Inhibitor PF-04691502 works as a senolytic to regulate cellular senescence.

Aging is a gradual process of natural change that occurs after reaching sexual maturity. It is also a known risk factor for many chronic diseases. Recent research has shown that senolytics can extend the lifespans and health spans of model organisms, and they have also been demonstrated effective in treating age-related diseases. In this study, we conducted a high-throughput screening of 156 drugs that targeted the PI3K/AKT/mTOR pathway to identify potential senolytic medications. Among these drugs, PF-04691502 was selected for further investigation to understand its molecular mechanism of action. Our findings indicate that PF-04691502, a dual inhibitor of PI3K/AKT and mTOR, specifically eliminates senescent cells. It reduces the expression levels of key markers of cellular senescence, such as SA-ß-Gal, senescence-associated secretory phenotypes (SASPs) and p16INK4a. Additionally, PF-04691502 inhibits the phosphorylation of S6K and AKT, leading to the apoptosis of senescent cells. These results suggest that PF-04691502 holds promise as a new senolytic drug. This paper provides important insights into the potential application of PF-04691502 in the study of cell senescence.

Dynamic mRNA expression during chicken ovarian follicle development.

Ovarian follicle development is a complex and well-orchestrated biological process of great economic significance for poultry production. Specifically, understanding the molecular mechanisms underlying follicular development is essential for high-efficiency follicular development can benefit the entire industry. In addition, domestic egg-laying hens often spontaneously develop ovarian cancer, providing an opportunity to study the genetic, biochemical, and environmental risk factors associated with the development of this cancer. Here, we provide high-quality RNA sequencing data for chicken follicular granulosa cells across 10 developmental stages, which resulted in a total of 204.57 Gb of clean sequencing data (6.82 Gb on average per sample). We also performed gene expression, time-series, and functional enrichment analyses across the 10 developmental stages. Our study revealed that SWF (small while follicle), F1 (F1 hierarchical follicles), and POFs (postovulatory follicles) best represent the transcriptional changes associated with the prehierarchical, preovulatory, and postovulatory stages, respectively. We found that the preovulatory stage F1 showed the greatest divergence in gene expression from the POF stage. Our research lays a foundation for further elucidation of egg-laying performance of chicken and human ovarian disease.

Integrated Transcriptome Analysis Reveals mRNA-miRNA Pathway Crosstalk in Roman Laying Hens' Immune Organs Induced by AFB1.

Aflatoxin B1 (AFB1) is a widely distributed contaminant in moldy corn, rice, soybean, and oil crops. Many studies have revealed its adverse effects, such as carcinogenicity, immunotoxicity, and hepatotoxicity, on the health of humans and animals. To investigate the immunotoxic effects on chicken immune organs induced by AFB1, we integrated RNA and small-RNA sequencing data of the spleen and the bursa of Fabricius to elucidate the response of the differentially expressed transcriptional profiles and related pathways. AFB1 consumption negatively influenced egg quality, but no obvious organ damage was observed compared to that of the control group. We identified 3918 upregulated and 2415 downregulated genes in the spleen and 231 upregulated and 65 downregulated genes in the bursa of Fabricius. We confirmed that several core genes related to immune and metabolic pathways were activated by AFB1. Furthermore, 42 and 19 differentially expressed miRNAs were found in the spleen and the bursa of Fabricius, respectively. Differentially expressed genes and target genes of differentially expressed miRNAs were mainly associated with cancer progression and immune response. The predicted mRNA-miRNA pathway network illustrated the potential regulatory mechanisms. The present study identified the transcriptional profiles and revealed potential mRNA-miRNA pathway crosstalk. This genetic regulatory network will facilitate the understanding of the immunotoxicity mechanisms of chicken immune organs induced by high concentrations of AFB1.

Dietary supplementation of salidroside alleviates liver lipid metabolism disorder and inflammatory response to promote hepatocyte regeneration via PI3K/AKT/Gsk3-ß pathway.

Fatty liver hemorrhagic syndrome (FLHS) is a chronic hepatic disease which occurs when there is a disorder in lipid metabolism. FLHS is often observed in caged laying hens and characterized by a decrease in egg production and dramatic increase of mortality. Salidroside (SDS) is an herbal drug which has shown numerous pharmacological activities, such as protecting mitochondrial function, attenuating cell apoptosis and inflammation, and promoting antioxidant defense system. We aimed to determine the therapeutic effects of SDS on FLHS in laying hens and investigate the underlying mechanisms through which SDS operates these functions. We constructed oleic acid (OA)-induced fatty liver model in vitro and high-fat diet-induced FLHS of laying hens in vivo. The results indicated that SDS inhibited OA-induced lipid accumulation in chicken primary hepatocytes, increased hepatocyte activity, elevated the mRNA expression of proliferation related genes PCNA, CDK2, and cyclinD1 and increased the protein levels of PCNA and CDK2 (P < 0.05), as well as decreased the cleavage levels of Caspase-9, Caspase-8, and Caspase-3 and apoptosis in hepatocytes (P < 0.05). Moreover, SDS promoted the phosphorylation levels of PDK1, AKT, and Gsk3-ß, while inhibited the PI3K inhibitor (P < 0.05). Additionally, we found that high-fat diet-induced FLHS hens had heavier body weight, liver weight, and abdominal fat weight, and severe steatosis in histology, compared with the control group (Con). However, hens fed with SDS maintained lighter body weight, liver weight, and abdominal fat weight, as well as normal liver without hepatic steatosis. In addition, high-fat diet-induced FLHS hens had high levels of serum total cholesterol (TC), triglyceride (TG), alanine transaminase (ALT), and aspartate aminotransferase (AST) compared to the Con group, however, in the Model+SDS group, the levels of TC, TG, ALT, and AST decreased significantly, whereas the level of superoxide dismutase (SOD) increased significantly (P < 0.05). We also found that SDS significantly decreased the mRNA expression abundance of PPARγ, SCD, and FAS in the liver, as well as increased levels of PPARα and MTTP, and decreased the mRNA expression of TNF-α, IL-1ß, IL-6, and IL-8 in the Model+SDS group (P < 0.05). In summary, this study showed that 0.3 mg/mL SDS attenuated ROS generation, inhibited lipid accumulation and hepatocyte apoptosis, and promoted hepatocyte proliferation by targeting the PI3K/AKT/Gsk3-ß pathway in OA-induced fatty liver model in vitro, and 20 mg/kg SDS alleviated high-fat-diet-induced hepatic steatosis, oxidative stress, and inflammatory response in laying hens in vivo.

Dietary quercetin and vitamin E supplementation modulates the reproductive performance and antioxidant capacity of aged male breeder chickens.

Aged male chickens experience rapid declines in spermatogenesis, antioxidant capacity, immunity, and hormone synthesis. Vitamin E plays a significant role in reproduction, nervous system function, and disease resistance in animals. Quercetin also exerts many biological effects, such as antioxidant ability, immunostimulation, and protection of spermatozoal plasma membranes. This study evaluated the effects of combining dietary quercetin (Q) and vitamin E (VE) on sperm quality, antioxidant capacity, immunity, and expression of genes related to spermatogenesis, immunity, apoptosis, and inflammation in aged male chickens. A total of 120 Tianfu breeder male chickens (65 wk old) were randomly allotted to 4 treatments with 3 replicates (10 birds each). The birds were fed diets containing Q (0.4g/kg), VE (0.2g/kg), Q+VE (0.4g/kg + 0.2g/kg), and a basal diet for 11 wk. At the end of the experimental period, blood, semen, liver, testes, and spleen samples were collected from 2 birds per replicate. Serum hormones, antioxidant parameters, cytokines, and immunoglobulins were evaluated; and the mRNA expression of genes related to spermatogenesis, apoptosis, and inflammation are determined in the testes and liver tissues. The results showed that the combination quercetin and vitamin E significantly promoted the sperm count and motility, as well as elevated the levels of testosterone, follicle-stimulating hormone, and luteinizing hormone, antioxidant enzymes (Superoxide dismutase, Glutathione, and Total antioxidant capacity), and serum immunoglobulins (IgA and IgM) in the aged male chickens; also Q+VE showed protective effects on the liver against injury. In addition, Q+VE significantly increased the expression of genes related to spermatogenesis (AR, pgk2, Cyclin A1, and Cyclin A2), immunity (IFN-γ and IL-2), and anti-inflammatory cytokines (IL-10) (P < 0.05), whereas the expression of proinflammatory cytokines (IL-1ß and IL-6) was decreased (P < 0.05). Taken together, these data indicate that the combination of quercetin and vitamin E improved reproductive characteristics such as spermatogenesis, sperm quality, and hormone regulation, as well as promoted antioxidant defense, hepatoprotective capacity, and immune response in aged male chickens without any detrimental effects.

CircCCDC91 regulates chicken skeletal muscle development by sponging miR-15 family via activating IGF1-PI3K/AKT signaling pathway.

Circular RNAs (circRNAs) has been reported in various tissues of animals and associated with multiple biological processes. From our previous sequencing data, we found a novel circRNA, circCCDC91 which was generated from exon 2 to 8 of the CCDC91 gene. We observed that circCCDC91 was differentially expressed in the chicken breast muscle among 4 different embryonic developmental time points (embryonic day 10 [E10], E13, E16, and E19). Therefore, we assumed that circCCDC91 have a potential function in chicken skeletal muscle development. In this study, we firstly verify the annular structure and expression pattern of circCCDC91, and further investigate on whether circCCDC91 could promote chicken skeletal development. Mechanistically, circCCDC91 could absorb miR-15a, miR-15b-5p, and miR-15c-5p to modulate the expression of Insulin receptor substrate1 (IRS1), as well as activate insulin-1ike growth factor 1-phosphatidylinositol 3-kinase/AKT (IGF1-PI3K/AKT) signaling pathway. In addition, circCCDC91 could rescue skeletal muscle atrophy by activating IGF1-PI3K/AKT pathway. Taken together, the findings in this study revealed that the newly identified circCCDC91 promotes myoblasts proliferation and differentiation, and alleviates skeletal muscle atrophy by directly binding to miR-15 family via activating IGF1-PI3K/AKT signaling pathway in chicken.

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.

Chicken interferon regulatory factor 7 (IRF7) can control ALV-J virus infection by triggering type I interferon production through affecting genes related with innate immune signaling pathway.

In order to breed new birds with strong disease resistance, it is necessary to first understand the mechanism of avian antiviral response. Interferon regulatory factor 7 (IRF7) is not only a member of type I interferons (IFNs) regulatory factor (IRFs) family, but also a major regulator of the IFN response in mammals. However, whether IRF7 is involved in the host innate immune response remains unclear in poultry, due to the absence of IRF3. Here, we first observed by HE stains that with the increase of the time of ALV-J challenge, the thymus was obviously loose and swollen, the arrangement of liver cell was disordered, and the bursa of fabricius formed vacuolated. Real-time PCR detection showed that the expression level of IRF7 gene and related immune genes in ALV-J group was significantly higher than that in control group (P < 0.05). To further study the role of chicken IRF7 during avian leukosis virus subgroup J (ALV-J) infection, we constructed an induced IRF7 overexpression and interfered chicken embryo fibroblasts (CEFs) cell and performed in vitro infection using low pathogenic ALV-J and virus analog poly(I:C). In ALV-J and poly(I:C) stimulated CEFs cells, the expression level of STAT1, IFN-α, IFN-ß, TLR3 and TLR7 were increased after IRF7 overexpressed, while the results were just the opposite after IRF7 interfered, which indicating that IRF7 may be associated with Toll-like receptor signaling pathway and JAK-STAT signaling pathway. These findings suggest that chicken IRF7 is an important regulator of IFN and is involved in chicken anti-ALV-J innate immunity.

Sexual Maturity Promotes Yolk Precursor Synthesis and Follicle Development in Hens via Liver-Blood-Ovary Signal Axis.

Several reproductive hormones were reported to be involved in regulating egg yolk precursor synthesis in chickens; however, the mechanism that shows how the liver-blood-ovary signal axis works in relation to age changes has not been reported yet. Therefore, in this study, we observe the morphology and histology of the liver and ovary and determine the serum biochemical parameters and the expression abundance of the critical genes from d90 to 153. Results show that the body weight and liver weight were significantly increased from d132, while the ovary weight increased from d139. Aside from the increase in weight, other distinct changes such as the liver color and an increased deposition of large amounts of yolk precursors into the ovarian follicles were observed. On d139, we observed small fatty vacuoles in the hepatocytes. The results of serum biochemical parameters showed a significant increase in the estradiol (E2) level, first on d125, and then it reached its peak on d132. Meanwhile, the levels of follicle-stimulating hormone (FSH) increased initially and then remained at a high level from d146 to d153, while the levels of luteinizing hormone (LH) increased significantly on d132 and reached the top level on d153. Moreover, the levels of lecithin (LEC), vitellogenin (VTG), very low density lipoprotein y (VLDLy), triglyceride (TG), and total cholesterol (TC) were significantly increased at d125 and were close from d146 to d153. The mRNA and protein expression of estrogen receptor-alpha (ER-α) and E2 levels in the liver and serum, respectively, showed similar changes. Moreover, with reference to an increase in serum E2 level, the mRNA expression of genes related to yolk precursor synthesis (very low density apolipoprotein-II, ApoVLDL-II) and vitellogenin-II (VTG-II), lipogenesis (fatty acid synthase, FAS), and lipid transport (microsomal triglyceride transport protein, MTTP) in the liver showed up-regulation. These results suggest that the correlation between liver-blood-ovary alliances regulate the transport and exchange of synthetic substances to ensure synchronous development and functional coordination between the liver and ovary. We also found that E2 is an activator that is regulated by FSH, which induces histological and functional changes in the hepatocytes through the ER-α pathway.

High Expression of miR-204 in Chicken Atrophic Ovaries Promotes Granulosa Cell Apoptosis and Inhibits Autophagy.

Chicken atrophic ovaries have decreased volume and are indicative of ovarian failure, presence of a tumor, or interrupted ovarian blood supply. Ovarian tumor is accompanied by an increase in follicular atresia, granulosa cell (GC) apoptosis, and autophagy. In a previous study, we found using high throughput sequencing that miR-204 is highly expressed in chicken atrophic ovaries. Thus, in the present study, we further investigated its function in GC apoptosis and autophagy. We found that overexpression of miR-204 reduced mRNA and protein levels of proliferation-related genes and increased apoptosis-related genes. Cell counting kit-8 (CCK-8), 5-ethynyl-2-deoxyuridine (EdU), and flow cytometry assays revealed that miR-204 inhibited GC proliferation and promoted apoptosis. Furthermore, we confirmed with reporter gene assays that Forkhead box K2 (FOXK2) was directly targeted by miR-204. FOXK2, as a downstream regulator of phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signal pathways, promoted GC proliferation and inhibited apoptosis. Subsequently, we observed that miR-204 was involved in GC autophagy by targeting Transient Receptor Potential Melastatin 3 (TRPM3). The luciferase activities of the two binding sites of TRPM3 were decreased in response to treatment with a miR-204 mimic, and the autophagic flux was increased after miR-204 inhibition. However, overexpression of miR-204 had opposite results in autophagosomes and autolysosomes. miR-204 inhibits GC autophagy by suppressing the protein expression of TRPM3/AMP-activated protein kinase (AMPK)/ULK signaling pathway components. Inhibition of miR-204 enhanced autophagy by accumulating and degrading the protein levels of LC3-II (Microtubule Associated Protein Light Chain 3B) and p62 (Protein of 62 kDa), respectively, whereas miR-204 overexpression was associated with contrary results. Immunofluorescence staining showed that there was a significant reduction in the fluorescent intensity of LC3B, whereas p62 protein was increased after TRPM3 silencing. Collectively, our results indicate that miR-204 is highly expressed in chicken atrophic ovaries, which promotes GC apoptosis via repressing FOXK2 through the PI3K/AKT/mTOR pathway and inhibits autophagy by impeding the TRPM3/AMPK/ULK pathway.

Screening of immune biomarkers in different breeds of chickens infected with J subgroup of avian leukemia virus by proteomic.

Avian leucosis (AL) is a disease characterized by tumors and is caused by the avian leukosis virus (ALV). Because of the high variability of viruses and complex pathogenic mechanisms, screening and breeding J subgroup of ALV (ALV-J) resistant avian breeds is one of the strategies for prevention and treatment of AL, thus screening of significant immune markers is needed to promote the development of disease-resistant breeds. In this study, data-independent acquisition (DIA) technology was used to detect the DEPs of three breeds of chicken according to different comparison to investigate the potential markers. Results showed special DEPs for spleen development of each breed were detected, such as PCNT, DDB2, and ZNF62. These DEPs were involved in intestinal immune network used in production of IgA signaling pathways and related to immune response which can be used as potential markers for spleen development in different breeds. The DEPs such as RAB44 and TPN involved in viral myocarditis, transcriptional misregulation in cancer, and tuberculosis can be used as potential markers of spleen immune response after ALV-J infection in chickens. Pair-wise analysis was performed for the three breeds after the infection of ALV-J. The proteins such as RFX1, TAF10, and VH1 were differently expressed between three breeds. These DEPs involved in antigen processing and expression, acute myelogenous leukemia, and viral carcinogenesis can be used as potential immune markers after ALV-J infection of different genetic backgrounds. The screening of potential markers at protein level provides a strong theoretical research basis for disease resistance breeding in poultry.

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.

Inhibin A regulates follicular development via hormone secretion and granulosa cell behaviors in laying hens.

Inhibin A regulates follicular development, and its expression level is related to physiological activities, such as the recruitment, selection, and predominance during follicular development. Therefore, examining inhibin A and its regulatory effects on the reproductive performance of poultry is crucial. In this study, we measured the mRNA and protein abundances of INHA and INHBA in the chicken reproductive system and determined the hormone secretion and apoptosis of follicular granulosa cells (GCs) after being treated with inhibin A protein, and flow cytometry was performed to analyze GC apoptosis in INHA-specific small RNA interference (siRNA). We detected that INHA and INHBA were mainly expressed in chicken follicles. The highest INHA mRNA abundance was found in the fifth largest preovulatory follicle (F5) (P < 0.05). INHBA mRNA expression in the largest preovulatory follicle (F1) was significantly higher than those in other follicles (P < 0.05). Similar results were found for INHA and INHBA protein expression in those follicles (P < 0.05). Treatment with inhibin A protein increased the activity of GCs in a dose-dependent manner (P < 0.05), which was characterized by decreased gene expression of pro-apoptotic factors Bax and Caspase-3 (P < 0.05) and increased expression of proliferation genes Bcl-2 and PCNA (P < 0.05). Additionally, inhibin A significantly increased the secretion of progesterone and estradiol (P < 0.05). RNAi-mediated knockdown of INHA increased apoptosis in GCs via a Caspase-3-dependent mitochondrial 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.

The Regulatory Functions of Circular RNAs in Digestive System Cancers.

Circular ribonucleic acids (circRNAs), which are a type of covalently closed circular RNA, are receiving increasing attention. An increasing amount of evidence suggests that circRNAs are involved in the biogenesis and development of multiple diseases such as digestive system cancers. Dysregulated circRNAs have been found to act as oncogenes or tumour suppressors in digestive system cancers. Moreover, circRNAs are related to ageing and a wide variety of processes in tumour cells, such as cell apoptosis, invasion, migration, and proliferation. Moreover, circRNAs can perform a remarkable multitude of biological functions, such as regulating splicing or transcription, binding RNA-binding proteins to enable function, acting as microRNA (miRNA) sponges, and undergoing translated into proteins. However, in digestive system cancers, circRNAs function mainly as miRNA sponges. Herein, we summarise the latest research progress on biological functions of circRNAs in digestive system cancers. This review serves as a synopsis of potential therapeutic targets and biological markers for digestive system cancer.

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.

Peroxisome proliferator-activated receptor-coactivator 1-beta (PGC-1ß) modulates the expression of genes involved in adipogenesis during preadipocyte differentiation in chicken.

To study the influence of the PGC-1ß gene on chicken adipocyte proliferation and differentiation, we constructed RNA interference (RNAi) vectors that target the PGC-1ß gene and transfected these vectors into adipocytes. Oil Red O staining and a CCK-8 cell kit were used to determine cell triglyceride accumulation status and cell proliferation after transfection, respectively. The mRNA abundances of PGC-1ß and adipocyte-differentiation-related genes (PPARγ, C/EBPα, SREBP-1c, FAS, and A-FABP) were detected by real-time PCR. The results showed that the mRNA and protein abundances of PGC-1ß in PGC-1ß-shRNA transfected adipocytes were significantly lower than those in the control. Interference decreased cell differentiation, but did not depress the cell proliferation. PGC-1ß interference impeded the triglyceride accumulation, the mRNA expression levels of nuclear receptors PPARγ and SREBP-1c, and fatty acid synthetase (FAS), and both proteins PPARγ and SREBP-1c, and the fatty acids transporting protein A-FABP. Generally, PGC-1ß modulated the cell differentiation and triglyceride accumulation in chicken adipocytes.

Exosomes Transmit Viral Genetic Information and Immune Signals may cause Immunosuppression and Immune Tolerance in ALV-J Infected HD11 cells.

Avian leukosis virus (ALV) is oncogenic retrovirus that not only causes immunosuppression but also enhances the host's susceptibility to secondary infection. Exosomes play vital role in the signal transduction cascades that occur in response to viral infection. We want to explore the function of exosomes in the spread of ALV and the body's subsequent immunological response. RNA-sequencing and the isobaric tags for relative and absolute quantitation (iTRAQ) method were used to detect differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) in exosomes secreted by macrophage cells in response to injection with ALV subgroup J (ALV-J). RNA-sequencing identified 513 DEGs in infected cells, with specific differential regulation in mRNA involved in tight junction signaling, TNF signaling, salmonella infection response, and immune response, among other important cellular processes. Differential regulation was observed in 843 lncRNAs, with particular enrichment in those lncRNA targets involved in Rap1 signaling, HTLV-I infection, tight junction signaling, and other signaling pathways. A total of 50 DEPs were identified in the infected cells by iTRAQ. The proteins enriched are involved in immune response, antigen processing, the formation of both MHC protein and myosin complexes, and transport. Combined analysis of the transcriptome and proteome revealed that there were 337 correlations between RNA and protein enrichment, five of which were significant. Pathways that were enriched on both the RNA and protein levels were involved in pathways in cancer, PI3K-Akt signaling pathway, Endocytosis, Epstein-Barr virus infection. These data show that exosomes are transmitters of intercellular signaling in response to viral infection. Exosomes can carry both viral nucleic acids and proteins, making it possible for exosomes to be involved in the viral infection of other cells and the transmission of immune signals between cells. Our sequencing results confirme previous studies on exosomes and further find exosomes may cause immunosuppression and immune tolerance.

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.