High expression circRALGPS2 in atretic follicle induces chicken granulosa cell apoptosis and autophagy via encoding a new protein.

BACKGROUND: The reproductive performance of chickens mainly depends on the development of follicles. Abnormal follicle development can lead to decreased reproductive performance and even ovarian disease among chickens. Chicken is the only non-human animal with a high incidence of spontaneous ovarian cancer. In recent years, the involvement of circRNAs in follicle development and atresia regulation has been confirmed. RESULTS: In the present study, we used healthy and atretic chicken follicles for circRNA RNC-seq. The results showed differential expression of circRALGPS2. It was then confirmed that circRALGPS2 can translate into a protein, named circRALGPS2-212aa, which has IRES activity. Next, we found that circRALGPS2-212aa promotes apoptosis and autophagy in chicken granulosa cells by forming a complex with PARP1 and HMGB1. CONCLUSIONS: Our results revealed that circRALGPS2 can regulate chicken granulosa cell apoptosis and autophagy through the circRALGPS2-212aa/PARP1/HMGB1 axis.

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.

Circular RNA circFNDC3AL Upregulates BCL9 Expression to Promote Chicken Skeletal Muscle Satellite Cells Proliferation and Differentiation by Binding to miR-204.

Skeletal muscle is a heterogeneous tissue that is essential for initiating movement and maintaining homeostasis. The genesis of skeletal muscle is an integrative process that lasts from embryonic development to postnatal stages, which is carried out under the modulation of many factors. Recent studies have shown that circular RNAs (circRNAs), a class of non-coding RNAs, are involved in myogenesis. However, more circRNAs and their mechanisms that may regulate skeletal muscle development remain to be explored. Through in-depth analysis of our previous RNA-Seq data, circFNDC3AL was found to be a potentially functional circRNA highly expressed during embryonic development of chicken skeletal muscle. Therefore, in this study, we investigated the effect of circFNDC3AL on skeletal muscle development in chickens and found that circFNDC3AL promoted chicken skeletal muscle satellite cell (SMSC) proliferation and differentiation. To gain a thorough understanding of the exact modulatory mechanisms of circFNDC3AL in chicken skeletal muscle development, we performed target miRNA analysis of circFNDC3AL and found that circFNDC3AL has a binding site for miR-204. Subsequently, we demonstrated that miR-204 inhibited chicken SMSC proliferation and differentiation, which showed the opposite functions of circFNDC3AL. Furthermore, we identified the miR-204 target gene B-cell CLL/lymphoma 9 (BCL9) and validated that miR-204 had an inhibitory effect on BCL9, while the negative effect could be relieved by circFNDC3AL. In addition, we verified that BCL9 performed the same positive functions on chicken SMSC proliferation and differentiation as circFNDC3AL, as opposed to miR-204. In conclusion, our study identified a circRNA circFNDC3AL that upregulates BCL9 expression to promote the proliferation and differentiation of chicken SMSCs by binding to miR-204.

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.

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.

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.

The Autophagy Regulatory Molecule CSRP3 Interacts with LC3 and Protects Against Muscular Dystrophy.

CSRP3/MLP (cysteine-rich protein 3/muscle Lim protein), a member of the cysteine-rich protein family, is a muscle-specific LIM-only factor specifically expressed in skeletal muscle. CSRP3 is critical in maintaining the structure and function of normal muscle. To investigate the mechanism of disease in CSRP3 myopathy, we performed siRNA-mediated CSRP3 knockdown in chicken primary myoblasts. CSRP3 silencing resulted in the down-regulation of the expression of myogenic genes and the up-regulation of atrophy-related gene expressions. We found that CSRP3 interacted with LC3 protein to promote the formation of autophagosomes during autophagy. CSRP3-silencing impaired myoblast autophagy, as evidenced by inhibited autophagy-related ATG5 and ATG7 mRNA expression levels, and inhibited LC3II and Beclin-1 protein accumulation. In addition, impaired autophagy in CSRP3-silenced cells resulted in increased sensitivity to apoptosis cell death. CSRP3-silenced cells also showed increased caspase-3 and caspase-9 cleavage. Moreover, apoptosis induced by CSRP3 silencing was alleviated after autophagy activation. Together, these results indicate that CSRP3 promotes the correct formation of autophagosomes through its interaction with LC3 protein, which has an important role in skeletal muscle remodeling and maintenance.