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Cholesterol 7alpha-hydroxylase (CYP7A1) is the rate limiting enzyme of the bile acid biosynthetic pathway to convert cholesterol to bile acids, which is a major output pathway for cholesterol catabolism. In this study, we aimed to assess the potential regulatory mechanisms of microRNA-185 (miR-185) in cholesterol and bile acid homeostasis.Eight-week-old male ApoE KO mice fed a high-fat diet (HFD) were injected with lentiviruses encoding antisense miR-185 (miR-185-inh). Microarrays were applied to profile miR-185-regulated genes involved in bile acid metabolism. The expression of potential targets of miR-185 was validated using qPCR and Western blotting assay in human hepatoma HepG2 cells.The administration of miR-185-inh correlated with decreased serum total bile acids levels in ApoE KO mice. Microarray gene profiling revealed that inhibition of miR-185 upregulated hepatic CYP7A1 expression in vivo, which was further validated in HepG2 cells and primary hepatic cells in vitro by overexpression or inhibition of miR-185. Furthermore, it was revealed that miR-185 regulated CYP7A1 expression via a FoxO1-involved indirect pathway and that miR-185 directly modulated FoxO1 expression by binding to its mRNA 3'UTR in a traditional post-transcriptional manner. Besides, we also observed that miR-185 regulated CYP7A1 expression by increasing p-AKT/AKT level, which induced the phosphorylation of FoxO1 and promoted FoxO1 degradation at a post-translational level.This study provides convincing evidence on the critical role of miR-185 in FoxO1 modulation at both post-transcriptional and post-translational levels, which accounts for the effects on CYP7A1 gene and its mediated cholesterol-bile acid metabolism. These results suggest an important role of miR-185 as a novel atherosclerosis-protective target for drug discovery.

Excessive hepatic glucose production (HGP) is a key factor promoting hyperglycemia in diabetes. Hepatic cryptochrome 1 (CRY1) plays an important role in maintaining glucose homeostasis by suppressing forkhead box protein O1 (FOXO1)-mediated HGP. Although downregulation of hepatic CRY1 appears to be associated with increased HGP, the mechanism(s) by which hepatic CRY1 dysregulation confers hyperglycemia in diabetic subjects is largely unknown. In this study, we demonstrate that a reduction in hepatic CRY1 protein is stimulated by elevated E3 ligase F-box and leucine-rich repeat protein 3 (FBXL3)-dependent proteasomal degradation in diabetic mice. In addition, we found that GSK3b-induced CRY1 phosphorylation potentiates FBXL3-dependent CRY1 degradation in the liver. Accordingly, in diabetic mice, GSK3b inhibitors effectively decreased HGP by facilitating the effect of CRY1-mediated FOXO1 degradation on glucose metabolism. Collectively, these data suggest that tight regulation of hepatic CRY1 protein stability is crucial for maintaining systemic glucose homeostasis.

The current study aimed to examine the effects of echinacoside on cognitive impairment in mice after exposure to sevoflurane. To examine the role of FOXO1, si-FOXO1 and si-con were injected into the hippocampus through the left lateral cerebral ventricles. Sevoflurane-induced mice had serious cognitive dysfunction. However, pretreatment with echinacoside alleviated the cognitive dysfunction, as measured by a shortened escape latency time, and increased platform crossing times, the percentage of distance in the target quadrant and Y-maze spontaneous alternations. In addition, we found that echinacoside elevated FOXO1 expression in the hippocampus; increased the expression of autophagy-related proteins including Beclin 1, ATG5, ATG7, and LC3; and reduced P62 expression. Silencing of FOXO1 aggravated the cognitive deficits and reduced expression of the autophagy-related markers, whereas the effects of si-FOXO1 on memory were abrogated by echinacoside. Echinacoside attenuated the cognitive impairment in sevoflurane-induced mice through FOXO1-mediated autophagy.

Purpose: Circular RNA (circRNA) has been proved to play a vital role in atherosclerosis (AS) progression, and vascular smooth muscle cells (VSMCs) are involved in the progression of AS. However, the in-depth mechanism by which circRNA regulates VSMC proliferation and migration remains to be elusive. Methods: We used tumor necrosis factor-alpha (TNF-α) to treat VSMCs to establish a cell model of AS. We used Cell Counting Kit-8, terminal-deoxynucleoitidyl transferase–mediated nick end labeling, and transwell assays to assess the proliferation, apoptosis, and migration in TNF-α-induced VSMCs. Moreover, the interaction between molecules was measured by RNA-binding protein immunoprecipitation, RNA pull-down, and luciferase reporter assays. Results: Our study found that a novel circRNA hsa_circ_0030042, which is derived from its host gene forkhead box O1 (FOXO1), was upregulated in TNF-α-induced VSMCs. Silencing of hsa_circ_0030042 inhibited proliferation and migration while promoting apoptosis in TNF-α-induced VSMCs. Mechanically, hsa_circ_0030042 positively regulated FOXO1 expression via sponging miR-514a-3p. Conclusions: Our study stated the vital role of the hsa_circ_0030042/miR-514a-3p/FOXO1 axis and provides a profound understanding about the circRNA in AS.

Literature evidence reveals that natural compounds are potential candidates for ameliorating obesity-associated non-alcoholic fatty liver disease (NAFLD) by targeting forkhead box O1 (FOXO1) transcription factor. FOXO1 has a dual and complex role in regulating both increase and decrease in lipid accumulation in hepatocytes and adipose tissues (AT) at different stages of NAFLD. In insulin resistance (IR), it is constitutively expressed, resulting in increased hepatic glucose output and lipid metabolism irregularity. The studies on different phytochemicals indicate that dysregulation of FOXO1 causes disturbance in cellular nutrients homeostasis, and the natural entities have an enduring impact on the mitigation of these abnormalities. The current review communicates and evaluates certain phytochemicals through different search engines, targeting FOXO1 and its downstream cellular pathways to find lead compounds as potential therapeutic agents for treating NAFLD and related metabolic disorders. The findings of this review confirm that polyphenols, flavonoids, alkaloids, terpenoids, and anthocyanins are capable of modulating FOXO1 and associated signaling pathways, and they are potential therapeutic agents for NAFLD and related complications. HIGHLIGHTS: • FOXO1 has the potential to be targeted by novel drugs from natural sources for the treatment of NAFLD and obesity. • FOXO1 regulates cellular autophagy, inflammation, oxidative stress, and lipogenesis through alternative mechanisms. • Phytochemicals treat NAFLD by acting on FOXO1 or SREBP1c and PPARγ transcription factor signaling pathways.

The sensitivity of cells to chemotherapeutic agents has a major effect on disease outcome in breast cancer patients. Unfortunately, there are numerous factors involved in the regulation of chemosensitivity, and the mechanisms need to be further investigated. Autophagy/Beclin 1 regulator 1 (Ambra1) is a key protein in the crosstalk between autophagy and apoptosis. It controls the switch between these two processes, which determines whether cells survive or die. Induction of apoptosis is the primary mechanism by which most chemotherapeutic drugs eliminate cancer cells. Recently, Ambra1 has been shown to modulate paclitaxel-induced apoptosis in breast cancer cells via the Bim/mitochondrial pathway, thereby modifying the sensitivity of cells to paclitaxel. However, how Ambra1 regulates Bim expression remains unclear. Here, we further confirmed that Bim plays an indispensable role in Ambra1's regulation of apoptosis and chemosensitivity in breast cancer cells. Furthermore, Ambra1 was found to regulate Bim expression at the transcriptional level through the Akt-FoxO1 pathway. Therefore, we propose a novel pathway, Ambra1-Akt-FoxO1-Bim, which regulates apoptosis and chemosensitivity in breast cancer cells. Thus, Ambra1 may represent a potential target for breast cancer treatment.

Alzheimer’s disease (AD) and Huntington’s disease (HD) are destructive worldwide diseases. Efforts have been made to elucidate the process of these two diseases, yet the pathogenesis remains elusive as it involves a combination of multiple factors, including genetic and environmental ones. To explore the potential role of forkhead box O1 (FOXO1) in the development of AD and HD, we identified 1,853 differentially expressed genes (DEGs) from 19,414 background genes in both the AD&HD/control and FOXO1-low/high groups. Four coexpression modules were predicted by the weighted gene coexpression network analysis (WGCNA), among which blue and turquoise modules had the strongest correlation with AD&HD and high expression of FOXO1. Functional enrichment analysis showed that DEGs in these modules were enriched in phagosome, cytokine-cytokine receptor interaction, cellular senescence, FOXO signaling pathway, pathways of neurodegeneration, GABAergic synapse, and AGE-RAGE signaling pathway in diabetic complications. Furthermore, the cross-talking pathways of FOXO1 in AD and HD were jointly determined in a global regulatory network, such as the FOXO signaling pathway, cellular senescence, and AGE-RAGE signaling pathway in diabetic complications. Based on the performance evaluation of the area under the curve of 85.6%, FOXO1 could accurately predict the onset of AD and HD. We then identified the cross-talking pathways of FOXO1 in AD and HD, respectively. More specifically, FOXO1 was involved in the FOXO signaling pathway and cellular senescence in AD; correspondingly, FOXO1 participated in insulin resistance, insulin, and the FOXO signaling pathways in HD. Next, we use GSEA to validate the biological processes in AD&HD and FOXO1 expression. In GSEA analysis, regulation of protein maturation and regulation of protein processing were both enriched in the AD&HD and FOXO1-high groups, suggesting that FOXO1 may have implications in onset and progression of these two diseases through protein synthesis. Consequently, a high expression of FOXO1 is a potential pathogenic factor in both AD and HD involving mechanisms of the FOXO signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and cellular senescence. Our findings provide a comprehensive perspective on the molecular function of FOXO1 in the pathogenesis of AD and HD.

It was to investigate the role of Gegen Qinlian decoction (GQD) in the regulation of ABTB1 gene based on PI3K/AKT/FOXO1 signaling pathway in colorectal cancer (CRC) metastasis. In this study, 10 cases of the CRC mouse model were established by inoculating CT26 cells into the spleen of mice, which were divided into the experimental group and the control group, 5 cases in each group; the control group was intragastrically administered with normal saline 0.3 mL/d, and the experimental group was intragastrically administered with GQD 0.2 mL/d at a ratio of 0.2 g medicinal materials/10 g for 10 days and sacrificed, and pathological sections were made. The expression density of signaling pathway PI3K/AKT/FOXO1 as well as gene ABTB1 was detected in the sections of the two groups, and the mechanism of action of this gene in the two groups of mice was studied. It was found that the densities of p-PI3K, p-AKT, and p-FOXO1 in the experimental group of mice were 26.55 g/cm3, 70.2 g/cm3, and 24.36 g/cm3, respectively, which were significantly increased compared with the control group, P < 0.05 ; the density of ABTB1 was 35.4 g/cm3, which was significantly increased compared with the control group, P < 0.05 ; the proliferation and migration ability of CRC cells in the experimental group were significantly decreased, P < 0.05 . GQD can promote the expression of ABTB1 by activating the PI3K/AKT/FOXO1 signaling pathway, in order to inhibit the proliferation and growth ability of CRC cells.

Objective. To investigate the molecular function of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB) underlying Alzheimer’s disease (AD). Methods. RNA sequencing data were used to filtrate differentially expressed genes (DEGs) in AD/nondementia control and PIK3CB-low/high groups. An unbiased coexpression network was established to evaluate module-trait relationships by using weight gene correlation network analysis (WGCNA). Global regulatory network was constructed to predict the protein-protein interaction. Further cross-talking pathways of PIK3CB were identified by functional enrichment analysis. Results. The mean expression of PIK3CB in AD patients was significantly lower than those in nondementia controls. We identified 2,385 DEGs from 16,790 background genes in AD/control and PIK3CB-low/high groups. Five coexpression modules were established using WGCNA, which participated in apoptosis, axon guidance, long-term potentiation (LTP), regulation of actin cytoskeleton, synaptic vesicle cycle, FoxO, mitogen-activated protein kinase (MAPK), and vascular endothelial growth factor (VEGF) signaling pathways. DEGs with strong relation to AD and low PIK3CB expression were extracted to construct a global regulatory network, in which cross-talking pathways of PIK3CB were identified, such as apoptosis, axon guidance, and FoxO signaling pathway. The occurrence of AD could be accurately predicted by low PIK3CB based on the area under the curve of 71.7%. Conclusions. These findings highlight downregulated PIK3CB as a potential causative factor of AD, possibly mediated via apoptosis, axon guidance, and FoxO signaling pathway.

CD4+CD25+FOXP3+ regulatory T (Treg) cells control immunological tolerance. Treg cells are generated in the thymus (tTreg) or in the periphery. Their superior lineage fidelity makes tTregs the preferred cell type for adoptive cell therapy (ACT). How human tTreg cells develop is incompletely understood. By combining single-cell transcriptomics and flow cytometry, we in this study delineated three major Treg developmental stages in the human thymus. At the first stage, which we propose to name pre-Treg I, cells still express lineage-inappropriate genes and exhibit signs of TCR signaling, presumably reflecting recognition of self-antigen. The subsequent pre-Treg II stage is marked by the sharp appearance of transcription factor FOXO1 and features induction of KLF2 and CCR7, in apparent preparation for thymic exit. The pre-Treg II stage can further be refined based on the sequential acquisition of surface markers CD31 and GPA33. The expression of CD45RA, finally, completes the phenotype also found on mature recent thymic emigrant Treg cells. Remarkably, the thymus contains a substantial fraction of recirculating mature effector Treg cells, distinguishable by expression of inflammatory chemokine receptors and absence of CCR7. The developmental origin of these cells is unclear and warrants caution when using thymic tissue as a source of stable cells for ACT. We show that cells in the major developmental stages can be distinguished using the surface markers CD1a, CD27, CCR7, and CD39, allowing for their viable isolation. These insights help identify fully mature tTreg cells for ACT and can serve as a basis for further mechanistic studies into tTreg development.

Melatonin can regulate apoptosis and autophagy of mouse Leydig cells, but its specific mechanism is still unclear.In this study, we used the TM3 cell line as the research object, and used H2O2 to induce autophagy. After adding 10 ng/ml melatonin, we used qRT-PCR and western-blot to detect autophagy-related gene and protein expression, and flow cytometry to detect cellular ROS level.The results showed that melatonin can significantly inhibit the occurrence of autophagy, accompanied by a significant decrease in the expression of Becn1, LC3, and FOXO1 (P 0.05), a significant increase in the expression of p62 and pAKT (P 0.05), and a significant decrease in ROS level (P 0.05). After added the inhibitor of AKT perifosine, the effect of melatonin on inhibiting autophagy was reversed. On this basis, we used small RNA interference technology to knock down the expression of FOXO1, and found that there was no significant change of the expression of genes and proteins related to autophagy and ROS level.In summary, melatonin can inhibit H2O2-induced autophagy in TM3 cells through the AKT/FOXO1 pathway.

Neuroinflammation plays a key role in cerebrovascular disease (CVD). Neuropsychiatric disorders appear to share an epidemiological association with inflammation, but the mechanisms are unclear. Forkhead box 1 (FoxO1) regulates inflammatory signaling in diabetes and cardiovascular diseases, but its role in psychological stress-induced neuroinflammation remains unknown. Therefore, we investigated the potential involvement of FoxO1 in repeated social defeat stress (RSDS)-induced neuroinflammation. 6-week-old male C57BL/6 J mice were randomly divided into RSDS or control groups. In the RSDS group, mice (18-22 g) were individually subjected to social defeat by an 8-week-old CD-1 mouse (28-32 g) for 10 min daily for 10 consecutive days. At 24 h after this 10-day process, corticosterone (CORT), epinephrine (EPI), hydrogen peroxide, and inflammatory factors (TNF-α, IL-6, IL-1β, and VCAM-1) from serum and brain tissues were assayed using ELISA, real-time PCR, and Western blot. Iba-1 was determined by immunofluorescence (IF), and FoxO1 siRNA was transfected into BV2 cells to further analyze the expression of inflammatory factors.RSDS significantly increased the levels of TNF-α, IL-6, IL-1β, and VCAM-1 in the serum; it also increased both mRNA and protein expression of these in the brain. FoxO1 was significantly increased after stress, while its knockdown significantly suppressed stress-induced inflammation. Immunofluorescence demonstrated the activation of microglia in the setting of RSDS.RSDS induced a measurable inflammatory response in the blood and brain, and FoxO1 was demonstrated in vitro to aggravate stress-induced inflammation.

Superoxide dismutase 3 (SOD3), one of SOD isozymes, maintains extracellular redox homeostasis through the dismutation reaction of superoxide. Loss of SOD3 in tumor cells induces oxidative stress and exacerbates tumor progression; however, interestingly, overexpression of SOD3 also promotes cell proliferation through the production of hydrogen peroxide. In this study, we investigated the functional role of SOD3 in human breast cancer MDA-MB-231 cell migration and the molecular mechanisms involved in high expression of SOD3 in MDA-MB-231 cells and human monocytic THP-1 cells. The level of histone H3 trimethylation at lysine 27 (H3K27me3), a marker of gene silencing, was decreased in 12

Background: Infantile pneumonia is an acute inflammatory lesion of the lung caused by mycoplasma pneumonia. Indeed, Twist2 signaling pathway controls inflammatory reaction, oxidative stress, and other biological reaction. However, the regulation of Twist2 on the inflammation in infantile pneumonia remains unclear. This study explained that the function and mechanism of Twist2 in infantile pneumonia. Methods: The subjects included the serum samples of 12 patients with infantile pneumonia and normal healthy volunteers from Hunan Children’s Hospital. Besides, mice were given with lipopolysaccharide (LPS) into the lung. Moreover, RAW264.7 macrophages were stimulated with LPS for 4 h and added to the culture medium. Results: In present study, in serum of patients with infantile pneumonia or lung tissue of mice model with infantile pneumonia, TWIST2 expression was lessened. Apart from that, TWIST2 protein could reduce the inflammatory reaction in mice model with infantile pneumonia, resulting in an inhibition in lung injury. Conversely, over-expression of TWIST2 also decreased inflammatory reaction in macrophages model via the regulation of FOXO1/NLRP3 pathway. Downregulation of TWIST2 promoted the inflammation in macrophages model by the regulation of FOXO1/NLRP3 pathway. Conclusion: According to the findings, present study have identified that the TWIST2 could reduce the inflammation of infantile pneumonia by NLRP3 inflammasome through the regulation of mitochondrial permeability transition and the induction of FOXO1 expression.

Injury and dysfunction of endothelial cells (ECs) are closely related to the pathogenesis of steroid-induced osteonecrosis of the femoral head (ONFH), while MicroRNAs (miRNAs) play an essential role in the processes. Extracorporeal shockwave treatment (ESWT) has been used in the non-invasive treatment of various diseases including musculoskeletal and vascular disorders. In particular, ESWT with low energy levels showed a beneficial effect in ischemic tissues. However, there has been no comprehensive assessment of the effect of ESWT and miRNAs on steroid-induced ONFH. In the present study, we investigated the role and mechanism of ESWT and miRNAs both in vitro and in vivo. Using a steroid-induced ONFH rat model, we found that ESWT significantly enhances proliferation and angiogenesis as well as alleviates apoptosis. In two types of ECs, ESWT can promote cell proliferation and migration, enhance angiogenesis, and inhibit apoptosis. Notably, our study demonstrates that miR-135b is downregulated and modulated forkhead box protein O1 (FOXO1) in ECs treated with dexamethasone. Remarkably, both miR-135b knockdown and FOXO1 overexpression reversed the beneficial effect of ESWT on ECs. Additionally, our data suggest that ESWT activates the FOXO1-related pathway to impact proliferation, apoptosis, and angiogenesis. Taken together, this study indicates that ESWT relieves endothelial injury and dysfunction in steroid-induced ONFH via miR-135b targeting FOXO1.

Glioblastoma (GBM) is the most universal and devastating primary intracranial neoplasm in the central nervous system. Urolithin A (UA) possesses many pharmacological and biological activities, but its function in GBM is not clear. CCK-8 and colony formation test were used to measure the anti-proliferative potency of UA against GBM cells. Flow cytometry was applied to evaluate cell cycle arrest and apoptosis of U251 and U118 MG cells upon UA incubation. Quantitative real-time PCR and western blotting were conducted to test the regulatory effect of UA on the expression of Sirt1 and FOXO1. Immunodeficient mice were implanted with GBM cells for in vivo validation of the anti-cancer effect of UA. We found UA repressed the proliferation, migration and invasion of glioblastoma cells, while also inhibiting the induction of colony formation ability and epithelial to mesenchymal transition (EMT) in a time- or dose-dependent manner. The does-dependent relationship of UA inducing the cell cycle arrest and apoptosis of glioblastoma cells was identified. Furthermore, UA could enhance the expression levels of Sirt1 and FOXO1 and the knockdown of Sirt1 blocked the inhibitory effects of UA on the proliferation and migration of glioblastoma cells and correspondingly modified the expression level of FOXO1. Overexpression of Sirt1 restored the despaired inhibitory effect of UA induced by Sirt1 knockout on the proliferation and migration of glioblastoma cells. In animal experiments, UA decreased the tumor size and weight of glioblastoma in xenograft nude mice and promoted the expression of Sirt1 and FOXO1 in transplanted tumors. Our findings presented in this study indicate that UA exerts a repressive effect on glioblastoma cells in vivo and in vitro by regulating the Sirt1-FOXO1 axis via the ERK and AKT pathways, indicating that UA is a new novel therapeutic candidate for the treatment of glioblastoma.

Somatic point mutations of the FOXO1 transcription factor were reported in non-Hodgkin lymphoma including diffuse large B-cell lymphoma, follicular lymphoma and Burkitt lymphoma. These alterations were associated with a poor prognosis and resistance to therapy. Nearly all amino acid substitutions are localized in two major clusters, affecting either the N-terminal region (Nt mutations) or the forkhead DNA-binding domain (DBD mutations). While recent studies have focused on Nt mutations, we characterized FOXO1 DBD mutants. We analyzed their transcriptional activity, DNA binding, phosphorylation and protein–protein interaction. The majority of DBD mutants showed a decrease in activity and DNA binding, while preserving AKT phosphorylation and interaction with the cytoplasmic ATG7 protein. In addition, we investigated the importance of conserved residues of the α-helix 3 of the DBD. Amino acids I213, R214, H215 and L217 appeared to be crucial for FOXO1 activity. Our data underlined the key role of multiple amino-acid residues of the forkhead domain in FOXO1 transcriptional activity and revealed a new type of FOXO1 loss-of-function mutations in B-cell lymphoma.

Colorectal adenocarcinoma (COAD) is a prevalent malignant tumor. Cancer-associated fibroblasts (CAFs)-derived extracellular vesicles (EVs) (CAFs-EVs) are implicated in COAD treatment. This study explored the mechanism of CAFs-EVs in COAD. CAFs and normal fibroblast (NFs) were isolated from COAD tissues and adjacent normal tissues. Vimentin, α-SMA, and FAP expressions were detected. EVs were isolated from CAFs and identified. SW480 and HCT116 cells were co-incubated with EVs. The EV uptake and COAD cell malignant behaviors were assessed. EV-treated SW480 and HCT116 cells were co-cultured with human umbilical vein endothelial cells (HUVECs). Extensive analyses were conducted to examine HUVEC proliferation, migration, and angiogenesis, and miR-135b-5p expression in COAD cells, and SW480 and HCT116 cells. CAFs were transfected with the miR-135b-5p inhibitor. miR-135b-5p downstream targets were predicted. FOXO1 expression in the co-culture system was determined and then overexpressed to evaluate its role in HUVECs mediated by COAD cells. COAD mouse model was established by transplanting SW480 cells into nude mice and injecting with EVs. Tumor growth rate, volume, and weight were examined. Ki67, VEGF, CD34, FOXO1 expressions, and VEGF content were detected. CAFs-EVs promoted COAD cell malignant behaviors and COAD cells-mediated HUVEC proliferation, migration, and angiogenesis. CAFs-EVs delivered miR-135b-5p into COAD cells. miR-135b-5p targeted FOXO1. Inhibition of miR-135b-5p in EVs or overexpression of FOXO1 partially reversed the effect of EVs on promoting COAD-induced angiogenesis. CAFs-EVs promoted tumor proliferation and angiogenesis of COAD in vivo. CAFs-EVs delivered miR-135b-5p into COAD cells to downregulate FOXO1 and promote HUVECs proliferation, migration, and angiogenesis.

Rhabdomyosarcomas comprise the single largest category of soft tissue sarcomas in children and adolescents in the United States, occurring in 4.5 million persons aged less than 20 years. Based on the clinic-pathologic features and genetic abnormalities identified, the rhabdomyosarcomas are classified into embryonal, alveolar, spindle cell / sclerosing and pleomorphic subtypes. Each subtype shows distinctive morphology and has characteristic genetic abnormalities. This review discusses the evolution of the classification of rhabdomyosarcoma to the present day, along with a discussion of key histomorpholgic and genetic features of each subtype and the diagnostic approach to these tumors.

The abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development and progression of diabetic vascular complications. In high-glucose (HG) conditions, endothelial cells (ECs) act as the first barrier to damaging stimuli and trigger a multi-response, including EC and VSMC crosstalk. However, the crosstalk pathways between ECs and VSMCs under HG conditions remain unclear. This study aimed to explore the roles and underlying mechanism of exosomes derived from ECs in the crosstalk between ECs and VSMCs. Our results showed that mouse aortic endothelial cell (MAEC)–secreted exosomes could promote the proliferation and inhibit the apoptosis of VSMCs induced by HG. Furthermore, we isolated the exosomes secreted by MAECs and found that exosomes derived from MAECs that were exposed to HG could transfer circHIPK3, which is enriched in MAEC-derived exosomes, to VSMCs. Exosomal circHIPK3 promoted the proliferation and inhibited the apoptosis of VSMCs. circHIPK3 sponged miR-106a-5p to relieve its repression of forkhead box O1 (Foxo1) expression. The increased expression of Foxo1 acted as a transcription factor to promote Vcam1 expression, thus facilitating the uptake of MAEC-derived exosomes by VSMCs. The results of this study suggested that exosomal circHIPK3 derived from MAECs promotes the proliferation of VSMCs induced by HG via the miR-106a-5p/Foxo1/Vcam1 pathway.

Sarcopenia is characterized by the loss of muscle mass and strength, and one of its major molecular mechanisms is muscle protein turnover. Quinoa, the grain-like food crop, is a health nutrient used to treat diseases that predispose individuals to muscle wasting, including cardiovascular disorders, diabetes mellitus, and cancer. Quinoa secondary metabolites have recently been demonstrated to regulate protein turnover (including protein synthesis and degradation), a main biological process within muscle cells, through diverse signals (such as the p38 MAPK, TNF-α, and IGF-1/PI3K/Akt/FOXO pathways). Here, we describe how quinoa functions in the main pathway of protein synthesis and degradation, screen promising pharmacological components in nutritional applications, and provide guidance for the effects of quinoa products in sarcopenia.

Type 2 diabetes (T2D) is a multifactorial and polygenetic disease, although its exact etiology remains poorly understood. The objective of this study was to identify key biomarkers and potential molecular mechanisms in the development of T2D. Human RNA-Seq datasets across different tissues (GSE18732, GSE41762, and GSE78721) were collected from the Gene Expression Omnibus (GEO) database and differentially expressed genes (DEGs) between T2D and controls were identified using differential analysis. A total of 90 overlapping DEGs were identified, among which YTHDF2, DDX21, and MDM2 were considered as key genes due to their central positions in the PPI network and the same regulatory pattern in T2D. Logistic regression analysis showed that low expression of the key genes increased the risk of T2D. Enrichment analysis revealed that the key genes are involved in various important biological functions and signaling pathways including Notch, Fork head box O (FOXO), and phosphoinositide 3-kinase (PI3K)-Akt. RT-qPCR and Western blot analysis showed that all three key genes were down-regulated in pancreatic islets of both prediabetic and diabetic mouse models. Finally, the insulin-sensitizer, pioglitazone was used to treat db/db mice and immunofluorescence analysis showed that the expression of all three key genes was significantly down-regulated in db/db islets, an effect that was overcome by pioglitazone treatment. Together, these results suggest that the identified key genes could be involved in the development of T2D and serve as potential biomarkers and therapeutic targets for this disease.

We investigated the effect of the long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) on hepatocellular carcinoma (HCC) tumorigenesis and progression by targeting miR-5195-3p and transcription factor forkhead box O1 (FOXO1) to identify a novel target for HCC treatment. HCC clinical samples were collected, and cell counting kit-8 (CCK-8), and transwell migration and invasion assays were performed. Furthermore, interaction was detected via double luciferase reporter and RNA pull-down assays. MEG3, miR-5195-3p, and FOXO1 expression was determined by quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting. Xenograft tumor models were established to investigate the effect of MEG3 in vivo. Compared with normal tissues, MEG3 expression was significantly downregulated in HCC tissues. MEG3 overexpression inhibited the viability and migration of HCC cells. Double luciferase reporter and RNA pull-down assays confirmed the binding between MEG3 and miR-5195-3p as well as between miR-5195-3p and FOXO1. RT-qPCR and Western blotting results showed that MEG3 inhibited the expression of miR-5195-3p and promoted that of FOXO1. Additionally, MEG3 overexpression inhibited HCC tumorigenesis and progression in xenograft tumor models while depletion of MEG3 exerted the opposite way. Therefore, the lncRNA MEG3 inhibits HCC tumorigenesis and progression through the miR-5195-3p/FOXO1 signaling axis.

As a highly regenerative organ, the intestine is a promising source for cellular reprogramming for replacing lost pancreatic β cells in diabetes. Gut enterochromaffin cells can be converted to insulin-producing cells by forkhead box O1 (FoxO1) ablation, but their numbers are limited. In this study, we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine. Accordingly, lineage-tracing experiments show that, upon genetic or pharmacologic FoxO1 ablation, the Paneth/goblet lineage can also undergo conversion to the insulin lineage. We designed a screening platform in gut organoids to accurately quantitate β-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process in mice and human adult intestinal organoids. We identified a triple blockade of FOXO1, Notch, and TGF-β that, when tested in insulin-deficient streptozotocin (STZ) or NOD diabetic animals, resulted in near normalization of glucose levels, associated with the generation of intestinal insulin-producing cells. The findings illustrate a therapeutic approach for replacing insulin treatment in diabetes.

Oncogenic FOXO1 gene fusions drive a subset of rhabdomyosarcoma (RMS) with poor survival; to date, these cancer drivers are therapeutically intractable. To identify new therapies for this disease, we undertook an isogenic CRISPR-interference screen to define PAX3-FOXO1-specific genetic dependencies and identified genes in the GATOR2 complex. GATOR2 loss in RMS abrogated aa-induced lysosomal localization of mTORC1 and consequent downstream signaling, slowing G1-S cell cycle transition. In vivo suppression of GATOR2 impaired the growth of tumor xenografts and favored the outgrowth of cells lacking PAX3-FOXO1. Loss of a subset of GATOR2 members can be compensated by direct genetic activation of mTORC1. RAS mutations are also sufficient to decouple mTORC1 activation from GATOR2, and indeed, fusion-negative RMS harboring such mutations exhibit aa-independent mTORC1 activity. A bisteric, mTORC1-selective small molecule induced tumor regressions in fusion-positive patient-derived tumor xenografts. These findings highlight a vulnerability in FOXO1 fusion-positive RMS and provide rationale for the clinical evaluation of bisteric mTORC1 inhibitors, currently in phase I testing, to treat this disease. Isogenic genetic screens can, thus, identify potentially exploitable vulnerabilities in fusion-driven pediatric cancers that otherwise remain mostly undruggable.

Background Skeletomuscular diseases result in significant muscle loss and decreased performance, paralleled by a loss in mitochondrial and oxidative capacity. Insulin and insulin‐like growth factor‐1 (IGF‐1) are two potent anabolic hormones that activate a host of signalling intermediates including the serine/threonine kinase AKT to influence skeletal muscle physiology. Defective AKT signalling is associated with muscle pathology, including cachexia, sarcopenia, and disuse; however, the mechanistic underpinnings remain unresolved. Methods To elucidate the role of AKT signalling in muscle mass and physiology, we generated both congenital and inducible mouse models of skeletal muscle‐specific AKT deficiency. To understand the downstream mechanisms mediating AKT's effects on muscle biology, we generated mice lacking AKT1/2 and FOXO1 (M‐AKTFOXO1TKO and M‐indAKTFOXO1TKO) to inhibit downstream FOXO1 signalling, AKT1/2 and TSC1 (M‐AKTTSCTKO and M‐indAKTTSCTKO) to activate mTORC1, and AKT1/2, FOXO1, and TSC1 (M‐QKO and M‐indQKO) to simultaneously activate mTORC1 and inhibit FOXO1 in AKT‐deficient skeletal muscle. Muscle proteostasis and physiology were assessed using multiple assays including metabolic labelling, mitochondrial function, fibre typing, ex vivo physiology, and exercise performance. Results Here, we show that genetic ablation of skeletal muscle AKT signalling resulted in decreased muscle mass and a loss of oxidative metabolism and muscle performance. Specifically, deletion of muscle AKT activity during development or in adult mice resulted in a significant reduction in muscle growth by 30–40% (P

Summary Objective The forkhead box O1 (FOXO1) transcription factor is a key regulator of autophagy. In chondrocytes, reduced FOXO1 expression with aging causes osteoarthritis due to dysfunction of autophagy, but the mechanisms underlying regulation of FOXO1 expression and the reduction in expression with aging remain unclear. We investigated the mechanism by which transforming growth factor β1 (TGFβ1) signaling regulates the FOXO1–autophagy axis. Methods Expression of FOXO1 was measured in chondrocytes after TGFβ1 treatment. Immunohistochemistry was performed to estimate the levels of activin receptor-like kinase 5 (ALK5) and FOXO1 in the knee joints of young, middle-aged and old mice. The effects of the ALK5 inhibitor and SMAD3 or SMAD2 knockdown on FOXO1 expression were evaluated. The role of TGFβ1 in autophagy after hydrogen peroxide (H2O2) treatment was analyzed. The protective effect of TGFβ1 against H2O2 treatment was assessed by cell viability assay and TUNEL assay. Results TGFβ1 promoted the expression of FOXO1 mRNA and protein. Both ALK5 and FOXO1 expression decreased with aging. ALK5 inhibition and SMAD3 knockdown suppressed induction of FOXO1 expression by TGFβ1, whereas SMAD2 knockdown increased it. TGFβ1 promoted the expression of microtubule-associated proteins 1A/1B light chain 3B (LC3)-I protein via the SMAD3–FOXO1 pathway. Furthermore, under H2O2 treatment, TGFβ1 promoted expression of LC3-II. TGFβ1 pretreatment suppressed cell death of chondrocytes following H2O2 treatment, but this protective effect was abolished by FOXO1 knockdown. Conclusions TGFβ1 protects chondrocytes against oxidative stress via the FOXO1–autophagy axis, and a reduction in ALK5 expression might cause reduced FOXO1 expression with aging.

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20–30 cancer genes1–8. Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease9–13 than in normal liver13–16, which enables positive selection to shape the genomic landscape9–13. Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes17–19, and GPAM, which produces storage triacylglycerol from free fatty acids20,21, also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease. Whole-genome sequencing analysis of somatic mutations in liver samples from patients with chronic liver disease identifies driver mutations in metabolism-related genes such as FOXO1, and shows that these variants frequently exhibit convergent evolution.

Physalin B (PB), one of the major active steroidal constituents of Solanaceae Physalis plants, has a wide variety of biological activities. We found that PB significantly down-regulated β-amyloid (Aβ) secretion in N2a/APPsw cells. However, the underlying mechanisms are not well understood. In the current study, we investigated the changes in key enzymes involved in β-amyloid precursor protein (APP) metabolism and other APP metabolites by treating N2a/APPsw cells with PB at different concentrations. The results indicated that PB reduced Aβ secretion, which was caused by down-regulation of β-secretase (BACE1) expression, as indicated at both the protein and mRNA levels. Further research revealed that PB regulated BACE1 expression by inducing the activation of forkhead box O1 (FoxO1) and inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). In addition, the effect of PB on BACE1 expression and Aβ secretion was reversed by treatment with FoxO1 siRNA and STAT3 antagonist S3I-201. In conclusion, these data demonstrated that PB can effectively down-regulate the expression of BACE1 to reduce Aβsecretion by activating the expression of FoxO1 and inhibiting the phosphorylation of STAT3.

Ionizing radiation (IR)-induced excessive reactive oxygen species (ROS) is an important contributor of the injury of hematopoietic system. Grape seed proanthocyanidin extract (GSPE) is a new type of antioxidant, whereas whether it could ameliorate IR-induced hematopoietic injury remains unclear. Here, we show that GSPE treatment improves the survival of irradiated mice and alleviates IR-induced myelosuppression. Meanwhile, the hematopoietic reconstituting ability of hematopoietic stem cells (HSCs) in mice following irradiation exposure is significantly increased after GSPE treatment. Furthermore, GSPE treatment can reduce IR-induced ROS production and relieve DNA damage and apoptosis in hematopoietic stem progenitor cells (HSPCs). Interestingly, we find that a critical antioxidant-associated gene fokhead box transcription factor O1 (Foxo1) is significantly decreased in HSPCs after irradiation. Consistently, hematopoietic specific deletion of Foxo1 increases the radiosensitivity of mice. Further investigations reveal that GSPE treatment specifically upregulates the expression of Foxo1, as well as its target genes superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2) and catalase (CAT). Importantly, Foxo1 deficiency largely abolishes the radioprotection of GSPE on HSPCs. Collectively, our data demonstrate that GSPE plays an important role in ameliorating IR-induced HSPC injury via the Foxo1-mediated pathway. Therefore, GSPE may be used as a promising radioprotective agent.

Recent studies indicate that Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) can function as the signal of pattern recognition receptors, which play a pivotal role in the pathogenesis of the autoimmune disease. Systemic lupus erythematosus (SLE) is a classic autoimmune disease. Previous reports mainly focused on the potential role of TLRs in regulating the development of SLE, but little is known about the role of CLRs in the progression of SLE. Our previous studies showed that the inflammation-mediated accumulation of myeloid-derived suppressor cells (MDSCs) including granulocytic (G-MDSCs) and monocytic (M-MDSCs) participated in the pathogenesis of lupus. Mice deficient in Card9 (the downstream molecule of CLRs) were more susceptible to colitis-associated cancer via promoting the expansion of MDSCs. Whether the abnormal activation of CLRs regulates the expansion of MDSCs to participate in the pathogenesis of lupus remains unknown. In the present study, the expressions of CLRs were examined in both SLE patients and mouse models, revealing the expression of Dectin3 was positively correlated with SLEDAI. Dectin3 deficiency retarded the lupus-like disease by regulating the expansion and function of MDSCs. The mechanistic analysis revealed that Dectin3 deficiency promoted FoxO1-mediated apoptosis of MDSCs. Syk-Akt1-mediated nuclear transfer of FoxO1 increased in Dectin3-deficient MDSCs. Notedly, the accumulation of M-MDSCs mainly decreased in Dectin3−/− lupus mice, and the nuclear transfer of FoxO1 negatively correlated with the expression of LOX-1 on M-MDSCs. The silencing of FoxO1 expression in Dectin3−/− mice promoted the expansion of LOX-1+ M-MDSCs in vivo, and LOX-1+ M-MDSCs increased the differentiation of Th17 cells. Both LOX-1 expression on M-MDSCs and Dectin3 expression on MDSCs increased in patients with SLE. These data indicated that increased LOX-1+ M-MDSCs were related to the exacerbation of SLE development and might be potential target cells for the treatment of SLE.

B7-H3 (also known as CD276) is associated with aggressive characteristics in various cancers. Meanwhile, in alveolar rhabdomyosarcoma (ARMS), PAX3-FOXO1 fusion protein is associated with increased aggressiveness and poor prognosis. In the present study, we explored the relationship between PAX3-FOXO1 and B7-H3 and the biological roles of B7-H3 in ARMS. Quantitative real time PCR and flow cytometry revealed that PAX3-FOXO1 knockdown downregulated B7-H3 expression in all the selected cell lines (Rh-30, Rh-41, and Rh-28), suggesting that PAX3-FOXO1 positively regulates B7-H3 expression. Gene expression analysis revealed that various genes and pathways involved in chemotaxis, INF-γ production, and myogenic differentiation were commonly affected by the knockdown of PAX3-FOXO1 and B7-H3. Wound healing and transwell migration assays revealed that both PAX3-FOXO1 and B7-H3 were associated with cell migration. Furthermore, knockdown of PAX3-FOXO1 or B7-H3 induced myogenin expression in all cell lines, although myosin heavy chain induction varied depending on the cellular context. Our results indicate that PAX3-FOXO1 regulates B7-H3 expression and that PAX3-FOXO1 and B7-H3 are commonly associated with multiple pathways related to an aggressive phenotype in ARMS, such as cell migration and myogenic differentiation block.

TAZ, as a crucial effector of Hippo pathway, is required for spermatogenesis and fertilization, but little is known regarding its physiological function in uterine decidualization. In this study, we showed that TAZ was localized in the decidua, where it promoted stromal cell proliferation followed by accelerated G1/S phase transition via Ccnd3 and Cdk4 and induced the expression or activity of stromal differentiation markers Prl8a2, Prl3c1 and ALP, indicating the importance of TAZ in decidualization. Knockdown of TAZ impeded HB-EGF induction of stromal cell proliferation and differentiation. Under oxidative stress, TAZ protected stromal differentiation against oxidative damage by reducing intracellular ROS and enhancing cellular antioxidant capacity dependent on the Nrf2/ARE/Foxo1 pathway. TAZ strengthened the transcriptional activity of Nrf2 which directly bound to the antioxidant response element (ARE) of Foxo1 promoter region. Additionally, silencing TAZ caused accumulation of intracellular ROS through heightening NOX activity whose blockade by APO reversed the disruption in stromal differentiation. Further analysis revealed that TAZ might restore mitochondrial function, as indicated by the increase in ATP level, mtDNA copy number and mitochondrial membrane potential with the reduction in mitochondrial superoxide. Additionally, TAZ modulated the activities of mitochondrial respiratory chain complexes I and III whose suppression by ROT and AA resulted in the inability of TAZ to defend against oxidative damage to stromal differentiation. Moreover, TAZ prevented stromal cell apoptosis by upregulating Bcl2 expression and inhibiting Casp3 activity and Bax expression. In summary, TAZ might mediate HB-EGF function in uterine decidualization through Ccnd3 and ameliorate oxidative damage to stromal cell differentiation via Nrf2/ARE/Foxo1 pathway., Reproduction: Protective protein readies uterus for pregnancy A protein known to regulate cell proliferation plays a key role in preparing a woman’s uterus for pregnancy, a finding that could inform future treatments for female infertility. A team led by Zhan-Peng Yue and Bin Guo from Jilin University, Changchun, China, examined the role of a co-activator protein called TAZ in decidualization, the process in which the uterine lining changes hormonally and biochemically following ovulation. The researchers showed that TAZ levels build up in the mucosal lining of the uterus, where the protein works with various regulators of the cell cycle to promote the proliferation of connective tissue cells known as stromal cells, which support early embryonic development. The researchers demonstrated that in the face of oxidative stress TAZ helps orchestrate molecular detoxification mechanisms that protect these stromal cells from damage.

Dysfunctional autophagy is linked to neuronal damage in ischemia/reperfusion injury. The Ras-related protein 7 (Rab7), a member of the Rab family of small GTPases, appears crucial for the progression of the autophagic flux, and its activity is strictly interconnected with the histone deacetylase Silent information regulator 1 (Sirt1) and transcription factor Forkhead box class O1 (FoxO1). The present study assessed the neuroprotective role of melatonin in the modulation of the Sirt1/FoxO1/Rab7 axis in HT22 cells and organotypic hippocampal cultures exposed to oxygen-glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin re-established physiological levels of autophagy and reduced propidium iodide-positive cells, speeding up autophagosome (AP) maturation and increasing lysosomal activity. Our study revealed that melatonin modulates autophagic pathways, increasing the expression of both Rab7 and FoxO1 and restoring the Sirt1 expression affected by OGD/R. In addition, the Sirt1 inhibitor EX-527 significantly reduced Rab7, Sirt1, and FoxO1 expression, as well as autolysosomes formation, and blocked the neuroprotective effect of melatonin. Overall, our findings provide, for the first time, new insights into the neuroprotective role of melatonin against ischemic injury through the activation of the Sirt1/FoxO1/Rab7 axis.

Background: Hepatocellular carcinoma (HCC) is a very common neoplasm worldwide, and competitive endogenous RNA (ceRNA) plays an important role in the development of HCC. The purpose of this study is to investigate the molecular mechanisms of ceRNAs in HCC. Methods: This study detects potential ceRNAs from HCC through whole genome analysis of lncRNA, miRNA and mRNA expression. We then performed high-throughput sequencing of tissues from five hepatitis B related HCC patients to screen ceRNAs and those screened ceRNAs expressions were verified on tissues from an independent group of six patients. Finally, the function of ceRNAs of interest was illustrated in vitro. Result: Functional and pathway analysis of The Cancer Genome Atlas revealed ceRNA networks. The high-throughput sequencing identified 985 upregulated and 1612 downregulated lncRNAs and 887 upregulated and 1116 downregulated mRNAs in HCC patients. Differentially expressed genes were parallel to cancer-associated processes, comprising 18 upregulated and 35 downregulated significantly enriched pathways including alcoholism and viral carcinogenesis. Among them, a potential ceRNA network was detected and verified in six HCC patients. CeRNAs of the lncRNA MAGI2-AS3/miR-374-5p/FOXO1 pathway were significantly dysregulated in HCC, and validation in vitro showed that FOXO1 is positively regulated by MAGI2-AS3 through the induction of miR-374a/b-5p in HCC cells. In addition, the overexpression of FOXO1 is associated with proliferation, migration, and invasion of HCC cells and increases apoptosis of HCC cells. MiR-374a/b-5p caused an opposite effect by directly suppressing FOXO1 in HCC cells. Conclusion: CeRNA networks were found in HCC and aberrantly expressed ceRNAs of lncRNA MAGI2-AS3/miR-374-5p/FOXO1 plays a crucial role in HCC, assisting in diagnosis and providing a method for treatment.

Pregestational Diabetes Mellitus (PDM) during pregnancy constitutes an unfavorable embryonic and fetal development environment, with a high incidence of congenital malformations (CM). Neural tube defects are the second most common type of CM in children of diabetic mothers (CDM), who also have an elevated risk of developing neurodevelopmental disorders. The mechanisms that lead to these neuronal disorders in CDM are not yet fully understood. The present study aimed to know the effect of hyperglycemia on proliferation, neuronal differentiation percentage, and expression of neuronal differentiation mRNA markers in human umbilical cord Wharton's jelly mesenchymal stem cells (hUCWJMSC) of children from normoglycemic pregnancies (NGP) and PDM. We isolated and characterized hUCWJMSC by flow cytometry, immunofluorescence, RT-PCR and were induced to differentiate into adipocytes, osteocytes, and neurons. Proliferation assays were performed to determine the doubling time, and Nestin, TUBB3, FOXO1, KCNK2, LMO3, and MAP2 mRNA gene expression was assessed by semiquantitative RT-PCR. Hyperglycemia significantly decreased proliferation and neuronal differentiation percentage in NGP and PDM cells treated with 40 mM d-glucose. Nestin mRNA expression decreased under control glycemic conditions, while FOXO1, KCNK2, LMO3, and MAP2 mRNA expression increased during neuronal differentiation in both NGP and PDM cells. On the other hand, under hyperglycemic conditions, Nestin was significantly decreased in cells from NGP but not in cells from PDM, while mRNA expression of FOXO1 and LMO3 was significantly increased in cells from NGP, but not in cells from PDM. We found evidence that maternal PDM, with hyperglycemia in culture, affects the biological properties of fetal cells. All these results could be part of fetal programming.

The transcription factor FoxO1 (forkhead box O1) regulates genes that are involved in development, metabolism, cellular innovation, longevity, and stress responses. Assessment of FoxO1 activity is therefore critical to understand the regulatory network of this transcription factor. FoxO1 transactivation activity relies on its ability to bind to the promoters of target genes, which is controlled by posttranslational modifications (e.g., dephosphorylation or phosphorylation) that may promote nuclear translocation or exclusion of FoxO1. In this chapter we describe the protocols for FoxO1 activity assessment using Western blotting analysis of the posttranslational modification of FoxO1 in whole cell lysates and ELISA of DNA binding activity of FoxO1 in nuclear extracts.

Background: There is an increasing attention on microRNAs because of their functional effect on polarization of CD4+ T cells. This study explores the mechanism of miR-493-5p regulating Th9 cell differentiation in allergic asthma.Methods: The allergic airway inflammation is induced by ovalbumin (OVA) in mice. CD4+ T cells from normal mice are cultured under Th9 cell conditions. IL-9 levels in mice and CD4+ T cells are analyzed by RT-qPCR, ELISA, flow cytometry and western blot. The miR-493-5p level in mice and cells is detected by RT-qPCR. The interaction between FOXO1 and miR-493-5p is predicted by TargetScan and confirmed by dual luciferase assay. The pathological state is evaluated by H&E staining and lung resistance is measured in the allergic mice treated with miR-493-5p agomiR before stimulation.Results: The miR-493-5p expression decreases significantly in OVA-induced asthma mice, accompanied by a significant upregulation in the expression of IL-9, IRF4 and FOXO1 and the proportion of CD4+ Th9 cells. MiR-493-5p mimic inhibits the expression of IL-9, IRF4 and FOXO1 and Th9 cell differentiation, while the inhibitor restores these effects. MiR-493-5p mimic represses FOXO1 expression through interacting with 3'UTR of FOXO1 mRNA. The rescue experiment proves that miR-493-5p regulates the differentiation of Th9 cell and the expression of IL-9 by targeting FOXO1. In addition, we find that miR-493-5p agomiR treatment inhibits the FOXO1, IL-9 and IRF4 expression, decreases the proportion of CD4+ Th9 cells, and alleviates the pathological state of lung tissue and airway hyperreactivity in OVA-induced asthma mice.Conclusions: Our study confirmed that miR-493-5p inhibited Th9 cell differentiation in allergic asthma by targeting FOXO1.

Rhabdomyosarcoma is a soft tissue cancer that arises in skeletal muscle due to mutations in myogenic progenitors that lead to ineffective differentiation and malignant transformation. The transcription factors Pax3 and Pax7 and their downstream target genes are tightly linked with the fusion positive alveolar subtype, whereas the RAS pathway is usually involved in the embryonal, fusion negative variant. Here, we analyse the role of Pax3 in a fusion negative context, by linking alterations in gene expression in pax3a/pax3b double mutant zebrafish with tumour progression in kRAS-induced rhabdomyosarcoma tumours. Several genes in the RAS/MAPK signalling pathway were significantly down-regulated in pax3a/pax3b double mutant zebrafish. Progression of rhabdomyosarcoma tumours was also delayed in the pax3a/pax3b double mutant zebrafish indicating that Pax3 transcription factors have an unappreciated role in mediating malignancy in fusion negative rhabdomyosarcoma.

Integrin β6 (ITGB6), an epithelial-specific receptor, is downregulated in the gingival epithelium of periodontitis and is associated with inflammation response and periodontitis development. However, the transcriptional regulatory mechanism of ITGB6 downregulation in the human gingival epithelium remains unclear. Fibroblast-stimulating lipopeptide-1 (FSL-1), an oral biofilm component, promotes an epithelial cell-driven proinflammatory response in periodontitis partially by suppressing ITGB6 expression. The aim of the current study was to investigate the transcriptional regulatory mechanism of ITGB6 inhibition by FSL-1 in human epithelial cells (HaCaT and primary human gingival epithelial cells), and to delineate the transcriptional mechanism of ITGB6 suppression in periodontitis. We found that FSL-1 inhibited ITGB6 transcription through increasing forkhead box protein O1 (FOXO1) expression and inhibiting signal transducer and activator of transcription 3 (STAT3) activation. Furthermore, FOXO1 bound to STAT3 directly, leading to decreased STAT3 phosphorylation induced by FSL-1. Consequently, the binding of phosphorylated STAT3 to the ITGB6 promoter was decreased, and ITGB6 transcription was therefore downregulated following FSL-1 stimulation. The reciprocal action of STAT3 and FOXO1 on ITGB6 downregulation was also confirmed by the immunostaining of the inflammatory epithelium associated with periodontitis. Our findings suggest that the interaction of FOXO1–STAT3 may be a useful signal target for the treatment of periodontitis.

Oxidized low-density lipoprotein (oxLDL)-induced oxidative stress and apoptosis are considered as critical contributors to cardiovascular diseases. Methionine sulfoxide reductase A (MSRA) is a potent intracellular oxidoreductase and serves as an essential factor that protects cells against oxidative damage. Here, we firstly provide evidence that recombinant humanized IgG1 antibody treatment upregulated the expression of MSRA in THP-1 cells to defend against oxLDL-induced oxidative stress and apoptosis. It was also observed that the upregulation of MSRA is regulated by the forkhead box O transcription factor (FOXO1), and the acetylation of FOXO1 increased when exposed to oxLDL but declined when treated with recombinant humanized IgG1 antibody. In addition, we identified that silent information regulator 1 (SIRT1) suppresses FOXO1 acetylation. Importantly, SIRT1 or FOXO1 deficiency impaired the anti-oxidative stress and anti-apoptotic effect of recombinant humanized IgG1 antibody. Together, our results suggest that recombinant humanized IgG1 antibody exerts its anti-oxidative stress and anti-apoptotic function by upregulation of MSRA via the Sirt1-FOXO1 axis.

Lycopene, a natural bioactive component, has potential to reduce the risk of environmental factors inducing chronic diseases. It is important to explore lycopene's health benefits and its mechanism. The uncontrolled use of atrazine in agriculture causes critical environmental pollution issues worldwide. Exposure to atrazine through water and food chains is a risk to humans. In this study, mice were orally treated with lycopene and/or different concentrations of atrazine for 21 days to explore the influence of atrazine on the spleen and the role of lycopene's protection in atrazine exposure. The work found that atrazine exerted its toxic role in the B cell zone of the spleen by inducing Foxo1 deficiency. Atrazine caused ROS generation and Pink1/Parkin dysfunction via inducing Foxo1 deficiency, which led to apoptosis in the B cell zone. Additionally, the work revealed that lycopene ameliorates atrazine-induced apoptosis in the B cell zone of the spleen via regulating the miR-27a-3p/Foxo1 pathway. The finding also underscored a novel target of lycopene in maintaining homeostasis during B cell maturation.

Vascular aging is associated with metabolic remodeling, and most studies focused on fatty acid and glucose metabolism. Based on our metabolomic data, leucine was significantly reduced in the aortas of aged mice. Whether leucine supplementation can reverse aging-induced vascular remodeling remains unknown. To investigate the effectiveness of leucine, male mice at 15 or 18 months were supplemented with leucine (1.5%) for 3 months. All the aged mice, with or without leucine, were sacrificed at 21 months. Blood pressure and vascular relaxation were measured. H&E, Masson’s trichrome, and Elastica van Gieson staining were used to assess aortic morphology. Vascular inflammation, reactive oxidative stress (ROS), and vascular smooth muscle cell (VSMC) phenotype were also measured in mouse aortas. Compared with the 21-month-old mice without leucine, leucine supplementation from 15 months significantly improved vascular relaxation, maintained the contractile phenotype of VSMCs, and repressed vascular inflammation and ROS levels. These benefits were not observed in the mice supplemented with leucine starting from 18 months, which was likely due to the reduction in leucine transporters Slc3a2 or Slc7a5 at 18 months. Furthermore, we found benefits from leucine via activating the Sirt1-induced Foxo1 deacetylation. Our findings indicated that leucine supplementation in middle-aged mice improved aging-induced vascular remodeling and dysfunction.

Mesenchymal stromal cells (MSCs) are attractive candidates for treating hepatic disorders given their potential to enhance liver regeneration and function. The paracrine paradigm may be involved in the mechanism of MSC-based therapy, and exosomes (Exo) play an important role in this paracrine activity. Hypoxia significantly improves the effectiveness of MSC transplantation. However, whether hypoxia preconditioned MSCs (Hp-MSCs) can enhance liver regeneration, and whether this enhancement is mediated by Exo, are unknown. In this study, mouse bone marrow-derived MSCs (BM-MSCs) and secreted Exo were injected through the tail vein. We report that Hp-MSCs promote liver regeneration after partial hepatectomy in mice through their secreted exosomes. Interestingly, MSC-Exo were concentrated in liver 6 h after administration and mainly taken up by macrophages, but not hepatocytes. Compared with normoxic MSC-Exo (N-Exo), hypoxic MSC-Exo (Hp-Exo) enhanced M2 macrophage polarization both in vivo and in vitro. Microarray analysis revealed significant enrichment of microRNA (miR)-182-5p in Hp-Exo compared with that in N-Exo. In addition, miR-182-5p knockdown partially abolished the beneficial effect of Hp-Exo. Finally, Hp-MSC-derived exosomal miR-182-5p inhibited theprotein expression of forkhead box transcription factor 1 (FOXO1) in macrophages, which inhibited toll-like receptor 4 (TLR4) expression and subsequently induced an anti-inflammatory response. These results highlight the therapeutic potential of Hp-Exo in liver regeneration and suggest that miR-182-5p from Hp-Exo facilitates macrophage polarization during liver regeneration by modulating the FOXO1/TLR4 signaling pathway.

Sirtuin1 (Sirt1)/forkhead box O1 (FoxO1) axis has been reported as a crucial regulator involved in chondral homeostasis of healthy or osteoarthritis (OA) cartilage. In our study, the aim is to investigate whether dioscin functions as an activator of Sirt1/FoxO1 to protect against mechanical stress-induced chondrocyte dysfunction in vitro and in vivo models. HERB and PubChem databases were implemented to predict dioscin-related gene targets. Cell and mouse models of OA were established to determine the pharmacological value of dioscin, a steroidal saponin. Cartilage loss in the knee joint was detected by Safranin O staining. Phosphorylation and nucleocytoplasmic shuttling of FoxO1 was observed in mechanical stress-stimulated chondrocyte and anterior cruciate ligament transection-induced cartilage injury. However, dioscin treatment repressed FoxO1 phosphorylation and cytoplasmic transfer and elevated Sirt1 protein expression. Dioscin treatment reversed mechanical stress-induced growth inhibition and apoptosis of chondrocytes and improved cartilage degradation and bone loss in the epiphysis of the distal femur. Moreover, dioscin could maintain the normal phenotype of chondrocytes via mediating multiple gene expressions. Dioscin inhibited apoptosis and metabolic disorders in OA-like chondrocytes via maintaining the transcriptional activity of FoxO1 and enhancing Sirt1 expression. Dioscin might be a potential Sirt1 activator providing a novel therapeutic schedule for the treatment of OA.