Forkhead Box O1 mRNA Research Articles

METTL3-regulated m6A modification impairs the decidualization of endometrial stromal cells by regulating YTHDF2-mediated degradation of FOXO1 mRNA in endometriosis-related infertility

BackgroundEndometriosis-related infertility is a common worldwide reproductive health concern. Despite ongoing research, the causes of infertility remain unclear. Evidence suggests that epigenetic regulation is crucial in reproduction. However, the role of N6-methyladenosine (m6A) modification of RNA in endometriosis-related infertility requires further investigation.MethodsWe examined the expression of m6A and methyltransferase-like 3 (METTL3) in endometrial samples taken from normal fertile women in the proliferative phase (the NP group) or the mid-secretory phase (the NS group) or from women with endometriosis-related infertility at the mid-secretory phase (the ES group). We treated primary endometrial stromal cells (ESCs) with medroxyprogesterone acetate and 8-Bromo-cyclic adenosine monophosphate for in vitro decidualization and detected the expression of m6A, METTL3, and decidual markers. We analyzed the expression of m6A, METTL3, and forkhead box O1 (FOXO1) in ESCs from normal fertile women (the ND group) or women with endometriosis-related infertility (the ED group). We also assessed the expression of m6A, METTL3, and decidual markers, as well as the embryo adhesion rate, upon METTL3 overexpression or knockdown. Additionally, we investigated the role of METTL3 in embryo implantation in vivo by applying mice with endometriosis. Furthermore, we performed RNA stability assays, RNA immunoprecipitation (RIP), and methylated RIP assays to explore the mechanisms underlying the regulation of FOXO1 by METTL3-mediated m6A.ResultsThe expression of m6A and METTL3 was reduced only in the NS group; the NP and ES groups demonstrated increased m6A and METTL3 levels. m6A and METTL3 levels decreased in ESCs with prolonged decidual treatment. Compared to the ND group, m6A and METTL3 levels in the ED group increased after decidual treatment, whereas the expression of FOXO1 decreased. METTL3 overexpression suppressed the expression of decidual markers and embryo implantation in vitro; METTL3 knockdown exhibited the opposite effect. Inhibition of METTL3 promoted embryo implantation in vivo. Furthermore, we observed that METTL3-mediated m6A regulated the degradation of FOXO1 mRNA through YTHDF2, a m6A binding protein.ConclusionsMETTL3-regulated m6A promotes YTHDF2-mediated decay of FOXO1 mRNA, thereby affecting cellular decidualization and embryo implantation. These findings provide novel insights into the development of therapies for women with endometriosis-related infertility.

Skeletal muscle angiogenic, regulatory, and heat shock protein responses to prolonged passive hyperthermia of the human lower limb.

Passive hyperthermia induces a range of physiological responses including augmenting skeletal muscle mRNA expression. This experiment aimed to examine gene and protein responses to prolonged passive leg hyperthermia. Seven young participants underwent 3 h of resting unilateral leg heating (HEAT) followed by a further 3 h of rest, with the contralateral leg serving as an unheated control (CONT). Muscle biopsies were taken at baseline (0 h), and at 1.5, 3, 4, and 6 h in HEAT and 0 and 6 h in CONT to assess changes in selected mRNA expression via qRT-PCR, and HSP72 and VEGFα concentration via ELISA. Muscle temperature (Tm) increased in HEAT plateauing from 1.5 to 3 h (+3.5 ± 1.5°C from 34.2 ± 1.2°C baseline value; P < 0.001), returning to baseline at 6 h. No change occurred in CONT. Endothelial nitric oxide synthase (eNOS), Forkhead box O1 (FOXO-1), Hsp72, and VEGFα mRNA increased in HEAT (P < 0.05); however, post hoc analysis identified that only Hsp72 mRNA statistically increased (at 4 h vs. baseline). When peak change during HEAT was calculated angiopoietin 2 (ANGPT-2) decreased (-0.4 ± 0.2-fold), and C-C motif chemokine ligand 2 (CCL2) (+2.9 ± 1.6-fold), FOXO-1 (+6.2 ± 4.4-fold), Hsp27 (+2.9 ± 1.7-fold), Hsp72 (+8.5 ± 3.5-fold), Hsp90α (+4.6 ± 3.7-fold), and VEGFα (+5.9 ± 3.1-fold) increased from baseline (all P < 0.05). At 6 h Tm were not different between limbs (P = 0.582; CONT = 32.5 ± 1.6°C, HEAT = 34.3 ± 1.2°C), and only ANGPT-2 (P = 0.031; -1.3 ± 1.4-fold) and VEGFα (P = 0.030; 1.1 ± 1.2-fold) differed between HEAT and CONT. No change in VEGFα or HSP72 protein concentration were observed over time; however, peak change in VEGFα did increase (P < 0.05) in HEAT (+140 ± 184 pg·mL-1) versus CONT (+7 ± 86 pg·mL-1). Passive hyperthermia transiently augmented ANGPT-2, CCL2, eNOS, FOXO-1, Hsp27, Hsp72, Hsp90α and VEGFα mRNA, and VEGFα protein.

MiR-29a-3p Inhibits Proliferation and Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells via Targeting FOXO3 and Repressing Wnt/β-Catenin Signaling in Steroid-Associated Osteonecrosis.

Background and ObjectivesThis study was to investigate the role of microRNA-29a-3p (miR-29a-3p) in human bone marrow mesenchymal stem cells (hBMSCs), and its relationship with steroid-associated osteonecrosis.Methods and ResultsThe online tool GEO2R was used to screen out the differentially expressed genes (DEGs) in GSE123568 dataset. Quantitative real time-polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-29a-3p, forkhead box O3 (FOXO3), alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (OCN) and RUNX family transcription factor 2 (Runx2) in the hBMSCs isolated from the patients with steroid-associated osteonecrosis. CCK-8 assay was executed to measure cell viability; western blot assay was utilized to detect FOXO3, ALP, Runx2, OCN and β-catenin expression. Cell apoptosis and cell cycle were detected by flow cytometry. Immunofluorescence assay was used to detect the sub-cellular localization of β-catenin. Bioinformatics analysis and luciferase reporter gene assay were performed to confirm whether miR-29a-3p can combine with FOXO3 3’UTR. MiR-29a-3p was markedly up-regulated in the hBMSCs of patients with steroid-associated osteonecrosis, while FOXO3 mRNA was significantly down-regulated. Transfection of miR-29a-3p mimics significantly inhibited the hBMSCs’ proliferation, osteogenic differentiation markers’ expressions, including ALP, Runx2, OCN, and repressed the ALP activity, as well as promoted cell apoptosis and cell-cycle arrest. FOXO3 was identified as a target gene of miR-29a-3p, and miR-29a-3p can inhibit the expression of FOXO3 and β-catenin, and inhibition of miR-29a-3p promoted translocation of β-catenin to the nucleus.ConclusionsMiR-29a-3p can modulate FOXO3 expression and Wnt/β-catenin signaling to inhibit viability and osteogenic differentiation of hBMSCs, thereby promoting the development of steroid-associated osteonecrosis.

Effect of circRNA_FOXO3 rs12196996 polymorphism and FOXO3 rs2232365 polymorphism on survival rate and severity of intensive care unit-acquired sepsis

ABSTRACT The expression of circRNA_FOXO3 was found to be positively associated with the expression of Forkhead Box O3 (FOXO3), which is targeted and regulated by miR-23a. Polymorphisms in rs12196996 and rs2232365 have been reported in various diseases. In this study, we recruited intensive care unit (ICU)-acquired sepsis patients and grouped them according to their genotypes of rs12196996 and rs2232365. Quantitative real-time PCR was performed to analyze the expression of circRNA_FOXO3, FOXO3 mRNA, and miR-23a. ELISA was carried out to evaluate the abundance of cytokines and luciferase assay was used to explore the inhibitory role of miR-23a on circRNA_FOXO3 and FOXO3. Accordingly, we found that rs12196996 GG and rs2232365 AA were significantly correlated with prolonged survival of ICU-acquired sepsis patients. Rs12196996 GG and rs2232365 AA were also correlated with increased level of miR-23a, IL-10 and decreased level of TNF, IL-2, IFN, IL-6 and IL-1β in the peripheral blood cell samples of patients with ICU-acquired sepsis. The luciferase activity of wild-type (WT) circRNA_FOXO3 and FOXO3 were severely reduced by miR-23a. MiR-23a precursors could effectively suppress the expression of circRNA_FOXO3 and FOXO3 in the cells. Moreover, LPS-induced cell viability loss and dysregulation of cytokines were effectively restored by the knockdown of FOXO3 or circRNA_FOXO3 siRNA in the cells. This study revealed that the minor allele of rs12196996 polymorphism and rs2232365 polymorphism collaboratively contributed to the increased survival and suppressed severity of ICU-acquired sepsis.

TGFβ1 signaling protects chondrocytes against oxidative stress via FOXO1–autophagy axis

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. 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. 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. TGFβ1 protects chondrocytes against oxidative stress via the FOXO1-autophagy axis, and a reduction in ALK5 expression might cause reduced FOXO1 expression with aging.

Effects of Rehmannia glutinosa polysaccharides on bone tissue structure and skeletal muscle atrophy in rats with disuse

ABSTRACTPurposeTo study effects of Rehmannia glutinosa polysaccharides (RGP) on bone tissue structure and skeletal muscle atrophy in rats with disuse.MethodsA rat model of disuse osteoporosis combined with muscle atrophy was established by removing the bilateral ovaries of rats and fixing their hind limbs for a long time. Forty SD rats were administered intragastrically for 12 weeks. The bone histomorphometry parameters and the level of oxidative stress were measured. In addition, the changes of muscle atrophy F-box (MAFbx), muscle RING-finger protein-1 (MuRF1), forkhead box O1 (FOXO1) mRNA expression in skeletal muscle of rats were observed.ResultsRGP significantly increased the percentage of fluorescence perimeter and bone mineralization deposition rate of the second lumbar vertebrae of rats. It also significantly increased the wet weight ratio and muscle fiber cross-sectional area of the gastrocnemius muscle of rats. At the same time, RGP significantly increased the levels of super oxide dismutase (SOD) and catalase (CAT) in the skeletal muscle of rats, and reduced the content of malondialdehyde (MDA). Rehmannia glutinosa polysaccharides also significantly reduced the expression levels of FOXO1, MAFbx and MuRF1 mRNA in rat skeletal muscle.ConclusionsRGP could improve the bone structure of osteoporotic rats. It could also improve muscle that atrophy may be related to the inhibition of FOXO1-mediated ubiquitin-proteasome pathway.

Down-Regulated FOXO1 in Refractory/Relapse Childhood B-Cell Acute Lymphoblastic Leukemia.

Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with an overall prevalence of 4/100,000, accounting for 25–30% of all childhood cancers. With advances in childhood ALL treatment, the cure rate for childhood ALL has exceeded 80% in most countries. However, refractory/relapsed ALL remains a leading cause of treatment failure and subsequent death. Forkhead box O1 (FOXO1) belongs to the forkhead family of transcription factors, but its role in B-cell ALL (B-ALL) has not been determined yet.Procedures: RNA sequencing was applied to an ALL case with induction failure (IF) to identify the possible genetic events. A cytokine-dependent growth assay in Ba/F3 cells was used to test the leukemic transformation capacity of MEIS1–FOXO1. The propidium iodide (PI) staining method was used to evaluate the effect of MEIS1–FOXO1 on cycle distribution. FOXO1 transactivity was examined using a luciferase reporter assay. FOXO1 mRNA expression levels were examined using real-time quantitative PCR among 40 children with B-ALL treated with the CCCG-ALL-2015 protocol. Association analysis was performed to test the correlation of FOXO1 transcription with childhood B-ALL prognosis and relapse in a series of GEO datasets. An MTT assay was performed to test the drug sensitivity.Results: In this ALL case with IF, we identified a novel MEIS1–FOXO1 fusion gene. The transactivity of MEIS1–FOXO1 was significantly lower than that of wild-type FOXO1. MEIS1–FOXO1 potentiated leukemia transformation and promoted Ba/F3 cell cycle S-phase entry. Low FOXO1 transcription levels were found to be strongly associated with unfavorable ALL subtype, minimal residual disease (MRD) positivity, and relapse. Lower FOXO1 expression was associated with prednisone and cyclophosphamide resistance.Conclusions: Low FOXO1 transcription was associated with high-risk stratification and relapse in children with B-ALL, probably due to multi-drug resistance.

High expression of FOXO3 is associated with poor prognosis in patients with hepatocellular carcinoma.

The role of forkhead box O3 (FOXO3) as a tumor suppressor gene and its association with the human lifespan is well documented. However, several studies have indicated that high expression of FOXO3 is also significantly associated with tumorigenesis. The aim of the present study was to determine the clinical significance of FOXO3 in the development and prognosis of hepatocellular carcinoma (HCC). mRNA expression data of FOXO3 from The Cancer Genome Atlas database was analyzed through the UALCAN online tool to compare the expression of FOXO3 between HCC and normal liver tissues. Subsequently, the expression of FOXO3 at the protein level was investigated via immunohistochemical staining of 314 HCC and 150 non-cancerous liver tissue samples. The association between protein expression and clinicopathological parameters was analyzed using the χ2 test, and the effect of FOXO3 expression on survival was assessed via Kaplan-Meier analysis. The expression of FOXO3 mRNA was significantly higher in HCC in comparison with healthy tissues. High FOXO3 protein expression was revealed in 43/150 non-cancerous liver tissues, and in 238/314 HCC samples. A significant association was demonstrated between FOXO3 expression and metastasis, Tumor-Node-Metastasis stage, Edmondson grade, α-fetoprotein level and overall survival. In conclusion, the high expression of FOXO3 predicts a poor prognosis in patients with HCC, indicating this protein as a potential therapeutic target in HCC.

METTL14 aggravates endothelial inflammation and atherosclerosis by increasing FOXO1 N6-methyladeosine modifications.

Aims: The N6-methyladenosine (m6A) modification plays an important role in various biological processes, but its role in atherosclerosis remains unknown. The aim of this study was to investigate the role and mechanism of m6A modification in endothelial cell inflammation and its influence on atherosclerosis development.Methods: We constructed a stable TNF-α-induced endothelial cell inflammation model and assessed the changes in the expression of m6A modification-related proteins to identify the major factors involved in this process. The m6A-modified mRNAs were identified by methylated RNA immunoprecipitation (RIP) sequencing and forkhead box O1 (FOXO1) was selected as a potential target. Through cytological experiments, we verified whether methyltransferase-like 14 (METTL14) regulates FOXO1 expression by regulating m6A-dependent mRNA and protein interaction. The effect of METTL14 on atherosclerosis development in vivo was verified using METTL14 knockout mice.Results: These findings confirmed that METTL14 plays major roles in TNF-α-induced endothelial cell inflammation. During endothelial inflammation, m6A modification of FOXO1, an important transcription factor, was remarkably increased. Moreover, METTL14 knockdown significantly decreased TNF-α-induced FOXO1 expression. RIP assay confirmed that METTL14 directly binds to FOXO1 mRNA, increases its m6A modification, and enhances its translation through subsequent YTH N6-methyladenosine RNA binding protein 1 recognition. Furthermore, METTL14 was shown to interact with FOXO1 and act directly on the promoter regions of VCAM-1 and ICAM-1 to promote their transcription, thus mediating endothelial cell inflammatory response. In vivo experiments showed that METTL14 gene knockout significantly reduced the development of atherosclerotic plaques.Conclusion: METTL14 promotes FOXO1 expression by enhancing its m6A modification and inducing endothelial cell inflammatory response as well as atherosclerotic plaque formation. Decreased expression of METTL14 can inhibit endothelial inflammation and atherosclerosis development. Therefore, METTL14 may serve as a potential target for the clinical treatment of atherosclerosis.

MicroRNA Let-7f-1-3p attenuates smoke-induced apoptosis in bronchial and alveolar epithelial cells in vitro by targeting FOXO1

Bronchial and alveolar epithelial cell apoptosis is a vital step in smoke-induced lung injury. We investigated whether and how microRNA (miRNA) Let-7f-1-3p would regulate smoke-induced apoptosis in bronchial and alveolar epithelial cells. Human small airway epithelial cells (HSAEC) and human pulmonary alveolar epithelial cells (HPAEpiC) were cultured using an air-liquid interface cell culture system. These cells were treated with Let-7f-1-3p agomir or antagomir for 24 h before smoke exposure or sham operation, after which the cells were rinsed and cultured for 24 h before cell viability, apoptosis, cytolysis, Caspase-9/8/3 activity assays, quantitative real-time polymerase chain reaction and Western blot. Bioinformatic and luciferase reporter assays were performed to predict or verify the target gene of Let-7f-1-3p. We found that smoke exposure significantly reduced Let-7f-1-3p expression level in HSAEC and HPAEpiC. Let-7f-1-3p agomir significantly attenuated cell apoptosis, cytolysis and Caspase-3, -8 and -9 activation while rescuing cell viability of smoke-exposed HSAEC and HPAEpiC. Let-7f-1-3p agomir downregulated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), Fas ligand (FasL) and B-cell lymphoma-2 (Bcl2)-like protein 11 (Bim) protein level in HSAEC and HPAEpiC. Forkhead box-O1 (FOXO1) was verified as a putative regulatory target of Let-7f-1-3p. Smoke exposure increased FOXO1 mRNA and protein level in HSAEC and HPAEpiC, which was attenuated by Let-7f-1-3p agomir treatment. FOXO1 inhibition by small-molecule drug partially attenuated the increase in smoke-exposed HSAEC and HPAEpiC apoptosis, cytolysis and the decrease in cell viability caused by Let-7f-1-3p antagomir treatment. We concluded Let-7f-1-3p attenuated smoke-induced apoptosis in HSAEC and HPAEpiC by targeting FOXO1.

Atrasentan alleviates high glucose-induced podocyte injury by the microRNA-21/forkhead box O1 axis

Diabetic nephropathy (DN) is the most common complication of diabetes mellitus. Atrasentan (Atr) has potential therapeutic values for DN. MicroRNAs (miRNAs) function as vital regulators in the pathophysiology of kidney diseases including DN. Our present study aimed to further explore whether Atr could alleviate kidney injury by regulating microRNA-21(miR-21)/forkhead box O1 (FOXO1) in DN mouse models and cell models. Blood glucose concentration and ACR ratio were determined by matching commercial kits. MiR-21 and FOXO1 mRNA level was measured by RT-qPCR assay. Protein levels of FOXO1, LC3Ⅰ, LC3Ⅱ and p62 were measured by western blot assay. Cell apoptotic index was examined by flow cytometry. The interaction of miR-21 and FOXO1 was tested by bioinformatics analysis, luciferase assay and RIP assay. We found that Atr alleviated kidney injury by inhibiting miR-21 expression and promoting autophagy in DN mice. Moreover, miR-21 loss suppressed apoptosis and induced autophagy in high glucose (HG)-treated podocytes. And, Atr inhibited cell apoptosis and improved cell autophagic activity by downregulating miR-21 in HG-cultured podocytes. Moreover, FOXO1 was identified as a target of miR-21. MiR-21 exerted its pro-apoptosis and anti-autophagy effects by targeting FOXO1 in HG-cultured podocytes. Atr enhanced FOXO1 expression by downregulating miR-21 in HG-cultured podocytes. We concluded that Atr mitigated kidney injury in DN mice and alleviated HG-mediated apoptosis increase and autophagy inhibition in podocytes by regulating miR-21/FOXO1 axis, further elucidating the molecular basis by which Atr hampered DN progression.

Forkhead box O3 promotes cell proliferation and inhibits myotube differentiation in chicken myoblast cells

ABSTRACT1. In the poultry industry, growth performance is important due to its effects on economic value. Much effort has been put forth to achieve introgression of specific genes and DNA markers related to muscle proliferation and differentiation in selective breeding approaches.2. This study investigated the biological functions of the gene Forkhead box O3 (FOXO3) during myogenic differentiation in chicken myoblast cells. FOXO3 was downregulated in primary chicken myoblast (pCM) cells by the piggyBac transposon-mediated microRNA (miRNA) knock-down (KD) system.3. The pCM cells that were stably integrated into the FOXO3 KD expression vector showed significant downregulation of FOXO3 protein and mRNA levels. Expression levels of paired box protein Pax7 (Pax7) and target genes such as CCAAT/enhancer binding protein beta and serum response element decreased in FOXO3 KD pCM cells. In addition, in the undifferentiated myoblast stage, there were no significant differences in cell morphology; however, proliferation rate in FOXO3 KD pCM cells was significantly lower during d 4 and 5 of in vitro culture. By contrast, when myotube differentiation was induced, FOXO3 KD pCM cells exhibited rapid initiation of myotube formation, higher expression of myogenin and desmin as myogenic indicators and a further differentiated phenotype than observed in regular pCM cells.4. These results demonstrated that FOXO3 promotes cell proliferation and inhibits myotube differentiation in chicken myoblast cells. Therefore, the regulation of FOXO3 could be applied to improve muscle differentiation in commercial poultry.

Peroxiredoxin 1 silencing inhibited the growth and promoted apoptosis of pancreatic cancer cells via targeting FOXO3 gene.

ObjectiveOur study aimed to investigate the interaction between peroxiredoxin 1 (Prx1) and forkhead box O3 (FOXO3) and to explore the role of PI3K/AKT pathway in the development of pancreatic cancer.Material and methodsHuman pancreatic normal cells HPDE6-C7 and pancreatic cancer cells PANC-1 were randomly divided into control group, Prx1-silencing (si-Prx1) group, Prx1/FOXO3 dual-silencing (si-Prx1/FOXO3) group, and negative control group. Cell proliferation assay, clone formation assay, and cell apoptosis assay were performed to investigate the effects of Prx1 silencing and FOXO3 silencing on the proliferation and apoptosis ability of pancreatic cancer cells. qRT-PCR and Western blot were performed to study the Prx1 and FOXO3 mRNA in the two cells and FOXO3 protein expression in PANC-1 cells.ResultWe found Prx1 silencing could inhibit growth and promote apoptosis of PANC-1 cells. And Prx1 silencing could decrease the Prx1 mRNA level and increase FOXO3 mRNA level. To further explore the role of Prx1 in PI3K/AKT, we study the cell proliferation and apoptosis ability after adding the PI3K inhibitor and PI3K activator. We observed that PI3K inhibitor could inhibit tumor cell growth and promote cell apoptosis. And PI3K inhibitor also downregulated Prx1 protein expression.ConclusionWe concluded that the Prx1 silencing inhibited the growth and promoted apoptosis of pancreatic cancer cells via modulation of PI3K/AKT pathway by targeting FOXO3 gene.

Ultrafine Particle Exposure Reveals the Importance of FOXO1/Notch Activation Complex for Vascular Regeneration.

Redox active ultrafine particles (UFP, d < 0.2 μm) promote vascular oxidative stress and atherosclerosis. Notch signaling is intimately involved in vascular homeostasis, in which forkhead box O1 (FOXO1) acts as a co-activator of the Notch activation complex. We elucidated the importance of FOXO1/Notch transcriptional activation complex to restore vascular regeneration after UFP exposure. In a zebrafish model of tail injury and repair, transgenic Tg(fli1:GFP) embryos developed vascular regeneration at 3 days post amputation (dpa), whereas UFP exposure impaired regeneration (p < 0.05, n = 20 for control, n = 28 for UFP). UFP dose dependently reduced Notch reporter activity and Notch signaling-related genes (Dll4, JAG1, JAG2, Notch1b, Hey2, Hes1; p < 0.05, n = 3). In the transgenic Tg(tp1:GFP; flk1:mCherry) embryos, UFP attenuated endothelial Notch activity at the amputation site (p < 0.05 vs. wild type [WT], n = 20). A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) inhibitor or dominant negative (DN)-Notch1b messenger RNA (mRNA) disrupted the vascular network, whereas notch intracellular cytoplasmic domain (NICD) mRNA restored the vascular network (p < 0.05 vs. WT, n = 20). UFP reduced FOXO1 expression, but not Master-mind like 1 (MAML1) or NICD (p < 0.05, n = 3). Immunoprecipitation and immunofluorescence demonstrated that UFP attenuated FOXO1-mediated NICD pull-down and FOXO1/NICD co-localization, respectively (p < 0.05, n = 3). Although FOXO1 morpholino oligonucleotides (MOs) attenuated Notch activity, FOXO1 mRNA reversed UFP-mediated reduction in Notch activity to restore vascular regeneration and blood flow (p < 0.05 vs. WT, n = 5). Innovation and Conclusion: Our findings indicate the importance of the FOXO1/Notch activation complex to restore vascular regeneration after exposure to the redox active UFP. Antioxid. Redox Signal. 28, 1209-1223.

Experimental evidences for miR-30b as a negative regulator of FOXO3 upregulated by kynurenine.

Molecular constituents regulated by the microenvironment components profoundly influence the propensity of cancer metastasis, a key event of estimating therapeutic actions of anticancer drugs. On one hand, kynurenine (Kyn), one of the microenvironment components, plays key roles in the metastasis of cancer cells in vivo; on the other hand, forkhead box O3 (FOXO3) can serve as a therapeutic target of various anticancer drugs. However, the effect of Kyn on FOXO3 is still not clear. The current study demonstrated that the selected dose of Kyn significantly upregulated the FOXO3 protein rather than FOXO3 messenger RNA (mRNA) in the lung cancer 95D cells. However, only 50μM Kyn markedly reduced miR-30b expression at the same time. Furthermore, the direct interaction between FOXO3 mRNA and miR-30b was also confirmed by dual-luciferase assay system. More importantly, miR-30b-induced suppression of FOXO3 protein was significantly attenuated on Kyn treatment, which demonstrated that Kyn-mediated increase of FOXO3 depended at least partly on miR-30b. In addition, Kyn-mediated upregulation of migration, a method of measuring cancer metastasis, was markedly decreased on miR-30b treatment, in vitro, which was altered in some degree via regulating FOXO3. These results suggest that miR-30b plays important roles in Kyn-induced increase of FOXO3 expression.

MicroRNA-9 promotes the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1.

The objective of the study was to investigate the role of microRNA-9 (miR-9) targeting forkhead box O1 (FOXO1) in the proliferation, migration, and invasion of breast cancer cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expressions of miR-9 and FOXO1 mRNA in breast cancer tissues, normal breast tissues, breast cancer cell lines, and normal breast epithelial cells. After the up-regulation of miR-9 expression, qRT-PCR and Western blotting were used to determine the expression of FOXO1. The luciferase reporter gene assay was used to validate the target gene. The CCK-8 assay, scratch-wound healing assay, and Transwell invasion assay were used to investigate the changes in the proliferation, migration, and invasion of breast cancer cells, respectively. MicroRNA-9 expression was significantly up-regulated in breast cancer tissues and breast cancer cell lines when compared with normal breast tissues and normal breast epithelial cells (both P<0.05). FOXO1 mRNA and protein expressions were substantially down-regulated in breast cancer tissues and breast cancer cell lines when compared with normal breast tissues and normal breast epithelial cells (both P<0.05). There can be a negative correlation between miR-9 and FOXO1 mRNA in breast cancer. Luciferase reporter gene assay indicated that miR-9 can down-regulate FOXO1 expression at a post-transcriptional level through binding specifically to FOXO1 3'UTR. The results of CCK-8 assay, scratch-wound healing assay, and Transwell invasion assay revealed that the inhibition of miR-9 can suppress MCF7 cell proliferation, migration, and invasion. Additionally, the expression of miR-9 increased significantly whilst that of FOXO1 decreased substantially as the disease progressed (P<0.05). Our study provides evidence that miR-9 can promote the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1.

Metformin inhibits estrogen-dependent endometrial cancer cell growth by activating the AMPK-FOXO1 signal pathway.

Metformin is an oral biguanide commonly used for treating type II diabetes and has recently been reported to possess antiproliferative properties that can be exploited for the prevention and treatment of a variety of cancers. The mechanisms underlying this effect have not been fully elucidated. Our study shows a marked loss of AMP‐activated protein kinase (AMPK) phosphorylation and nuclear human Forkhead box O1 (FOXO1) protein in estrogen‐dependent endometrial cancer (EC) tumors compared to normal control endometrium. Metformin treatment suppressed EC cell growth in a time‐dependent manner in vitro; this effect was cancelled by cotreatment with an AMPK inhibitor, compound C. Metformin decreased FOXO1 phosphorylation and increased FOXO1 nuclear localization in Ishikawa and HEC‐1B cells, with non‐significant increase in FOXO1 mRNA expression. Moreover, compound C blocked the metformin‐induced changes of FOXO1 and its phosphorylation protein, suggesting that metformin upregulated FOXO1 activity by AMPK activation. Similar results were obtained after treatment with insulin. In addition, transfection with siRNA for FOXO1 cancelled metformin‐inhibited cell growth, indicating that FOXO1 mediated metformin to inhibit EC cell proliferation. A xenograft mouse model further revealed that metformin suppressed HEC‐1B tumor growth, accompanied by downregulated ki‐67 and upregulated AMPK phosphorylation and nuclear FOXO1 protein. Taken together, these data provide a novel mechanism of antineoplastic effect for metformin through the regulation of FOXO1, and suggest that the AMPK–FOXO1 pathway may be a therapeutic target to the development of new antineoplastic drugs.

Molecular cloning, characterisation and functional analysis of the duck Forkhead box O3 (FOXO3) gene

1. The Forkhead box O3 (FOXO3) transcription factor is a crucial regulator of cell fate that controls proliferation, apoptosis and differentiation. However, the role of FOXO3 regulation in duck myoblasts is not fully understood.2. The aim of this study was to clone and determine the complete coding sequence (CDS) of the duck FOXO3 gene and to assess its function in myoblasts.3. Primers specific for the predicted duck FOXO3 gene were designed using the public mallard duck reference sequence in GenBank. The CDS was cloned by RT-PCR and double digested to generate the expression vector pEGFP-N1-FOXO3.4. Sequence analysis showed that the full-length FOXO3 CDS is 1467 bp, encoding 488 amino acids and is highly conserved across many bird species. Amino acid sequence analysis revealed a DNA-binding domain (aa 1–77).5. Myoblast transfection with pEGFP-N1-FOXO3 showed that FOXO3 mRNA expression at 24 h was elevated in pEGFP-N1-FOXO3-transfected myoblasts compared to pEGFP-N1-transfected cells or controls. MRF4, MyoD, MyoG, Myf5 and PAX7 mRNA expression in the pEGFP-N1-FOXO3 group was lowest. However, myostatin (MSTN) and PAX3 mRNA expression did not differ.6. These results suggest that FOXO3 plays a critical role in the proliferation and differentiation of duck myoblasts.

Effect of acute and chronic eccentric exercise on FOXO1 mRNA expression as fiber type transition factor in rat skeletal muscles

Skeletal muscle is a highly elastic tissue which can respond to various functional demands by altering fiber-type composition. Exercise affects muscle fiber phenotype. One of the transcription factors that induce fiber-type transition is forkhead box O1 (FOXO1). Since eccentric contraction considered an essential part of exercise, so we are interested to see the effects of eccentric exercise (acute/chronic) on FOXO1 as an important factor of fiber-type transition in rat skeletal muscles. Twenty-four Sprague–Dawley rats (190–235g) were divided to 3 groups of 8 rats: 1) chronic eccentric exercise (CEE), 2) acute eccentric exercise (AEE), and 3) control (C). The exercise groups underwent downhill running protocol. CEE was running on treadmill in 3days of week for 9weeks, that slope and duration gradually managed from −4° to −16° and 15 to 90min, respectively. AEE group was running with 16m/min on −16° slope for 3 consecutive days that included 18 sets of 5min with rest interval of 2min in between. Soleus and super vastus lateralis (SVL) muscles mRNA were analyzed by real-time RT-PCR. SVL FOXO1 mRNA levels increased by 3.92-fold in the AEE and decreased 0.56-fold in the CEE group and were not significant in soleus muscle. In soleus muscle, myosin heavy chain (MHC) IIa, IIx, and IIb decreased in the AEE group and MHC IIa and IIx decreased in the CEE group. In SVL muscle, MHC I, IIa, and IIx increased in the AEE group and MHC IIa and IIX increased in the CEE group. In summary, both acute and chronic eccentric exercise could lead to change in FOXO1 mRNA only in fast SVL muscle of rat and so could induce fiber-type transition in both muscles regardless of changes in expression of FOXO1. So, oxidative stress can play important role in change of FOXO1.

Post-transcriptional repression of FOXO1 by QKI results in low levels of FOXO1 expression in breast cancer cells

The RNA-binding protein Quaking (QKI) is known to be essential for embryonic development and postnatal myelination. Forkhead box O1 (FOXO1) is a critical tumor suppressor for cell proliferation control. Dysregulation of FOXO1 expression has been observed in a variety of cancers. In the present study, we demonstrated that QKI decreased FOXO1 mRNA expression at the post-transcriptional level. QKI was able to bind the 3'UTR of FOXO1 mRNA directly and decreased its mRNA stability. To determine whether QKI-mediated post-transcriptional repression of FOXO1 indeed plays a role in cancer cells, we first detected both QKI and FOXO1 expression in four breast cancer cell lines. FOXO1 expression was extremely low in these cell lines, whereas QKI expression was relative high. Knockdown of QKI significantly restored FOXO1 expression. ATRA, an inducer of apoptosis or differentiation, dramatically enhanced FOXO1 expression while it repressed QKI expression. Importantly, the ATRA-induced increase in FOXO1 expression was dependent on QKI-mediated post-transcriptional regulation. Consistently, 5-FU, a widely used chemotherapeutic agent, increased FOXO1 expression via inhibition of QKI. In summary, our study provides initial evidence demonstrating that QKI-mediated repression of FOXO1 may be one of the factors contributing to the oncogenesis and progression of breast carcinoma, which suggests that targeting QKI may serve as a novel strategy to sensitize breast cancers to chemotherapy.