ABSTRACT
Natural Nicotinamide Adenine Dinucleotide (NAD+) precursors have attracted much attention due to their positive effects in promoting ovarian health. However, their target tissue, synthesis efficiency, advantages, and disadvantages are still unclear. This review summarizes the distribution of NAD+ at the tissue, cellular and subcellular levels, discusses its biosynthetic pathways and the latest findings in ovary, include: (1) NAD+ plays distinct roles both intracellularly and extracellularly, adapting its distribution in response to requirements. (2) Different precursors differs in target tissues, synthetic efficiency, biological utilization, and adverse effects. Importantly: tryptophan is primarily utilized in the liver and kidneys, posing metabolic risks in excess; nicotinamide (NAM) is indispensable for maintaining NAD+ levels; nicotinic acid (NA) constructs a crucial bridge between intestinal microbiota and the host with diverse functions; nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) increase NAD+ systemically and can be influenced by delivery route, tissue specificity, and transport efficiency. (3) The biosynthetic pathways of NAD+ are intricately intertwined. They provide multiple sources and techniques for NAD+ synthesis, thereby reducing the dependence on a single molecule to maintain cellular NAD+ levels. However, an excess of a specific precursor potentially influencing other pathways. In addition, Protein expression analysis suggest that ovarian tissues may preferentially utilize NAM and NMN. These findings summarize the specific roles and potential of NAD+ precursors in enhancing ovarian health. Future research should delve into the molecular mechanisms and intervention strategies of different precursors, aiming to achieve personalized prevention or treatment of ovarian diseases, and reveal their clinical application value.
Subject(s)
NAD , Niacinamide , Ovary , Humans , NAD/metabolism , NAD/biosynthesis , Ovary/metabolism , Female , Animals , Niacinamide/metabolism , Niacinamide/biosynthesis , Biosynthetic Pathways , Nicotinamide Mononucleotide/metabolismABSTRACT
Understanding how skeletal muscle fiber proportions are regulated is essential for understanding muscle function and improving the quality of mutton. While circular RNA (circRNA) has a critical function in myofiber type transformation, the specific mechanisms are not yet fully understood. Prior evidence indicates that circular ubiquitin-specific peptidase 13 (circUSP13) can promote myoblast differentiation by acting as a ceRNA, but its potential role in myofiber switching is still unknown. Herein, we found that circUSP13 enhanced slow myosin heavy chain (MyHC-slow) and suppressed MyHC-fast expression in goat primary myoblasts (GPMs). Meanwhile, circUSP13 evidently enhanced the remodeling of the mitochondrial network while inhibiting the autophagy of GPMs. We obtained fast-dominated myofibers, via treatment with rotenone, and further demonstrated the positive role of circUSP13 in the fast-to-slow transition. Mechanistically, activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway significantly impaired the slow-to-fast shift in fully differentiated myotubes, which was restored by circUSP13 or IGF1 overexpression. In conclusion, circUSP13 promoted the fast-to-slow myofiber type transition through MAPK/ERK signaling in goat skeletal muscle. These findings provide novel insights into the role of circUSP13 in myofiber type transition and contribute to a better understanding of the genetic mechanisms underlying meat quality.
Subject(s)
Goats , MAP Kinase Signaling System , Muscle Fibers, Fast-Twitch , Muscle Fibers, Slow-Twitch , Myosin Heavy Chains , RNA, Circular , Animals , Autophagy/physiology , Cell Differentiation , Cells, Cultured , Extracellular Signal-Regulated MAP Kinases/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , MAP Kinase Signaling System/physiology , Muscle Development/genetics , Muscle Fibers, Fast-Twitch/metabolism , Muscle Fibers, Slow-Twitch/metabolism , Myoblasts/metabolism , Myosin Heavy Chains/metabolism , Myosin Heavy Chains/genetics , RNA, Circular/metabolismABSTRACT
RUNX1T1 (Runt-related transcription factor 1, translocated to 1) plays a wide-ranging and diverse role in cellular development, including hematopoiesis and adipogenesis. However, little is known about the function of RUNX1T1 in the skeletal muscle development. Here, we assessed the impact of RUNX1T1 on the proliferation and myogenic differentiation of goat primary myoblasts (GPMs). It was observed that RUNX1T1 is highly expressed during the early stages of myogenic differentiation and the fetal stage. Moreover, the knockdown of RUNX1T1 promotes the proliferation and inhibits myogenic differentiation and mitochondrial biogenesis of GPMs. RNA sequencing analysis revealed that significantly differentially expressed genes in RUNX1T1 knockdown cells were enriched in the calcium signaling pathway. Additionally, we discovered that RUNX1T1 regulates alternative splicing (AS) events involved in myogenesis. We also show that silencing RUNX1T1 blocked the Ca2+ -CAMK signaling pathway and reduced the expression levels of muscle-specific isoforms of recombinant rho associated coiled coil containing crotein kinase 2 (ROCK2) during myogenic differentiation, partially explaining why RUNX1T1 deficiency leads to the impairment of myotube formation. These findings suggest that RUNX1T1 is a novel regulator of myogenic differentiation that regulates the calcium signaling pathway and AS of ROCK2. Overall, our results highlight the critical role of RUNX1T1 in myogenesis and broaden our understanding of myogenic differentiation.
Subject(s)
Alternative Splicing , Calcium Signaling , Cell Differentiation/genetics , Muscle Development/genetics , Muscle Fibers, Skeletal/metabolism , Goats , AnimalsABSTRACT
MicroRNAs (miRNAs) are evolutionarily conserved endogenous small non-coding RNAs that play critical roles in skeletal muscle development. In this study, we identified putative miRNAs that were differentially expressed in the longissimus dorsi muscle between fetus (75 days of pregnancy) and lamb (1 day of age). We detected 1208 miRNAs, 313 of which were differentially expressed. In particular, we found that miR-145-5p was differentially and highly expressed in lamb skeletal muscle. In addition, our results demonstrated that overexpression of miR-145-5p inhibited the differentiation and apoptosis of goat primary myoblasts (GPMs), whereas knockdown of miR-145-5p had the opposite effect. The coding domain sequence (CDS) of ubiquitin-specific peptidase 13 (USP13) was predicted and validated as a target of miR-145-5p. We also demonstrated that the influence as a key regulator of GPMs differentiation is primarily mediated by targeting and inhibiting USP13. Taken together, these results revealed a novel pathway in skeletal muscle development in which miR-145-5p targets CDS region of USP13 to regulate differentiation and apoptosis of GPMs.
Subject(s)
Goats , MicroRNAs , Animals , Cell Line , Cell Proliferation/physiology , Goats/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Myoblasts/metabolism , Sheep , Ubiquitin-Specific Proteases/metabolismABSTRACT
Circular RNAs (circRNAs) are an indispensable element of post-transcriptional gene regulation, influencing a variety of biological processes including myogenic differentiation; however, little is known about the function of circRNA in goat myogenic differentiation. Using RNA-sequencing data from our laboratory, we explored the influences of circUSP13, as a candidate circRNA, on myoblast differentiation since its expression is higher in myoblasts of lamb (first day of age) than that of the fetus (75th day of pregnancy). In in vitro experiments, circUSP13 significantly promoted differentiation and inhibited apoptosis in goat primary myoblasts. Mechanistically, circUSP13 localized with miR-29c in the cytoplasm of goat myoblasts to regulate IGF1 expression. We further demonstrated that circUSP13 sponges miR-29c, promoting IGF1 expression that upregulated the expression of MyoG and MyHC. Thus, our results identified circUSP13 as a molecular marker for breeding programs of mutton production, as well as the circUSP13-miR-29c-IGF1 axis as a potential therapeutic target for combating muscle wasting.
Subject(s)
Apoptosis , Cell Differentiation , Insulin-Like Growth Factor I/metabolism , MicroRNAs/metabolism , Myoblasts/metabolism , RNA, Circular/metabolism , Animals , GoatsABSTRACT
The FDA Bacteriological Analytical Manual (BAM) Salmonella culture method takes at least 3 days for a presumptive positive result. The FDA developed a quantitative PCR (qPCR) method to detect Salmonella from 24-h preenriched cultures, using ABI 7500 PCR system. The qPCR method has been evaluated as a rapid screening method for a broad range of foods by single laboratory validation (SLV) studies. The present multi-laboratory validation (MLV) study was aimed to measure the reproducibility of this qPCR method and compare its performance with the culture method. Sixteen laboratories participated in two rounds of MLV study to analyze twenty-four blind-coded baby spinach test portions each. The first round yielded â¼84% and â¼82% positive rates across laboratories for the qPCR and culture methods, respectively, which were both outside the fractional range (25%-75%) required for fractionally inoculated test portions by the FDA's Microbiological Method Validation Guidelines. The second round yielded â¼68% and â¼67% positive rates. The relative level of detection (RLOD) for the second-round study was 0.969, suggesting that qPCR and culture methods had similar sensitivity (p > 0.05). The study demonstrated that the qPCR yields reproducible results and is sufficiently sensitive and specific for the detection of Salmonella in food.
Subject(s)
Food Microbiology , Spinacia oleracea , Real-Time Polymerase Chain Reaction/methods , Laboratories , Reproducibility of Results , Salmonella/geneticsABSTRACT
DNA methylation has crucial roles in regulating the expression of genes involved in skeletal muscle development. However, the DNA methylation pattern of lncRNA during sheep skeletal muscle development remains unclear. This study investigated previous WGBS and LncRNA data in skeletal muscle of sheep (fetus and adult). We then focused on LncRNA GTL2, which is differentially expressed in skeletal muscle and has multiple DMRs. We found that the expression level of GTL2 decreased with age. GTL2 DMRs methylation levels were significantly higher in adult muscle than in fetal muscle. After 5AZA treatment, GTL2 expression was significantly increased in a dose-dependent manner.The dCas9-DNMT3A-sgRNA significantly reduced the expression level of GTL2 in cells, but increased GTL2 DMR methylation levels. The above studies indicate that dCas9-DNMT3A can effectively increase the methylation level in the DMR region of GTL2, the expression level of GTL2 is regulated by DNA methylation during muscle development.
Subject(s)
DNA Methylation , RNA, Long Noncoding , Animals , Genomic Imprinting , Muscle Development/genetics , Muscle, Skeletal , RNA, Long Noncoding/genetics , Sheep/geneticsABSTRACT
N6-Methyladenosine (m6A) modification is the most abundant chemical modification in mRNA, and it participates in various biological processes, such as cell differentiation and proliferation. However, little is known about the function of m6A demethylase fat mass and obesity-associated (FTO) in myoblast proliferation. Here, we demonstrated that knockdown of FTO can significantly inhibit myoblast proliferation and promote apoptosis. RNA sequencing analysis revealed that a lot of downregulated genes in FTO knockdown cells are associated with cell cycle and apoptosis. Furthermore, silencing FTO drastically decreased cyclin D1 (CCND1) expression through YTHDF2-mediated mRNA degradation, thereby delaying the progression of G1 phase, and leading to impaired myoblast proliferation. These findings unraveled that FTO regulates myoblast proliferation by controlling CCND1 expression in an m6A-YTHDF2-dependent manner, which highlights the critical roles of m6A modification in myoblast proliferation.
Subject(s)
Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Cyclin D1/genetics , RNA Stability/genetics , RNA-Binding Proteins/genetics , Adenosine/analogs & derivatives , Adenosine/genetics , Apoptosis/genetics , Cell Cycle/genetics , Cell Differentiation/genetics , Cell Proliferation/genetics , G1 Phase/genetics , Humans , Myoblasts/metabolismABSTRACT
BACKGROUND: DNA methylation is an epigenetic regulatory form that plays an important role in regulating the gene expression and the tissues development.. However, DNA methylation regulators involved in sheep muscle development remain unclear. To explore the functional importance of genome-scale DNA methylation during sheep muscle growth, this study systematically investigated the genome-wide DNA methylation profiles at key stages of Hu sheep developmental (fetus and adult) using deep whole-genome bisulfite sequencing (WGBS). RESULTS: Our study found that the expression levels of DNA methyltransferase (DNMT)-related genes were lower in fetal muscle than in the muscle of adults. The methylation levels in the CG context were higher than those in the CHG and CHH contexts, and methylation levels were highest in introns, followed by exons and downstream regions. Subsequently, we identified 48,491, 17, and 135 differentially methylated regions (DMRs) in the CG, CHG, and CHH sequence contexts and 11,522 differentially methylated genes (DMGs). The results of bisulfite sequencing PCR (BSP) correlated well with the WGBS-Seq data. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation analysis revealed that some DMGs were involved in regulating skeletal muscle development and fatty acid metabolism. By combining the WGBS-Seq and previous RNA-Seq data, a total of 159 overlap genes were obtained between differentially expressed genes (DEGs) and DMGs (FPKM > 10 and fold change > 4). Finally, we found that 9 DMGs were likely to be involved in muscle growth and metabolism of Hu sheep. CONCLUSIONS: We systemically studied the global DNA methylation patterns of fetal and adult muscle development in Hu sheep, which provided new insights into a better understanding of the epigenetic regulation of sheep muscle development.
Subject(s)
DNA Methylation , Epigenome , Muscle Development/genetics , Muscle, Skeletal/growth & development , Animals , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , Sequence Analysis, DNA , Sheep , Sulfites/chemistryABSTRACT
The more complex 3' UTR in higher organisms may have the function of increasing post-transcriptional gene regulation. Recent RNA sequencing technologies have provided us with the possibility to capture the complete 3' UTR landscape of different species and cells. However, no systematic analysis of sheep-related 3' UTR has been performed. Here, we conducted a detailed analysis of the 3' UTR with the primary goal of identifying intact 3' UTR landscapes in the sheep muscles of the three developmental stages. Based on strand-specific RNA sequencing (ssRNA-seq) data, we found that thousands of genes in sheep muscle are continuously transcribed after the UTR of the reference genome (Oar_v4.0). More than 66% of the 3' UTR extensions exhibit similar expression trends to their upstream gene exons. These 3' UTR extensions strongly enrich thousands of conserved microRNA binding sites. The 3' UTR extension-associated RNA of PFKM (PuaRNA) was predicted to be derived from the 3' UTR of PFKM. In sheep myocytes, myotubes and various tissues, the expression pattern of PuaRNA is positively correlated with that of PFKM. Taken together, these new 3' UTR annotations greatly extend the range of mammalian post-transcriptional regulatory networks, which have a particular impact on the regulation of sheep muscle development.
Subject(s)
3' Untranslated Regions/genetics , Muscle Development/genetics , Sheep, Domestic/genetics , Transcriptome , Animals , Muscles/metabolism , Sequence Analysis, RNA/veterinary , Sheep, Domestic/growth & developmentABSTRACT
The current study aimed to examine the effects of dietary spirulina supplementation in high-energy (HE) diets on fatty acid metabolism in sheep, and preliminarily explored the potential mechanisms underlying the associated autophagy-mediated regulation of lipid metabolism. In a 2 × 3 factorial design, including six treatment combinations of two metabolisable energy diets (10 and 11 MJ/kg DM), three spirulina supplementation levels (0, 1%, and 3%) were used. Serum alanineaminotransferase (ALT) (p = 0.003) and aspartatetransaminase (AST) (p = 0.002) activities increased, whereas total PUFA content (p < 0.001) decreased in the liver of lambs fed a HE diet. With the addition of spirulina, serum ALT (p = 0.037) and AST (p = 0.014) activities decreased, whereas EPA (p = 0.004), GLA (p = 0.019), n-6 PUFA (p = 0.005), and total PUFA contents (p = 0.019) increased. Moreover, the crude protein content in the Longissimus thoracis et lumborum (LTL) increased (p = 0.013), the expression of PPARα and PPARγ was up-regulated, while ELOVL2 was down-regulated in liver and LTL (p < 0.05). Spirulina supplementation increased mRNA expression levels of autophagy-associated genes, including that of Beclin-1, AMPK, and ULK1 (p < 0.05). In conclusion, spirulina supplementation in a HE diet exerted a protective effect on the liver, increased PUFA content, and modulated expression levels of autophagy-related genes in growing lambs.
Subject(s)
Autophagy/drug effects , Diet/veterinary , Lipid Metabolism/drug effects , Liver/drug effects , Muscle, Skeletal/drug effects , Sheep, Domestic/physiology , Spirulina/chemistry , Animal Feed/analysis , Animals , Diet/classification , Dietary Supplements/analysis , Dose-Response Relationship, Drug , Liver/physiology , Male , Muscle, Skeletal/physiology , Random AllocationABSTRACT
Adipose tissue development is regulated by a serial of developmental signaling pathways. The Hippo pathway is a novel signaling cascade closely associated with adipogenesis. While most of Hippo pathway components had been verified that have a vital role in preadipocytes proliferation and differentiation, little is known about the function of Yes-associated protein 1 (YAP1) in mammalian adipose tissue development. Therefore, we investigated the role of YAP1 in ovine adipose tissue development by in vitro and in vivo experiments. We observed that the adipocyte size in subcutaneous adipose tissue increased with development. YAP1 expression increased during adipose tissue development, while decreased during the differentiation of ovine preadipocytes in vitro. YAP1 knockdown notably promoted lipid accumulation and suppressed ovine preadipocyte proliferation. In addition, we observed that YAP1 deficiency significantly upregulated peroxisome proliferator-activated receptor gamma (PPARG) and retinoid X receptor alpha (RXR alpha) expression. By contrast, overexpression of YAP1 led to the suppression of preadipocyte differentiation, lipid droplets formation, and PPARG expression. In brief, our findings demonstrated that YAP1 regulates the proliferation and differentiation of ovine preadipocyte via altering PPARG and RXR alpha expression.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adipocytes/cytology , Adipogenesis , Cell Differentiation , Cell Proliferation , PPAR gamma/metabolism , Retinoid X Receptors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adipocytes/metabolism , Animals , Cells, Cultured , PPAR gamma/genetics , Retinoid X Receptors/genetics , Sheep , Signal Transduction , Subcutaneous Fat/cytology , Subcutaneous Fat/metabolismABSTRACT
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) is a central regulator of mitochondrial biogenesis and metabolism, and its expression is closely related to embryo development. To gain insights into the possible mechanisms of PPARGC1A during early embryogenesis, the development potential, mitochondrial biogenesis, and the culture medium metabolomics of embryos were evaluated when PPARGC1A overexpressed or suppressed in rabbit zygotes. Results showed that different PPARGC1A levels in rabbit zygotes could affect blastocyst percentage, and the expressions of mitochondrial biogenesis and metabolic-related genes, as well as the glutathione and adenosine triphosphate levels during early embryo development. In addition, compared with the controls, 12 and 10 different metabolites involved in carbohydrate, amino acid, and fatty acid metabolism were screened in the 5 day's spent culture medium of PPARGC1A overexpressed and suppressed embryos by gas chromatography-mass spectrometer, respectively. Consistent with these metabolite changes, the transcriptions of genes encoding glucose transporters and fatty acid biosynthetic proteins in the embryos from different groups were regulated by PPARGC1A during rabbit embryo development. Taken together, these data provide evidence that PPARGC1A may regulate early rabbit embryo development through mitochondrial biogenesis and metabolism.
Subject(s)
Blastocyst/metabolism , Embryonic Development , Gene Expression Regulation, Developmental , Mitochondria/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/biosynthesis , Zygote/metabolism , Animals , Blastocyst/cytology , Female , Rabbits , Zygote/cytologyABSTRACT
Runt-related transcription factor 1 translocation partner 1 (RUNX1T1), a potential novel regulator of adipogenesis, exists in two splice variants: a long (RUNX1T1-L) and a short (RUNX1T1-S) isoform. However, there is no data showing the existence of RUNX1T1 in ovine subcutaneous fat at different stages of developmental and its role on ovine adipogenesis. Therefore, the objectives of this study were to evaluate the presence of RUNX1T1 in subcutaneous fat of five-day-old to 24-month-old sheep and to investigate the role of RUNX1T1 in ovine adipogenesis. In this study, we detected a 1829 bp cDNA fragment of RUNX1T1 which contains a 1815 bp coding sequence that encodes 602-amino acid and 14 bp of 5' untranslated region, respectively. The amino acid sequence of RUNX1T1 has 31.18â»94.21% homology with other species' protein sequences. During fat development, the RUNX1T1 protein expression was higher in subcutaneous fat of 24-month-old Hu sheep. In addition, the expression of RUNX1T1-L mRNA decreased first, then subsequently increased during ovine preadipocyte differentiation. Knockdown of RUNX1T1-L in ovine preadipocytes promoted preadipocyte differentiation and lipid accumulation. Taken together, our data suggests that RUNX1T1 is an important functional molecule in adipogenesis. Moreover, it showed for the first time that RUNX1T1-L was negatively correlated with the ovine preadipocyte differentiation.
Subject(s)
Adipocytes/metabolism , Adipogenesis , RUNX1 Translocation Partner 1 Protein/metabolism , Adipocytes/cytology , Animals , Cells, Cultured , Female , RUNX1 Translocation Partner 1 Protein/chemistry , RUNX1 Translocation Partner 1 Protein/genetics , Sheep , Subcutaneous Fat/growth & development , Subcutaneous Fat/metabolismABSTRACT
Myoblast differentiation depends on fatty acid oxidation (FAO)ï¼and its rate-limiting enzyme acetyl-CoA carboxylase 2 (ACC2) participate in the regulation skeletal muscle development. However, the precise regulatory mechanism is still unknown. Using previous RNA-sequencing data from our laboratory, we explored the effect of ACC2 on myoblast differentiation, as a candidate gene, since its expression is higher in myoblasts of lamb (first day of age) than that of the fetus (75th day of pregnancy). Our findings show that siACC2 inhibited myoblast proliferation, promoted differentiation, and boosted mitochondrial and fatty acid oxidation activities. The effect of ACC2 on goat muscle cell differentiation was modulated by Etomoxir, a CPT1A inhibitor. Notably, the AMPK/ACC2 pathway was found to regulate fatty acid oxidation and goat muscle cell differentiation. Inhibiting the AMPK/ACC2 pathway significantly reduced CPT1A expression. These findings indicate that AMPK/ACC2 regulate goat myoblast differentiation via fatty acid oxidation, contributing to understanding the mechanism of goat skeletal muscle development.
Subject(s)
AMP-Activated Protein Kinases , Acetyl-CoA Carboxylase , Cell Differentiation , Fatty Acids , Goats , Myoblasts , Oxidation-Reduction , Animals , Fatty Acids/metabolism , Myoblasts/metabolism , Myoblasts/cytology , Acetyl-CoA Carboxylase/metabolism , Acetyl-CoA Carboxylase/genetics , AMP-Activated Protein Kinases/metabolism , Cell Proliferation , Epoxy Compounds/pharmacology , Signal TransductionABSTRACT
Despite previous investigations elucidating the regulatory mechanisms of long non-coding RNAs (lncRNAs) in endometrial function and reproductive disorders, the precise pathways through which lncRNAs impact endometrial functions and fertility remain unclear. In this study, we performed an expression profile analysis of lncRNAs in the endometrial tissue of Hu sheep with different prolificacy, identifying 13,707 lncRNAs. We discovered a bidirectional lncRNA, designated lncRNA12097.1, exhibiting significant up-regulation exclusively in the endometrium of Hu sheep with high fecundity. Functional analyses revealed lncRNA12097.1 significantly enhanced proliferation and cell cycle progression in both endometrial epithelial cell (EEC) and stromal cells (ESC), while inhibiting apoptosis in these cell types. Mechanistically, we demonstrated a directly interaction between lncRNA12097.1 and miR-145-5p, with YES proto-oncogene 1 (YES1) being identified as a validated target of miR-145-5p. Interference with lncRNA12097.1 resulted in suppressed cell growth through down-regulation of YES1 expression, which could be rescued by miR-145-5p. Furthermore, lncRNA12097.1 functions as a competitive endogenous RNA (ceRNA) for miR-145-5p in ESCs, sequestering miR-145-5p and preventing its binding to the 3'UTR of YES1 mRNA. This interaction led to increased expression of YES1 and subsequent activation of downstream ß-catenin signaling, thereby promoting ESC growth in Hu sheep. These findings provide novel molecular insights into the mechanisms underlying prolificacy in sheep.
Subject(s)
MicroRNAs , RNA, Long Noncoding , Female , Animals , Sheep , MicroRNAs/genetics , MicroRNAs/metabolism , beta Catenin/genetics , beta Catenin/metabolism , Cell Line, Tumor , RNA, Long Noncoding/genetics , Cell Proliferation/genetics , Endometrium/metabolism , Cell Cycle/genetics , Gene Expression Regulation, NeoplasticABSTRACT
Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), a significant member of the conserved RNA-binding protein family, plays various roles in numerous physiological and pathological processes. However, the specific function of IGF2BP2 in regulating endometrial function in sheep remains largely unknown. In this study, we observed a significant upregulation in IGF2BP2 mRNA abundance in the endometrium during the luteal phase compared to the follicular phase in Hu sheep. The knockdown of IGF2BP2 resulted in accelerated cell proliferation and migration of Hu sheep endometrial stromal cells (ESCs). Moreover, RNA sequencing analysis revealed that genes with significantly altered expression in IGF2BP2 knockdown cells were predominantly enriched in endometrial receptivity-related signaling pathways, such as cytokine-cytokine receptor interaction, NOD-like receptor, PI3K-AKT, and JAK-STAT signaling pathway. Additionally, the knockdown of IGF2BP2 significantly increased the expression of matrix metalloprotein 9 (MMP9), vascular endothelial growth factor, and prolactin (PRL) in ESCs. The knockdown of IGF2BP2 was also observed to stimulate the PI3K/AKT/mTOR pathway by upregulating integrin ß4 (ITGB4) expression. Notably, the downregulation of ITGB4 attenuates IGF2BP2 knockdown-induced facilitation of proliferation and migration of Hu sheep ESCs by inhibiting the PI3K/AKT/mTOR pathway. Collectively, these findings highlight the important role of IGF2BP2 in regulating endometrial function, particularly through the modulation of ESC proliferation and migration via the PI3K/AKT/mTOR pathway.
The maintenance of normal physiological functionality of the endometrium is crucial for successful embryo implantation. Endometrial stromal cells (ESCs), as the principal components of the endometrium, play a key role in establishing optimal endometrial receptivity for embryo implantation. Despite the well-established role of IGF2BP2 in the pathogenesis of endometriosis in women, its functional impact on endometrial activity in ruminants, particularly in ovine species, remains undefined. In this study, we investigated the expression pattern of IGF2BP2 in the reproductive organs of female sheep and evaluated the potential roles and underlying mechanisms of IGF2BP2 in the function of sheep ESCs. This experiment confirmed the important role of IGF2BP2 in regulating endometrial function by modulating the proliferation and migration of Hu sheep ESCs.
Subject(s)
Cell Movement , Cell Proliferation , Endometrium , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Signal Transduction , Stromal Cells , TOR Serine-Threonine Kinases , Animals , Female , Endometrium/metabolism , Endometrium/cytology , Stromal Cells/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/genetics , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/genetics , Sheep , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Gene Knockdown TechniquesABSTRACT
An emerging research focus is the role of m6A modifications in mediating the post-transcriptional regulation of mRNA during mammalian development. Recent evidence suggests that m6A methyltransferases and demethylases play critical roles in skeletal muscle development. Ythdf2 is a m6A "reader" protein that mediates mRNA degradation in an m6A-dependent manner. However, the specific function of Ythdf2 in skeletal muscle development and the underlying mechanisms remain unclear. Here, we observed that Ythdf2 expression was significantly upregulated during myogenic differentiation, whereas Ythdf2 knockdown markedly inhibited myoblast proliferation and differentiation. Combined analysis of high-throughput sequencing, Co-IP, and RIP assay revealed that Ythdf2 could bind to m6A sites in STK11 mRNA and form an Ago2 silencing complex to promote its degradation, thereby regulating its expression and consequently, the AMPK/mTOR pathway. Furthermore, STK11 downregulation partially rescued Ythdf2 knockdown-induced impairment of proliferation and myogenic differentiation by inhibiting the AMPK/mTOR pathway. Collectively, our results indicate that Ythdf2 mediates the decay of STK11 mRNA, an AMPK activator, in an Ago2 system-dependent manner, thereby driving skeletal myogenesis by suppressing the AMPK/mTOR pathway. These findings further enhance our understanding of the molecular mechanisms underlying RNA methylation in the regulation of myogenesis and provide valuable insights for conducting in-depth studies on myogenesis.
Subject(s)
AMP-Activated Protein Kinases , TOR Serine-Threonine Kinases , Animals , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Transcription Factors , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA Stability , Muscle Development/genetics , Mammals/geneticsABSTRACT
Recent evidence suggested that N6-methyladenosine (m6A) methylation can determine m6A-modified mRNA fate and play an important role in skeletal muscle development. It was well known that transforming growth factor beta 1 (TGFß1) is involved in a variety of cellular processes, such as proliferation, differentiation, and apoptosis. However, little is known about the m6A-mediated TGFß1 regulation in myogenesis. Here, we observed an increase in endogenous TGFß1 expression and activity during myotube differentiation. However, the knockdown of TGFß1 inhibits the proliferation and induces cell apoptosis of myoblast. Moreover, we found that m6A in 5'-untranslated regions (5'UTR) of TGFß1 promote its decay and inhibit its expression, leading to the blockage of the TGFß1/SMAD2 signaling pathway. Furthermore, the targeted specific demethylation of TGFß1 m6A using dCas13b-FTO significantly increased the TGFß1-mediated activity of the SMAD2 signaling pathway, promoting myoblast proliferation. These findings suggest that TGFß1 is an essential regulator of myoblast growth that is negatively regulated by m6A. Overall, these results highlight the critical role of m6A-mediated post-transcriptional regulation in myogenesis.
Subject(s)
Myoblasts , Signal Transduction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Myoblasts/metabolism , Cell Proliferation/genetics , DemethylationABSTRACT
circRNAs have been found to be involved in the regulatory network of skeletal muscle development in studies. However, their precise functions and regulatory mechanisms remain unknown. The expression patterns and alterations of circRNAs in the longissimus dorsi muscle of two major developmental stages of goats (D75 fetus and D1 kid) were studied using high-throughput sequencing technology and bioinformatics tools in this study. In kid skeletal muscles, 831 differently expressed circRNAs were found, comprising 486 up-regulated circRNAs and 345 down-regulated circRNAs. In skeletal muscle, we focused on the highly expressed and variably expressed circUBE3A. CircUBE3A levels were discovered to be much higher in kid skeletal muscle and differentiated myoblasts. Knocking down circUBE3A resulted in a significant increase in cell proliferation and differentiation in goat myoblasts. CircUBE3A specifically binds to and inhibits miR-28-5p, boosting the expression of Hydroxyacyl Coenzyme A Dehydrogenase Beta (HADHB) and contributing to goat myoblast proliferation and differentiation, according to the mechanistic investigation. The above results indicated that circUBE3A could regulate HADHB expression by targeting miR-28-5p, consequently increasing goat myoblast proliferation and differentiation. Our findings offer fresh perspectives on goat breeding and growth regulation, as well as substantial theoretical basis.