Exosomal YB-1 facilitates ovarian restoration by MALAT1/miR-211-5p/FOXO3 axis.

Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. Mesenchymal stem cells-derived small extracellular vesicles (MSCs-sEVs) are attractive candidates for ovarian function restoration and folliculogenesis for POF due to their safety and efficacy, however, the key mediator in MSCs-sEVs that modulates this response and underlying mechanisms remains elusive. Herein, we reported that YB-1 protein was markedly downregulated in vitro and in vivo models of POF induced with H2O2 and CTX respectively, accompanied by granulosa cells (GCs) senescence phenotype. Notably, BMSCs-sEVs transplantation upregulated YB-1, attenuated oxidative damage-induced cellular senescence in GCs, and significantly improved the ovarian function of POF rats, but that was reversed by YB-1 depletion. Moreover, YB-1 showed an obvious decline in serum and GCs in POF patients. Mechanistically, YB-1 as an RNA-binding protein (RBP) physically interacted with a long non-coding RNA, MALAT1, and increased its stability, further, MALAT1 acted as a competing endogenous RNA (ceRNA) to elevate FOXO3 levels by sequestering miR-211-5p to prevent its degradation, leading to repair of ovarian function. In summary, we demonstrated that BMSCs-sEVs improve ovarian function by releasing YB-1, which mediates MALAT1/miR-211-5p/FOXO3 axis regulation, providing a possible therapeutic target for patients with POF.

Nuclear MAST4 Suppresses FOXO3 through Interaction with AKT3 and Induces Chemoresistance in Pancreatic Ductal Carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant, with a 5-year survival rate of less than 10%. Furthermore, the acquisition of anticancer drug resistance makes PDAC treatment difficult. We established MIA-GEM cells, a PDAC cell line resistant to gemcitabine (GEM), a first-line anticancer drug, using the human PDAC cell line-MIA-PaCa-2. Microtubule-associated serine/threonine kinase-4 (MAST4) expression was increased in MIA-GEM cells compared with the parent cell line. Through inhibitor screening, dysregulated AKT signaling was identified in MIA-GEM cells with overexpression of AKT3. MAST4 knockdown effectively suppressed AKT3 overexpression, and both MAST4 and AKT3 translocation into the nucleus, phosphorylating forkhead box O3a (FOXO3) in MIA-GEM cells. Modulating FOXO3 target gene expression in these cells inhibited apoptosis while promoting stemness and proliferation. Notably, nuclear MAST4 demonstrated higher expression in GEM-resistant PDAC cases compared with that in the GEM-sensitive cases. Elevated MAST4 expression correlated with a poorer prognosis in PDAC. Consequently, nuclear MAST4 emerges as a potential marker for GEM resistance and poor prognosis, representing a novel therapeutic target for PDAC.

FOXO3a deregulation in uterine smooth muscle tumors.

OBJECTIVE: The present study aimed to investigate FOXO3a deregulation in Uterine Smooth Muscle Tumors (USMT) and its potential association with cancer development and prognosis. METHODS: The authors analyzed gene and protein expression profiles of FOXO3a in 56 uterine Leiomyosarcomas (LMS), 119 leiomyomas (comprising conventional and unusual leiomyomas), and 20 Myometrium (MM) samples. The authors used techniques such as Immunohistochemistry (IHC), FISH/CISH, and qRT-PCR for the present analyses. Additionally, the authors conducted an in-silico analysis to understand the interaction network involving FOXO3a and its correlated genes. RESULTS: This investigation revealed distinct expression patterns of the FOXO3a gene and protein, including both normal and phosphorylated forms. Expression levels were notably elevated in LMS, and Unusual Leiomyomas (ULM) compared to conventional Leiomyomas (LM) and Myometrium (MM) samples. This upregulation was significantly associated with metastasis and Overall Survival (OS) in LMS patients. Intriguingly, FOXO3a deregulation did not seem to be influenced by EGF/HER-2 signaling, as there were minimal levels of EGF and VEGF expression detected, and HER-2 and EGFR were negative in the analyzed samples. In the examination of miRNAs, the authors observed upregulation of miR-96-5p and miR-155-5p, which are known negative regulators of FOXO3a, in LMS samples. Conversely, the tumor suppressor miR-let7c-5p was downregulated. CONCLUSIONS: In summary, the outcomes of the present study suggest that the imbalance in FOXO3a within Uterine Smooth Muscle Tumors might arise from both protein phosphorylation and miRNA activity. FOXO3a could emerge as a promising therapeutic target for individuals with Unusual Leiomyomas and Leiomyosarcomas (ULM and LMS), offering novel directions for treatment strategies.

[Identification of Osteoarthritis Inflamm-Aging Biomarkers by Integrating Bioinformatic Analysis and Machine Learning Strategies and the Clinical Validation]. / ç”Ÿç‰©ä¿¡æ¯å­¦å’Œæœºå™¨å­¦ä¹ ç­–ç•¥è¯†åˆ«éª¨å ³èŠ‚ç‚Žç‚Žæ€§è¡°è€ç”Ÿç‰©æ ‡å¿—ç‰©ä¸Žä¸´åºŠéªŒè¯.

Objective: To identify inflamm-aging related biomarkers in osteoarthritis (OA). Methods: Microarray gene profiles of young and aging OA patients were obtained from the Gene Expression Omnibus (GEO) database and aging-related genes (ARGs) were obtained from the Human Aging Genome Resource (HAGR) database. The differentially expressed genes of young OA and older OA patients were screened and then intersected with ARGs to obtain the aging-related genes of OA. Enrichment analysis was performed to reveal the potential mechanisms of aging-related markers in OA. Three machine learning methods were used to identify core senescence markers of OA and the receiver operating characteristic (ROC) curve was used to assess their diagnostic performance. Peripheral blood mononuclear cells were collected from clinical OA patients to verify the expression of senescence-associated secretory phenotype (SASP) factors and senescence markers. Results: A total of 45 senescence-related markers were obtained, which were mainly involved in the regulation of cellular senescence, the cell cycle, inflammatory response, etc. Through the screening with the three machine learning methods, 5 core senescence biomarkers, including FOXO3, MCL1, SIRT3, STAG1, and S100A13, were obtained. A total of 20 cases of normal controls and 40 cases of OA patients, including 20 cases in the young patient group and 20 in the elderly patient group, were enrolled. Compared with those of the young patient group, C-reactive protein (CRP), interleukin (IL)-6, and IL-1ß levels increased and IL-4 levels decreased in the elderly OA patient group (P<0.01); FOXO3, MCL1, and SIRT3 mRNA expression decreased and STAG1 and S100A13 mRNA expression increased (P<0.01). Pearson correlation analysis demonstrated that the selected markers were associated with some indicators, including erythrocyte sedimentation rate (ESR), IL-1ß, IL-4, CRP, and IL-6. The area under the ROC curve of the 5 core aging genes was always greater than 0.8 and the C-index of the calibration curve in the nomogram prediction model was 0.755, which suggested the good calibration ability of the model. Conclusion: FOXO3, MCL1, SIRT3, STAG1, and S100A13 may serve as novel diagnostic biomolecular markers and potential therapeutic targets for OA inflamm-aging.

Photobiomodulation therapy moderates cancer cachexia-associated muscle wasting through activating PI3K/AKT/FoxO3a pathway.

Cancer cachexia-associated muscle wasting as a multifactorial wasting syndrome, is an important factor affecting the long-term survival rate of tumor patients. Photobiomodulation therapy (PBMT) has emerged as a promising tool to cure and prevent many diseases. However, the effect of PBMT on skeletal muscle atrophy during cancer progression has not been fully demonstrated yet. Here, we found PBMT alleviated the atrophy of myotube diameter induced by cancer cells in vitro, and prevented cancer-associated muscle atrophy in mice bearing tumor. Mechanistically, the alleviation of muscle wasting by PBMT was found to be involved in inhibiting E3 ubiquitin ligases MAFbx and MuRF-1. In addition, transcriptomic analysis using RNA-seq and GSEA revealed that PI3K/AKT pathway might be involved in PBMT-prevented muscle cachexia. Next, we showed the protective effect of PBMT against muscle cachexia was totally blocked by AKT inhibitor in vitro and in vivo. Moreover, PBMT-activated AKT promoted FoxO3a phosphorylation and thus inhibiting the nucleus entry of FoxO3a. Lastly, in cisplatin-treated muscle cachexia model, PBMT had also been shown to ameliorate muscle atrophy through enhancing PI3K/AKT pathway to suppress MAFbx and MuRF-1 expression. These novel findings revealed that PBMT could be a promising therapeutic approach in treating muscle cachexia induced by cancer.

circRNA-PTPN4 mediated regulation of FOXO3 and ZO-1 expression: implications for blood-brain barrier integrity and cognitive function in uremic encephalopathy.

Uremic encephalopathy (UE) poses a significant challenge in neurology, leading to the need to investigate the involvement of non-coding RNA (ncRNA) in its development. This study employed ncRNA-seq and RNA-seq approaches to identify fundamental ncRNAs, specifically circRNA and miRNA, in the pathogenesis of UE using a mouse model. In vitro and in vivo experiments were conducted to explore the circRNA-PTPN4/miR-301a-3p/FOXO3 axis and its effects on blood-brain barrier (BBB) function and cognitive abilities. The research revealed that circRNA-PTPN4 binds to and inhibits miR-301a-3p, leading to an increase in FOXO3 expression. This upregulation results in alterations in the transcriptional regulation of ZO-1, affecting the permeability of human brain microvascular endothelial cells (HBMECs). The axis also influences the growth, proliferation, and migration of HBMECs. Mice with UE exhibited cognitive deficits, which were reversed by overexpression of circRNA-PTPN4, whereas silencing FOXO3 exacerbated these deficits. Furthermore, the uremic mice showed neuronal loss, inflammation, and dysfunction in the BBB, with the expression of circRNA-PTPN4 demonstrating therapeutic effects. In conclusion, circRNA-PTPN4 plays a role in promoting FOXO3 expression by sequestering miR-301a-3p, ultimately leading to the upregulation of ZO-1 expression and restoration of BBB function in mice with UE. This process contributes to the restoration of cognitive abilities.

Exosomal miR-122, miR-128, miR-200, miR-298, and miR-342 as novel diagnostic biomarkers in NAFL/NASH: Impact of LPS/TLR-4/FoxO3 pathway.

Nonalcoholic fatty liver disease (NAFLD) is a common liver disorder affecting a quarter of the global residents. Progression of NAFL into nonalcoholic steatohepatitis (NASH) may cause cirrhosis, liver cancer, and failure. Gut microbiota imbalance causes microbial components translocation into the circulation, triggering liver inflammation and NASH-related fibrosis. MicroRNAs (miRNAs) regulate gene expression via repressing target genes. Exosomal miRNAs are diagnostic and prognostic biomarkers for NAFL and NASH liver damage. Our work investigated the role of the gut microbiota in NAFLD pathogenesis via the lipopolysaccharide/toll-like receptor 4/Forkhead box protein O3 (LPS/TLR-4/FoxO3) pathway and certain miRNAs as noninvasive biomarkers for NAFL or its development to NASH. miRNA expression levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 50 NAFL patients, 50 NASH patients, and 50 normal controls. Plasma LPS, TLR-4, adiponectin, peroxisome proliferator-activated receptor γ (PPAR-γ), and FoxO3 concentrations were measured using enzyme-linked immunosorbent assay (ELISA). In NAFL and NASH patients, miR-122, miR-128, FoxO3, TLR-4, LPS, and PPAR-γ were upregulated while miR-200, miR-298, miR-342, and adiponectin were downregulated compared with the normal control. The examined miRNAs might distinguish NAFL and NASH patients from the normal control using receiver operating characteristic analysis. Our study is the first to examine these miRNAs in NAFLD. Our findings imply that these are potentially promising biomarkers for noninvasive early NAFL diagnosis and NASH progression. Understanding the LPS/TLR-4/FoxO3 pathway involvement in NAFL/NASH pathogenesis may aid disease management.

Effect of moxibustion on the SIRT1/FOXO3a signaling pathway in ApoE-/- mice with atherosclerosis. / 艾灸对ApoE-/-åŠ¨è„‰ç²¥æ ·ç¡¬åŒ–å°é¼ SIRT1/FOXO3a信号通路的影响.

OBJECTIVES: To observe the effects of moxibustion on blood lipid metabolism, pathological morphology of thoracic aorta, and the expression of silent information regulator 1 (SIRT1) and forkhead box transcription factor O3a (FOXO3a) in ApoE-/- atherosclerosis (AS) mice, so as to explore the potential mechanism of moxibustion in preventing and treating AS. METHODS: Ten C57BL/6J mice were fed a normal diet as the control group, and 30 ApoE-/- mice were fed a high-fat diet to establish the AS model, which were randomly divided into the model group, simvastatin group, and moxibustion group, with 10 mice in each group. From the first day of modeling, mice in the moxibustion group received mild moxibustion treatment at "Shenque"(CV8), "Yinlingquan"(SP9), bilateral "Neiguan"(PC6) and "Xuehai"(SP10) for 30 min per time；the mice in the simvastatin group were given simvastatin orally (2.5 mg·kg-1·d-1), with both treatments given once daily, 5 times a week, with a total intervention period of 12 weeks. The body weight and general condition of the mice were observed and recorded during the intervention period. After the intervention, the contents of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using an automated biochemistry analyzer. Hematoxylin eosin (HE) staining was used to observe the pathological morphology of the thoracic aorta. ELISA was used to measure the contents of serum oxidized low-density lipoprotein (ox-LDL) and superoxide dismutase (SOD) activity. Western blot and real-time fluorescent quantitative PCR analysis were used to detect the expression levels of SIRT1 and FOXO3a protein and mRNA in the thoracic aorta. RESULTS: Compared with the control group, body weight at the 8th and 12th week, serum TC, TG, LDL-C, and ox-LDL contents of the model group mice were significantly increased(P<0.05, P<0.01), while the HDL-C contents, SOD activity, and the expression levels of SIRT1 protein and mRNA in the thoracic aorta were significantly decreased(P<0.05, P<0.01). HE staining showed thickening of the aortic intima, endothelial cell degeneration, swelling, and shedding. Compared with the model group, body weight at the 8th and 12th week, serum TC, TG, LDL-C, and ox-LDL contents of mice in the simvastatin group and moxibustion group were significantly decreased(P<0.01), while the serum SOD activity, expression levels of SIRT1 protein and mRNA in the thoracic aorta were significantly increased(P<0.01). The HDL-C contents were significantly increased in the simvastatin group(P<0.05). The thoracic aortic structure was more intact in both groups, with a more regular lumen and orderly arrangement of the elastic membrane in the media, and a slight amount of endothelial cell degeneration and swelling in the intima. There was no significant difference in the evaluated indexes between the moxibustion group and the simvastatin group and the pathological changes in the thoracic aorta were similar between the two groups. CONCLUSIONS: Moxibustion can reduce the body weight of AS model mice, regulate lipid levels, repair vascular intima, and alleviate endothelial damage. Its mechanism of action may be related to the regulation of the SIRT1/FOXO3a signaling pathway to improve oxidative damage.

S-Adenosyl-l-Methionine Alleviates the Senescence of MSCs Through the PI3K/AKT/FOXO3a Signaling Pathway.

Cellular senescence significantly affects the proliferative and differentiation capacities of mesenchymal stem cells (MSCs). Identifying key regulators of senescence and exploring potential intervention strategies, including drug-based approaches, are active areas of research. In this context, S-adenosyl-l-methionine (SAM), a critical intermediate in sulfur amino acid metabolism, emerges as a promising candidate for mitigating MSC senescence. In a hydrogen peroxide-induced MSC aging model (100 µM for 2 hours), SAM (50 and 100 µM) was revealed to alleviate the senescence of MSCs, and also attenuated the level of reactive oxygen species and enhanced the adipogenic and osteogenic differentiation in senescent MSCs. In a premature aging mouse model (subcutaneously injected with 150 mg/kg/day d-galactose in the neck and back for 7 weeks), SAM (30 mg/kg/day by gavage for 5 weeks) was shown to delay the overall aging process while increasing the number and thickness of bone trabeculae in the distal femur. Mechanistically, activation of PI3K/AKT signaling and increased phosphorylation of forkhead box O3 (FOXO3a) was proved to be associated with the antisenescence role of SAM. These findings highlight that the PI3K/AKT/FOXO3a axis in MSCs could play a crucial role in MSCs senescence and suggest that SAM may be a potential therapeutic drug for MSCs senescence and related diseases.

FOXO3a-interacting proteins' involvement in cancer: a review.

Due to its role in apoptosis, differentiation, cell cycle arrest, and DNA damage repair in stress responses (oxidative stress, hypoxia, chemotherapeutic drugs, and UV irradiation or radiotherapy), FOXO3a is considered a key tumor suppressor that determines radiotherapeutic and chemotherapeutic responses in cancer cells. Mutations in the FOXO3a gene are rare, even in cancer cells. Post-translational regulations are the main mechanisms for inactivating FOXO3a. The subcellular localization, stability, transcriptional activity, and DNA binding affinity for FOXO3a can be modulated via various post-translational modifications, including phosphorylation, acetylation, and interactions with other transcriptional factors or regulators. This review summarizes how proteins that interact with FOXO3a engage in cancer progression.

FOXO3a functions as a transcriptional and co-transcriptional splicing regulator in vascular endothelial cell lines.

Recent studies have indicated a connection between Forkhead box O3a protein and coronary artery disease, yet the exact role of FOXO3a in the regulation of metabolic processes and apoptosis in vascular endothelial cells is still unknown. Therefore, we investigated the role of FOXO3a on target genes in a human vascular endothelial cell line. Through the utilization of high-throughput sequencing technology, we analyzed gene expression profiles and alternative splicing patterns in human vascular endothelial cells with FOXO3a over expression. This study identified 419 DEGs between FOXO3a-OE HUVEC model and control cells. KEGG analysis indicated that the upregulated genes were mainly enriched in inflammation-related signaling pathways, and the downregulated genes were enriched in lipid metabolism-related pathways.

FOXO3: at the crossroads of metabolic, inflammatory, and tumorigenic remodeling in the colon.

The Forkhead box O3 (FOXO3) transcription factor regulates the expression of genes critical for diverse cellular functions in homeostasis. Diminished FOXO3 activity is associated with human diseases such as obesity, metabolic diseases, inflammatory diseases, and cancer. In the mouse colon, FOXO3 deficiency leads to an inflammatory immune landscape and dysregulated molecular pathways, which, under various insults, exacerbates inflammation and tumor burden, mimicking characteristics of human diseases. This deficiency also results in dysregulated lipid metabolism, and consequently, the accumulation of intracellular lipid droplets (LDs) in colonic epithelial cells and infiltrated immune cells. FOXO3 and LDs form a self-reinforcing negative regulatory loop in colonic epithelial cells, neutrophils, and macrophages, which is associated with inflammatory bowel disease and colon cancer, particularly in the context of obesity.

Genetic Profile of FOXO3 Single-Nucleotide Polymorphism in Colorectal Cancer Patients.

INTRODUCTION: Colorectal cancer (CRC) heritability is determined by the composite relations between inherited variants and environmental factors. In developing countries like India, the incidence rates of CRC are especially increasing. In this study, we have focused on the distribution of the FOXO3 gene polymorphisms among the patients with CRC in North India. METHODS: A case-control study was conducted on 487 CRC patients and 487 age-matched controls. We genotyped single-nucleotide polymorphisms rs2253310 and rs4946936 through polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis and PCR-single-stranded conformation polymorphism procedure followed by sequence detection. RESULTS: A significantly increased risk of CRC was observed for the CC genotype of the rs4946936 polymorphism compared to the TT genotype (p = 0.02; odd ratio [OR] = 1.40, confidence interval [CI] = 1.05-1.87). GT haplotype appeared to be a "risk" haplotype (OR = 1.71, 95% CI = 0.82-2.19), while as other haplotypes CC (OR = 0.83, 95% CI = 0.32-1.54), CT (OR = 0.75, 95% CI = 0.25-1.01), and GC (OR = 0.98, 95% CI = 0.88-1.14) were found to be "protective" for developing CRC. CONCLUSION: This study suggests an association of increased risk of CRC with the rs4946936 polymorphism but not with the rs2253310 polymorphism.

Enhancer-mediated FOXO3 expression promotes MSC adipogenic differentiation by activating autophagy.

BACKGROUND: Mesenchymal stem cells (MSCs) are pluripotent stem cells capable of differentiating into osteocytes, adipocytes and chondrocytes. However, in osteoporosis, the balance of differentiation is tipped toward adipogenesis and the key mechanism is controversial. Researches have shown that, as upstream regulatory elements of gene expression, enhancers ar involved in the expression of identity genes. In this study, we identified enhancers-mediated gene FOXO3 promoting MSC adipogenic differentiation by activating autophagy. METHODS: We integrated data of RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and ATAC-sequencing (ATAC-seq) to find the identity gene FOXO3. The expression of FOXO3 protein, adipogenic transcription factors and the substrate of autophagy were measured by western blotting. The Oil Red O (ORO) staining was used to visualize the adipogenesis of MSCs. Immunohistochemistry was used to visualize the FOXO3 expression in adipocytes in bone marrow. Immunofluorescence was used to detect the expression of PPARγ and LC3B. RESULTS: During adipogenesis, enhancers redistribute to genes associated with adipogenic differentiation, among which we identified the pivotal identity gene FOXO3. FOXO3 could promote the expression of the adipogenic transcription factors PPARγ, CEBPα, and CEBPß during adipogenic differentiation, while PPARγ, CEBPα, and CEBPß could in turn bind to FOXO3 and continue to promote FOXO3 expression to form a positive feedback loop. Consistently elevated FOXO3 expression promotes autophagy by activating the PI3K-AKT pathway which mediates adipogenic differentiation. CONCLUSIONS: Pivotal identity gene FOXO3 promotes autophagy by activating PI3K-AKT pathway, which provokes adipogenic differentiation of MSCs. Enhancer-regulated adipogenic identity gene FOXO3 could be an attractive treatment for osteoporosis.

FOXO3 longevity genotype attenuates the impact of hypertension on cerebral microinfarct risk.

OBJECTIVE: The G -allele of FOXO3 SNP rs2802292 , which is associated with human resilience and longevity, has been shown to attenuate the impact of hypertension on the risk of intracerebral hemorrhage (ICH). We sought to determine whether the FOXO3 G -allele similarly attenuates the impact of hypertension on the risk of cerebral microinfarcts (CMI). METHODS: From a prospective population-based cohort of American men of Japanese ancestry from the Kuakini Honolulu Heart Program (KHHP) and Kuakini Honolulu-Asia Aging Study (KHAAS) that had brain autopsy data, age-adjusted prevalence of any CMI on brain autopsy was assessed. Logistic regression models, adjusted for age at death, cardiovascular risk factors, FOXO3 and APOE-ε4 genotypes, were utilized to determine the predictors of any CMI. Interaction of FOXO3 genotype and hypertension was analyzed. RESULTS: Among 809 men with complete data, 511 (63.2%) participants had evidence of CMI. A full multivariable model demonstrated that BMI [odds ratio (OR) 1.07, 95% confidence interval (CI) 1.01-1.14, P  = 0.015) was the only predictor of CMI, while hypertension was a borderline predictor (OR 1.44, 95% CI 1.00-2.08, P  = 0.052). However, a significant interaction between FOXO3 G -allele carriage and hypertension was observed ( P  = 0.020). In the stratified analyses, among the participants without the longevity-associated FOXO3 G -allele, hypertension was a strong predictor of CMI (OR 2.25, 95% CI 1.34-3.77, P  = 0.002), while among those with the longevity-associated FOXO3 G -allele, hypertension was not a predictor of CMI (OR 0.88, 95% CI 0.51-1.54, P  = 0.66). CONCLUSION: The longevity-associated FOXO3 G -allele mitigates the impact of hypertension on the risk of CMI.

Therapeutic Consequences of Targeting the IGF-1/PI3K/AKT/FOXO3 Axis in Sarcopenia: A Narrative Review.

The high prevalence of sarcopenia in an aging population has an underestimated impact on quality of life by increasing the risk of falls and subsequent hospitalization. Unfortunately, the application of the major established key therapeutic-physical activity-is challenging in the immobile and injured sarcopenic patient. Consequently, novel therapeutic directions are needed. The transcription factor Forkhead-Box-Protein O3 (FOXO3) may be an option, as it and its targets have been observed to be more highly expressed in sarcopenic muscle. In such catabolic situations, Foxo3 induces the expression of two muscle specific ubiquitin ligases (Atrogin-1 and Murf-1) via the PI3K/AKT pathway. In this review, we particularly evaluate the potential of Foxo3-targeted gene therapy. Foxo3 knockdown has been shown to lead to increased muscle cross sectional area, through both the AKT-dependent and -independent pathways and the reduced impact on the two major downstream targets Atrogin-1 and Murf-1. Moreover, a Foxo3 reduction suppresses apoptosis, activates satellite cells, and initiates their differentiation into muscle cells. While this indicates a critical role in muscle regeneration, this mechanism might exhaust the stem cell pool, limiting its clinical applicability. As systemic Foxo3 knockdown has also been associated with risks of inflammation and cancer progression, a muscle-specific approach would be necessary. In this review, we summarize the current knowledge on Foxo3 and conceptualize a specific and targeted therapy that may circumvent the drawbacks of systemic Foxo3 knockdown. This approach presumably would limit the side effects and enable an activity-independent positive impact on skeletal muscle.

Pro-carcinogenic actions of miR-155/FOXO3a in colorectal cancer development.

Colorectal cancer (CRC) ranks third in cancer incidence and second in cancer mortality globally. MicroRNAs (miRNAs) are promising biomarkers and therapeutic targets for CRC diagnosis and treatment. The miR-155 is reported to induce radiation resistance in CRC. In this study, we aimed to further clarify the role and underlying mechanism of the miR-155 in CRC cell malignancy. We found that miR-155 was significantly up-regulated in CRC tissues. The results of loss-of-function experiments revealed that miR-155 deficiency suppressed the proliferative capacity, invasion, and migration of CRC cells. Moreover, the downstream target genes of miR-155 were screened, and miR-155 was demonstrated to directly bind to FOXO3a in CRC cells to negatively regulate FOXO3a expression. FOXO3a was downregulated in CRC tissues and the expression of FOXO3a and miR-155 was in negative correlation in CRC tissues. FOXO3a overexpression alone was revealed to inhibit CRC cell growth, migration and invasion. Additionally, rescue assays showed that FOXO3a silencing significantly reversed the inhibitory effect of miR-155 deficiency on CRC cell malignant behaviors. In conclusion, miR-155 induces malignant phenotypes of CRC cells including cell proliferation, migration and invasion by targeting FOXO3a, which might provide clues for the targeted therapy of CRC.

Knockdown of the Long Noncoding RNA CRNDE Ameliorates Apoptosis and Inflammation in Ischemia-Reperfusion-Induced Brain Injury via the mir-489-3p/FOXO3 Pathway.

AIM: To examine the role and mechanism of colorectal tumor differential expression (CRNDE) in brain injury induced by ischemicreperfusion. MATERIAL AND METHODS: Sh-SY5Y cells were cultured, and oxygen and glucose deprivation/reperfusion (OGD/R) injury tests were performed. The effects on SH-SY5Y cells were evaluated by the Cell Counting Kit-8 (CCK-8) assay, qPCR, apoptosis analysis, western blot analysis, ELISA, a luciferase reporter assay, and an RNA pull-down assay. RESULTS: Knockdown of CRBDE ameliorated SH-SY5Y cell impairment induced by OGD/R. CRNDE, the target of mir-489-3p, was directly bound to FOXO3. Mir-489-3p knockdown partially reversed OGD/R-mediated impairment in CRBDE knockdown SH-SY5Y cells. CONCLUSION: The results indicate that knockdown of lncRNA CRNDE ameliorates apoptosis and the inflammatory response in ischemia-reperfusion-induced brain injury through the mir-489-3p/FOXO3 axis. LncRNA CRNDE may represent a novel therapeutic target for brain injury.

FoxO3 Modulates Circadian Rhythms in Neural Stem Cells.

Both FoxO transcription factors and the circadian clock act on the interface of metabolism and cell cycle regulation and are important regulators of cellular stress and stem cell homeostasis. Importantly, FoxO3 preserves the adult neural stem cell population by regulating cell cycle and cellular metabolism and has been shown to regulate circadian rhythms in the liver. However, whether FoxO3 is a regulator of circadian rhythms in neural stem cells remains unknown. Here, we show that loss of FoxO3 disrupts circadian rhythmicity in cultures of neural stem cells, an effect that is mediated via regulation of Clock transcriptional levels. Using Rev-Erbα-VNP as a reporter, we then demonstrate that loss of FoxO3 does not disrupt circadian rhythmicity at the single cell level. A meta-analysis of published data revealed dynamic co-occupancy of multiple circadian clock components within FoxO3 regulatory regions, indicating that FoxO3 is a Clock-controlled gene. Finally, we examined proliferation in the hippocampus of FoxO3-deficient mice and found that loss of FoxO3 delayed the circadian phase of hippocampal proliferation, indicating that FoxO3 regulates correct timing of NSC proliferation. Taken together, our data suggest that FoxO3 is an integral part of circadian regulation of neural stem cell homeostasis.

Foxo3 Knockdown Mediates Decline of Myod1 and Myog Reducing Myoblast Conversion to Myotubes.

Sarcopenia has a high prevalence among the aging population. Sarcopenia is of tremendous socioeconomic importance because it can lead to falls and hospitalization, subsequently increasing healthcare costs while limiting quality of life. In sarcopenic muscle fibers, the E3 ubiquitin ligase F-Box Protein 32 (Fbxo32) is expressed at substantially higher levels, driving ubiquitin-proteasomal muscle protein degradation. As one of the key regulators of muscular equilibrium, the transcription factor Forkhead Box O3 (FOXO3) can increase the expression of Fbxo32, making it a possible target for the regulation of this detrimental pathway. To test this hypothesis, murine C2C12 myoblasts were transduced with AAVs carrying a plasmid for four specific siRNAs against Foxo3. Successfully transduced myoblasts were selected via FACS cell sorting to establish single clone cell lines. Sorted myoblasts were further differentiated into myotubes and stained for myosin heavy chain (MHC) by immunofluorescence. The resulting area was calculated. Myotube contractions were induced by electrical stimulation and quantified. We found an increased Foxo3 expression in satellite cells in human skeletal muscle and an age-related increase in Foxo3 expression in older mice in silico. We established an in vitro AAV-mediated FOXO3 knockdown on protein level. Surprisingly, the myotubes with FOXO3 knockdown displayed a smaller myotube size and a lower number of nuclei per myotube compared to the control myotubes (AAV-transduced with a functionless control plasmid). During differentiation, a lower level of FOXO3 reduced the expression Fbxo32 within the first three days. Moreover, the expression of Myod1 and Myog via ATM and Tp53 was reduced. Functionally, the Foxo3 knockdown myotubes showed a higher contraction duration and time to peak. Early Foxo3 knockdown seems to terminate the initiation of differentiation due to lack of Myod1 expression, and mediates the inhibition of Myog. Subsequently, the myotube size is reduced and the excitability to electrical stimulation is altered.