on regulation of chicken growth

Identification, expression and variation of the GNPDA2 gene, and its association with body weight and fatness traits in chicken

Background. The GNPDA2 (glucosamine-6-phosphate deaminase 2) gene is a member of Glucosamine-6-phosphate (GlcN6P) deaminase subfamily, which encoded an allosteric enzyme of GlcN6P. Genome-wide association studies (GWAS) have shown that variations of human GNPDA2 are associated with body mass index and obesity risk, but its function and metabolic implications remain to be elucidated.The object of this study was to characterize the gene structure, expression, and biological functions of GNPDA2 in chickens. Methods. Variant transcripts of chicken GNPDA2 and their expression were investigated using rapid amplification of cDNA ends (RACE) system and real-time quantitative PCR technology. We detected the GNPDA2 expression in hypothalamic, adipose, and liver tissue of Xinghua chickens with fasting and high-glucose-fat diet treatments, and performed association analysis of variations of GNPDA2 with productive traits in chicken. The function of GNPDA2 was further studied by overexpression and small interfering RNA (siRNA) methods in chicken preadipocytes. Results.Four chicken GNPDA2 transcripts (cGNPDA2-a∼cGNPDA2-d) were identified in this study. The complete transcript GNPDA2-a was predominantly expressed in adipose tissue (subcutaneous fat and abdominal fat), hypothalamus, and duodenum. In fasting chickens, the mRNA level of GNPDA2 was decreased by 58.8% (P < 0.05) in hypothalamus, and returned to normal level after refeeding. Chicken fed a high-glucose-fat diet increased GNPDA2 gene expression about 2-fold higher in adipose tissue (P < 0.05) than that in the control (fed a basal diet), but decreased its expression in hypothalamus. Two single-nucleotide polymorphisms of the GNPDA2 gene were significantly associated with body weight and a number of fatness traits in chicken (P < 0.05). Conclusion. Our findings indicated that the GNPDA2 gene has a potential role in the regulation of body weight, fat and energy metabolism in chickens