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.

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.

Gga-miR-146b-3p promotes apoptosis and attenuate autophagy by targeting AKT1 in chicken granulosa cells.

The normal development of follicles determines the reproductive performance of females. Granulosa cells (GC) play crucial roles in follicular maturation. Numerous studies have shown that miRNAs are involved in the regulation of GC. According to our previous sequencing data, gga-miR-146b-3p was differentially expressed in normal and atretic chicken follicles. In this study, we verified that gga-miR-146b-3p attenuated proliferation and autophagy but promoted apoptosis in chicken GC. Threonine kinase1 (AKT1), a key member of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, was predicted to be a target gene of gga-miR-146b-3p via bioinformatic analysis. Dual-luciferase reporter gene assays were used to determine target relationships. Moreover, knockout of AKT1 decelerated proliferation and autophagy while accelerating the apoptosis of GC. However, overexpression of AKT1 reversed these results. In summary, our results demonstrated that gga-miR-146b-3p repressed the proliferation and autophagy of chicken GC while up-regulating apoptosis by targeting AKT1 through the PI3K/AKT signaling pathway. These findings may provide great insights for further exploration of the molecular regulation of gga-miR-146b-3p and AKT1 on the functions of GC during folliculogenesis.

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.

Knockdown of CSRP3 inhibits differentiation of chicken satellite cells by promoting TGF-ß/Smad3 signaling.

Muscle LIM protein (MLP/CSRP3/CRP3) is a microtubule-associated protein preferentially expressed in cardiac and skeletal muscle and has a central role during muscle development and for architectural maintenance of muscle cells. LIM-domain proteins act as both modulators and downstream targets of TGF-ß signaling, which is well documented to negatively regulate differentiation of myogenic precursor cells or myoblasts. Herein, we determined whether CSRP3 regulates chicken satellite cell proliferation and differentiation in vitro, and examined its mechanism of action by focusing on the TGF-ß signaling pathway. Interference of CSRP3 mRNA expression had no effect on the proliferation of satellite cells, but significantly inhibited satellite cell differentiation into myotubes at 24, 48, and 72 h after initiation of differentiation. However, CSRP3 overexpression did not affect the proliferation or differentiation of satellite cells. Moreover, knockdown of CSRP3 caused up-regulation of TGF-ß and Smad3 mRNA and protein levels. The phosphorylation of Smad3 in CSRP3-knockdown cells was greater than that in wild-type cells at 24, 48, and 72 h after initiation of differentiation. Collectively, knockdown of CSRP3 suppressed chicken satellite cell differentiation by regulating Smad3 phosphorylation in the TGF-ß signaling pathway. Our results indicate that CSRP3 might play an important role in promoting satellite cell differentiation in chicken.