MiRNA-21-5p induces chicken hepatic lipogenesis by targeting NFIB and KLF3 to suppress the PI3K/AKT signaling pathway.

The liver plays a critical role in metabolic activity and is the body's first immune barrier, and maintaining liver health is particularly important for poultry production. MicroRNAs (miRNAs) are involved in a wide range of biological activities due to their capacity as posttranscriptional regulatory elements. A growing body of research indicates that miR-21-5p plays a vital role as a modulator of liver metabolism in various species. However, the effect of miR-21-5p on the chicken liver is unclear. In the current study, we discovered that the fatty liver had high levels of miR-21-5p. Then the qPCR, Western blot, flow cytometry, enzyme-linked immunosorbent assay, dual-luciferase, and immunofluorescence assays were, respectively, used to determine the impact of miR-21-5p in the chicken liver, and it turned out that miR-21-5p enhanced lipogenesis, oxidative stress, and inflammatory responses, which ultimately induced hepatocyte apoptosis. Mechanically, we verified that miR-21-5p can directly target nuclear factor I B (NFIB) and kruppel-like factor 3 (KLF3). Furthermore, our experiments revealed that the suppression of NFIB promoted apoptosis and inflammation, and the KLF3 inhibitor accelerated lipogenesis and enhanced oxidative stress. Furthermore, the cotransfection results suggest that the PI3K/AKT pathway is also involved in the process of miRNA-21-5p-mediate liver metabolism regulation. In summary, our study demonstrated that miRNA-21-5p plays a role in hepatocyte lipogenesis, oxidative stress, inflammation, and apoptosis, via targeting NFIB and KLF3 to suppress the PI3K/AKT signal pathway in chicken.

miR-21-5p is a typical noncoding RNA that could inhibit messenger RNA expression by targeting the 3ʹ-untranslated region to participate in fatty liver-related disease formation and progression. We demonstrated that miRNA-21-5p plays a role in hepatocyte lipogenesis, oxidative stress, inflammation, and apoptosis, via targeting nuclear factor I B and kruppel-like factor 3 to suppress the PI3K/AKT signal pathway in chicken. This research established the regulatory network mechanisms of miR-21-5p in chicken hepatic lipogenesis and fatty liver syndrome.

Evolutionary conserved circular MEF2A RNAs regulate myogenic differentiation and skeletal muscle development.

Circular RNAs (circRNAs) have been recognized as critical regulators of skeletal muscle development. Myocyte enhancer factor 2A (MEF2A) is an evolutionarily conserved transcriptional factor that regulates myogenesis. However, it remains unclear whether MEF2A produces functional circRNAs. In this study, we identified two evolutionarily conserved circular MEF2A RNAs (circMEF2As), namely circMEF2A1 and circMEF2A2, in chicken and mouse muscle stem cells. Our findings revealed that circMEF2A1 promotes myogenesis by regulating the miR-30a-3p/PPP3CA/NFATC1 axis, whereas circMEF2A2 facilitates myogenic differentiation by targeting the miR-148a-5p/SLIT3/ROBO2/ß-catenin signaling pathway. Furthermore, in vivo experiments demonstrated that circMEF2As both promote skeletal muscle growth. We also discovered that the linear MEF2A mRNA-derived MEF2A protein binds to its own promoter region, accelerating the transcription of MEF2A and upregulating the expression of both linear MEF2A and circMEF2As, forming a MEF2A autoregulated positive feedback loop. Moreover, circMEF2As positively regulate the expression of linear MEF2A by adsorbing miR-30a-3p and miR-148a-5p, which directly contribute to the MEF2A autoregulated feedback loop. Importantly, we found that mouse circMEF2As are essential for the myogenic differentiation of C2C12 cells. Collectively, our results demonstrated the evolution, function, and underlying mechanisms of circMEF2As in animal myogenesis, which may provide novel insight for both the farm animal meat industry and human medicine.

CircLRRFIP1 promotes the proliferation and differentiation of chicken skeletal muscle satellite cells by sponging the miR-15 family via activating AKT3-mTOR/p70S6K signaling pathway.

Skeletal muscle is important for animal meat production, regulating movements, and maintaining homeostasis. Circular RNAs (circRNAs) have been founded to play vital role in myogenesis. However, the effects of the numerous circRNAs on growth and development of the skeletal muscle are yet to be uncovered. Herein, we identified circLRRFIP1, which is a novel circular RNA that is preferentially expressed in the skeletal muscle. To study the role of circLRRFIP1 in the skeletal muscle, the skeletal muscle satellite cells (SMSCs) was used to silenced or overexpressed circLRRFIP1. The results obtained in this study showed that circLRRFIP1 play a positive role in the proliferation and differentiation of SMSCs. The SMSCs were generated with stable knockdown and overexpression of circLRRFIP1, and the results showed that circLRRFIP1 exerts a stimulatory effect on the proliferation and differentiation of SMSCs. We further generated SMSCs with stable knockdown and overexpression of circLRRFIP1, and the results revealed that circLRRFIP1 exerts a stimulatory effect on the proliferation and differentiation of SMSCs. Mechanistically, circLRRFIP1 targets the myogenic inhibitory factor-miR-15 family to release the suppression of the miR-15 family to AKT3. The knockdown of AKT inhibits SMSC differentiation through the mTOR/p70S6K pathway. Taken together, the results obtained in this present study revealed the important role and the regulatory mechanisms of circLRRFIP1 in the development of chicken skeletal muscle. Therefore, this study provides an attractive target for molecular breeding to enhance meat production in the chicken industry.

Quantitative proteomic and phosphoproteomic analysis of chicken skeletal muscle during embryonic development.

Skeletal muscle also plays a vital role in regulating the movement energy storage and health of metabolism. In order to investigate the expression profile of protein and phosphor-proteins in chicken skeletal muscle during embryonic development, we performed phosphor-proteomics analysis by label-free and TiO2 enrichment strategy in chicken leg muscle tissues of at embryonic age embryo day 7(E7), E12, E17 and 3-day post-hatch (D3). The study led to the identification of 4332 proteins in the proteome and 1043 phosphorylation modification sites in the phosphorylated proteome, corresponding to 718 proteins (FC ≥ 2 or FC ≤ 0.5 and p < 0.05). The DEP-associated biological processes were involved in Focal adhesion, Glycolysis/gluconeogenesis, Arginine and proline metabolism by KEGG analysis. PPI analyses revealed that these DEPs TNNC1, TNNC2, TNNT2, TNNT3 and phosphorylated DEPs MYLPF interacted with involved pathways. Integrative analysis of proteome and phosphoproteome data found 324 common proteins, corresponding to 521 modification sites and Focal adhesion was the only pathway significantly enriched. These results provide a basis for further understanding the proteome and phosphoproteome and their regulatory biochemical pathways during the development of embryonic chicken skeletal muscle.

MyoG-enhanced circGPD2 regulates chicken skeletal muscle development by targeting miR-203a.

Circular RNAs (circRNAs) are a subclass of RNA macromolecules that are reported to be involved in the regulation of skeletal muscle development. However, the functions and regulatory mechanisms of circRNAs in chicken myogenesis are still largely unclear. Here, we identified a novel circRNA, circGPD2, an RNA macromolecule with a calculated molecular weight of 215 kDa. We discovered that circGPD2 is a muscle-specific circRNA and is strongly expressed in the breast muscle of broilers by utilizing the comparison model of layers and broilers. Functional analysis revealed circGPD2 has a positive role in the proliferation and differentiation of myoblasts, and circGPD2 performs function through the release of the inhibition effect of miR-203a on c-JUN and MEF2C. Besides, the myogenic regulatory factor MyoG enhanced the expression of circGPD2 by targeting the E-box element on the GPD2 promoter. Importantly, lentivirus-mediated circGPD2 knockdown resulted in the breast muscle mass loss of the chicks. Overall, we revealed the crucial role of circGPD2 in chicken myogenesis in vitro and in vivo, and analyzed the upstream and downstream regulation mechanisms of circGPD2. Our study provides an attractive target for molecular marker-assisted breeding to improve the meat yield in the chicken meat industry.

miR-10a-5p inhibits chicken granulosa cells proliferation and Progesterone(P4) synthesis by targeting MAPRE1 to suppress CDK2.

The proliferation and steroid hormone synthesis of granulosa cells (GCs) are essential for ovarian follicle growth and ovulation, which are necessary to support the normal function of the follicle. Numerous studies suggest that miRNAs play key roles in this process. In this study, we report a novel role for miR-10a-5p that inhibits ovarian GCs proliferation and progesterone (P4) synthesis in chicken. Specifically, we found that miR-10a-5p significantly decreased the P4 secretion by quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot. Moreover, we observed that miR-10a-5p can inhibit the proliferation of chicken GCs through the investigation of cell proliferation gene expression, cell counting kit 8 (CCK-8), cell cycle progression, and 5-ethynyl-2'-deoxyuridine (EdU) assay. Then we screened a target gene MAPRE1 of miR-10a-5p, which can promote P4 synthesis and proliferation of GCs. To explore how miR-10a-5p affects cell cycle by MAPRE1, we investigated the interaction between MAPRE1 and cyclin-dependent kinase 2 (CDK2) by Co-Immunoprecipitation (Co-IP), and then we found that MAPRE1 can form a complex with CDK2. In addition, miR-10a-5p was found to inhibit CDK2 expression by repressing the expression of MAPRE1. Overall, our results indicate that miR-10a-5p regulates the proliferation and P4 synthesis of chicken GCs by targeting MAPRE1 to suppress CDK2.

Effects of Small Peptide Supplementation on Growth Performance, Intestinal Barrier of Laying Hens During the Brooding and Growing Periods.

The growing period is a critical period for growth and development in laying hens. During this period, chicks grow rapidly, but are accompanied by unstable digestive function, incomplete organ development, and high mortality. Small peptide, a feed additive, which has been proved to promote intestinal development and immunity in poultry. In order to elucidate the effects of small peptides on growth performance, immunity, antioxidant capacity, and intestinal health of growing laying hens, a total of 900 Tianfu green shell laying hens (1-day-old) were randomly divided into 5 treatments with 6 replicates of 30 birds each in this 18-week trial. Dietary treatments included a corn-soybean meal-based diet supplemented with 0 g/kg, 1.5 g/kg, 3.0 g/kg, 4.5 g/kg and 6.0 g/kg small peptide, respectively. The results showed that the supplementation of small peptides significantly increased growth rate (P<0.05) in laying hens, as well as elevated the serum immunoglobulins (P<0.05) and antioxidant indices (P<0.05), however, it decreased inflammation parameters (P<0.05). The supplementation of small peptides enhanced the intestinal function by promoting gut development (P<0.05) and improving gut integrity (P<0.05), barrier function (P<0.05) and the diversity of gut microbiota (P<0.05) in the growing hens. The best performance was recorded among the hens fed 4.5 g/kg level of small peptide. Taken together, these results showed that small peptide supplementation could improve the economic value of growing hens by promoting growth rate, disease resistance, and the optimal amount of addition for Tianfu green shell laying hens was 4.5 g/kg.

Filamin C regulates skeletal muscle atrophy by stabilizing dishevelled-2 to inhibit autophagy and mitophagy.

FilaminC (Flnc) is a member of the actin binding protein family, which is preferentially expressed in the cardiac and skeletal muscle tissues. Although it is known to interact with proteins associated with myofibrillar myopathy, its unique role in skeletal muscle remains largely unknown. In this study, we identify the biological functions of Flnc in vitro and in vivo using chicken primary myoblast cells and animal models, respectively. From the results, we observe that the growth rate and mass of the skeletal muscle of fast-growing chickens (broilers) were significantly higher than those in slow-growing chickens (layers). Furthermore, we find that the expression of Flnc in the skeletal muscle of broilers was higher than that in the layers. Our results indicated that Flnc was highly expressed in the skeletal muscle, especially in the skeletal muscle of broilers than in layers. This suggests that Flnc plays a positive regulatory role in myoblast development. Flnc knockdown resulted in muscle atrophy, whereas the overexpression of Flnc promotes muscle hypertrophy in vivo in an animal model. We also found that Flnc interacted with dishevelled-2 (Dvl2), activated the wnt/ß-catenin signaling pathway, and controlled skeletal muscle development. Flnc also antagonized the LC3-mediated autophagy system by decreasing Dvl2 ubiquitination. Moreover, Flnc knockdown activated and significantly increased mitophagy. In summary, these results indicate that the absence of Flnc induces autophagy or mitophagy and regulates muscle atrophy.

Fibromodulin is involved in autophagy and apoptosis of granulosa cells affecting the follicular atresia in chicken.

Follicular atresia is an important cause of reproductive decline in egg-laying hens. Therefore, a better understanding of the regulation mechanism of follicle atresia in poultry is an important measure to maintain persistent high egg performance. However, how the role of the regulatory relationship between autophagy and apoptosis in the intrafollicular environment affects the follicular atresia of chickens is remain unclear. The objective of this study was to explore the regulatory molecular mechanisms in regard to follicular atresia. 20 white leghorn layers (32-wk-old) were equally divided into 2 groups. The control group was fed freely, and the experimental group induced follicular atretic by fasting for 5 d. The results showed that the expression of prolactin (PRL) levels was significantly higher in the fasted hens, while the levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were lower. Most importantly, RNA sequencing, qPCR, and Western blotting detected significantly elevated levels of autophagy and apoptosis markers in atresia follicles. Interestingly, we found that fibromodulin (FMOD) levels was significantly lower in follicles from fasted hens and that this molecule had an important regulatory role in autophagy. FMOD silencing significantly promoted autophagy and apoptosis in granulosa cells, resulting in hormonal imbalance. FMOD was found to regulate autophagy via the transforming growth factor beta (TGF-ß) signaling pathway. Our results suggest that the increase in autophagy and the imbalance in internal homeostasis cause granulosa cell apoptosis, leading to follicular atresia in the chicken ovary. This finding could provide further insight into broodiness in chicken and provide avenues for further improvements in poultry production.

Zearalenone Induces Apoptosis and Cytoprotective Autophagy in Chicken Granulosa Cells by PI3K-AKT-mTOR and MAPK Signaling Pathways.

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. ZEA causes reproductive disorders, genotoxicity, and testicular toxicity in animals. However, little is known about the functions of apoptosis and autophagy after exposure to ZEA in granulosa cells. This study investigated the effects of ZEA on chicken granulosa cells. The results show that ZEA at different doses significantly inhibited the growth of chicken granulosa cells by inducing apoptosis. ZEA treatment up-regulated Bax and downregulated Bcl-2 expression, promoted cytochrome c release into the cytosol, and triggered mitochondria-mediated apoptosis. Consequently, caspase-9 and downstream effector caspase-3 were activated, resulting in chicken granulosa cells apoptosis. ZEA treatment also upregulated LC3-II and Beclin-1 expression, suggesting that ZEA induced a high level of autophagy. Pretreatment with chloroquine (an autophagy inhibitor) and rapamycin (an autophagy inducer) increased and decreased the rate of apoptosis, respectively, in contrast with other ZEA-treated groups. Autophagy delayed apoptosis in the ZEA-treated cells. Therefore, autophagy may prevent cells from undergoing apoptosis by reducing ZEA-induced cytotoxicity. In addition, our results further show that the autophagy was stimulated by ZEA through PI3K-AKT-mTOR and MAPK signaling pathways in chicken granulosa cells.

Plectin regulates Wnt signaling mediated-skeletal muscle development by interacting with Dishevelled-2 and antagonizing autophagy.

Skeletal muscle is the most abundant tissue in the human and animal body, loss of its function can lead to muscle aging and various myogenic diseases. The skeletal muscle development is a complex and tightly regulated process, which is driven by a variety of many factors, signaling pathways and regulatory mechanisms. Plectin (Plec), a cytolinker protein, is ubiquitously expressed in various tissues such as skin, muscle, plasma membrane, and most types of cells. Although known isoforms of Plec is well-characterized in muscle dystrophy, very little is known on the function of Plec in the skeletal muscle development. Here, we found that Plec plays a vital role in promoting C2C12 myoblasts differentiation and proliferation, but inhibits their apoptosis. Also, Plec regulates the expression of atrophy-related genes (atrogin-1 and muRF-1) to rescue muscle atrophy. Furthermore, we have demonstrated that Plec binds to Dishevelled-2 (Dvl-2) and forms a protein complex, which is then activate the canonical Wnt signaling. We also observed that Plec resists ubiquitination by stabilizing Dvl-2 and reduces the level of LC3-labeled Dvl-2 and antagonizes the autophagy system. In conclusion, our findings suggest that Plec regulates canonical Wnt signaling mediated skeletal development by stabilizing Dvl-2 and downregulating the cellular autophagic degradation system.

Erratum: Zhao, J.; Shen, X.; Cao, X.; He, H.; Han, S.; Chen, Y.; Cui, C.; Wei, Y.; Wang, Y.; Li, D.; Zhu, Q.; Yin, H. HDAC4 Regulates the Proliferation, Differentiation and Apoptosis of Chicken Skeletal Muscle Satellite Cells. Animals 2020, 10, 84.

The authors wish to make the following corrections to their paper [...].

Circular RNA CircFAM188B Encodes a Protein That Regulates Proliferation and Differentiation of Chicken Skeletal Muscle Satellite Cells.

Circular RNAs (circRNAs) are recognized as functional non-coding transcripts; however, emerging evidence has revealed that some synthetic circRNAs generate functional peptides or proteins. Additionally, the diverse biological functions of circRNAs include acting as miRNA-binding sponges, RNA-binding protein regulators, and protein translation templates. Previously, we found that circular RNA circFAM188B is a stable circular RNA and differentially expressed between broiler chickens and layers during embryonic skeletal muscle development. In this study, we found that circFAM188B exhibited a unique pattern of sharply decreased expression from embryonic day 10 (E10) to Day 35 (D35) after hatching. Our experimental results showed that circFAM188B promotes the proliferation, but inhibits the differentiation of chicken skeletal muscle satellite cells (SMSCs). Bioinformatic analysis revealed circFAM188B contain an opening reading frame (ORF) which translate into circFAM188B-103aa, internal ribosome entry site (IRES) analysis further confirmed the coding potential of circFAM188B. In addition, western blot assay detected a flag tagged circFAM188B-103aa, and several peptides of circFAM188B-103aa were detected by LC-MS/MS analysis. We further verified that the role of circFAM188B-103aa in chicken myogenesis is consistent with that of its parent transcript circFAM188B, which facilitates proliferation, but represses differentiation of chicken SMSC. Taken together, these results suggested that a novel protein circFAM188B-103aa encoded by circFAM188B that promotes the proliferation but inhibits the differentiation of chicken SMSCs.

Fibromodulin Modulates Chicken Skeletal Muscle Development via the Transforming Growth Factor-ß Signaling Pathway.

Fibromodulin (Fmod), which is an extracellular matrix protein, belongs to the extracellular matrix small-leucine-rich proteoglycan family. Fmod is abundantly expressed in muscles and connective tissues and is involved in biological regulation processes, including cell apoptosis, cell adhesion, and modulation of cytokine activity. Fmod is the main regulator of myostatin, which controls the development of muscle cells, but its regulatory path is unknown. Chicken models are ideal for studying embryonic skeletal muscle development; therefore, to investigate the mechanism of Fmod in muscle development, Fmod-silenced and Fmod-overexpressed chicken myoblasts were constructed. The results showed that Fmod plays a positive role in differentiation by detecting the expression of myogenic differentiation markers, immunofluorescence of MyHC protein, and myotube formation in myoblasts. Fmod regulates expression of atrophy-related genes to alleviate muscle atrophy, which was confirmed by histological analysis of breast muscles in Fmod-modulated chicks in vivo. Additionally, genes differentially expressed between Fmod knockdown and normal myoblasts were enriched in the signaling pathway of transforming growth factor ß (TGF-ß). Both Fmod-silenced and Fmod-overexpressed myoblasts regulated the expression of TGFBR1 and p-Smad3. Thus, Fmod can promote differentiation but not proliferation of myoblasts by regulating the TGF-ß signaling pathway, which may serve a function in muscular atrophy.

ISLR regulates skeletal muscle atrophy via IGF1-PI3K/Akt-Foxo signaling pathway.

Immunoglobulin superfamily containing leucine-rich repeat (Islr) contains an Ig-like domain, an LRR motif, and a transmembrane domain and is highly expressed in various chicken tissues. Although Islr has known roles in muscle regeneration, its role in the regulation of muscle atrophy has not been studied. In this study, we constructed Islr-silenced or Islr-overexpressed myoblasts to investigate its role during the differentiation of myoblasts into myotubes. The results showed that Islr was highly expressed in chicken skeletal muscle tissue and regulated myoblast differentiation, but not proliferation. Islr regulated the expression of atrophy-related genes including atrogin-1 and MuRF-1, and could rescue dexamethasone-induced atrophy in myoblasts and myotubes. Western blot analysis indicated that Islr participates in myoblast atrophy through IGF/PI3K/AKT-FOXO signaling. Meanwhile, the expression of caspase-8 and caspase-9 increased in Islr-silenced groups, indicating its role in cell viability. Taken together, these data suggested that Islr plays an important role in myoblasts differentiation, and which can alleviate skeletal muscle atrophy and prevents muscle cell apoptosis via IGF/PI3K/AKT-FOXO signaling pathway.

MiR-148a-3p Regulates Skeletal Muscle Satellite Cell Differentiation and Apoptosis via the PI3K/AKT Signaling Pathway by Targeting Meox2.

As bioinformatic approaches have been developed, it has been demonstrated that microRNAs (miRNAs) are involved in the formation of muscles and play important roles in regulation of muscle cell proliferation and differentiation. Previously, it has been demonstrated that miR-148a-3p is one of the most abundant miRNAs in chicken skeletal muscle. Here, we build on that work and demonstrate that miR-148a-3p is important in the control of differentiation of chicken skeletal muscle satellite cells (SMSCs). Elevated expression of miR-148a-3p significantly promoted differentiation and inhibited apoptosis of SMSCs but did not affect proliferation. Furthermore, it was observed that the mesenchyme homeobox 2 (Meox2) is a target gene of miR-148a-3p and that miR-148a-3p can down-regulate expression of Meox2, which promote differentiation of SMSCs and suppress apoptosis. Furthermore, miR-148a-3p overexpression encouraged activation of the PI3K/AKT signaling pathway, which could be recovered by overexpression of Meox2. Overall, these findings suggest that microRNA-148a-3p is a potent promoter of myogenesis via direct targeting of Meox2 and increase of the PI3K/AKT signaling pathway in chicken SMSCs.

MicroRNA Profiling Reveals an Abundant miR-200a-3p Promotes Skeletal Muscle Satellite Cell Development by Targeting TGF-ß2 and Regulating the TGF­ß2/SMAD Signaling Pathway.

MicroRNAs (miRNAs) are evolutionarily conserved, small noncoding RNAs that play critical post-transcriptional regulatory roles in skeletal muscle development. Chicken is an optimal model to study skeletal muscle formation because its developmental anatomy is similar to that of mammals. In this study, we identified potential miRNAs in the breast muscle of broilers and layers at embryonic day 10 (E10), E13, E16, and E19. We detected 1836 miRNAs, 233 of which were differentially expressed between broilers and layers. In particular, miRNA-200a-3p was significantly more highly expressed in broilers than layers at three time points. In vitro experiments showed that miR-200a-3p accelerated differentiation and proliferation of chicken skeletal muscle satellite cells (SMSCs) and inhibited SMSCs apoptosis. The transforming growth factor 2 (TGF-ß2) was identified as a target gene of miR-200a-3p, and which turned out to inhibit differentiation and proliferation, and promote apoptosis of SMSCs. Exogenous TGF-ß2 increased the abundances of phosphorylated SMAD2 and SMAD3 proteins, and a miR-200a-3p mimic weakened this effect. The TGFß2 inhibitor treatment reduced the promotional and inhibitory effects of miR-200a-3p on SMSC differentiation and apoptosis, respectively. Our results indicate that miRNAs are abundantly expressed during embryonic skeletal muscle development, and that miR-200a-3p promotes SMSC development by targeting TGF-ß2 and regulating the TGFß2/SMAD signaling pathway.

Molecular characterization, tissue distribution, and functional analysis of the STAC3 gene in chicken.

The Src homology 3 and cysteine-rich domain 3 gene (STAC3) encodes a protein containing both a cysteine-rich domain and two Src (sarcoma) homology 3 domains (SH3). STAC3 is specifically expressed in skeletal muscle and plays an important role in skeletal muscle development, but the explicit sequence and function of chicken SATC3 remain unknown. In the current study, we found the full-length chicken STAC3 cDNA to be 1383 bp long, with a 1092 bp open reading frame that harbors one cysteine-rich C1 domain and two SH3 domains. Tissue distribution analysis reveals chicken STAC3 mRNA only in skeletal muscle among 12 chicken tissues examined by reverse transcription PCR. Both cholecystokinin octapeptide analysis and a 5-ethynyl-2'-deoxyuridine assay suggest that neither STAC3 overexpression nor knockdown has any effect on the proliferation of chicken skeletal muscle satellite cells. However, STAC3 knockdown significantly increases the mRNA expression of MyoG, MyoD, Mb, and MyHC, and the protein abundance of MyHC and MyoG, whereas the opposite result is found in STAC3 overexpressed cells. We conclude that the STAC3 gene is expressed specifically in skeletal muscle and is a negative regulator of skeletal muscle satellite cell differentiation in chicken.

miR-9-5p Inhibits Skeletal Muscle Satellite Cell Proliferation and Differentiation by Targeting IGF2BP3 through the IGF2-PI3K/Akt Signaling Pathway.

MicroRNAs are evolutionarily conserved, small non-coding RNAs that play critical post-transcriptional regulatory roles in skeletal muscle development. We previously found that miR-9-5p is abundantly expressed in chicken skeletal muscle. Here, we demonstrate a new role for miR-9-5p as a myogenic microRNA that regulates skeletal muscle development. The overexpression of miR-9-5p significantly inhibited the proliferation and differentiation of skeletal muscle satellite cells (SMSCs), whereas miR-9-5p inhibition had the opposite effect. We show that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is a target gene of miR-9-5p, using dual-luciferase assays, RT-qPCR, and Western Blotting, and that it promotes proliferation and differentiation of SMSCs. In addition, we found that IGF2BP3 regulates IGF-2 expression, using overexpression and knockdown studies. We show that Akt is activated by IGF2BP3 and is essential for IGF2BP3-induced cell development. Together, our results indicate that miR-9-5p could regulate the proliferation and differentiation of myoblasts by targeting IGF2BP3 through IGF-2 and that this activity results in the activation of the PI3K/Akt signaling pathway in skeletal muscle cells.

T-2 Toxin Induces Oxidative Stress, Apoptosis and Cytoprotective Autophagy in Chicken Hepatocytes.

T-2 toxin is type A trichothecenes mycotoxin, which produced by fusarium species in cereal grains. T-2 toxin has been shown to induce a series of toxic effects on the health of human and animal, such as immunosuppression and carcinogenesis. Previous study has proven that T-2 toxin caused hepatotoxicity in chicken, but the regulatory mechanism is unclear. In the present study, we assessed the toxicological effect of T-2 toxin on apoptosis and autophagy in hepatocytes. The total of 120 1-day-old healthy broilers were allocated randomly into four groups and reared for 21 day with complete feed containing 0 mg/kg, 0.5 mg/kg, 1 mg/kg or 2 mg/kg T-2 toxin, respectively. The results showed that the apoptosis rate and pathological changes degree hepatocytes were aggravated with the increase of T-2 toxin. At the molecular mechanism level, T-2 toxin induced mitochondria-mediated apoptosis by producing reactive oxygen species, promoting cytochrome c translocation between the mitochondria and cytoplasm, and thus promoting apoptosomes formation. Meanwhile, the expression of the autophagy-related protein, ATG5, ATG7 and Beclin-1, and the LC3-II/LC3-I ratio were increased, while p62 was downregulated, suggesting T-2 toxin caused autophagy in hepatocytes. Further experiments demonstrated that the PI3K/AKT/mTOR signal may be participated in autophagy induced by T-2 toxin in chicken hepatocytes. These data suggest a possible underlying molecular mechanism for T-2 toxin that induces apoptosis and autophagy in chicken hepatocytes.