Forkhead Box Protein O1 Expression Research Articles

Palmitic acid reduces the methylation of the FOXO1 promoter to suppress the development of diffuse large B-cell lymphoma via modulating the miR-429/DNMT3A axis

Diffuse large B-cell lymphoma (DLBCL) is characterized by significant treatment resistance. Palmitic acid (PA) has shown promising antitumor properties. This study aims to elucidate the molecular mechanisms by which PA influences DLBCL progression. We quantified the expression levels of microRNAs (miRNAs), Forkhead box protein O1 (FOXO1), and DNA methyltransferase 3A (DNMT3A) in both untreated and PA-treated DLBCL tumors and cell lines. Assessments were made of cell viability, apoptosis, and autophagy-related protein expression following PA administration. Interaction analyses among miR-429, DNMT3A, and FOXO1 were conducted using luciferase reporter assays and methylation-specific (MSP) Polymerase chain reaction (PCR). After transfecting the miR-429 inhibitor, negative control (NC) inhibitor, shRNA against DNMT3A (sh-DNMT3A), shRNA negative control (sh-NC), overexpression vector for DNMT3A (oe-DNMT3A), or overexpression negative control (oe-NC), we evaluated the effects of miR-429 and DNMT3A on cell viability, mortality, and autophagy-related protein expression in PA-treated DLBCL cell lines. The efficacy of PA was also tested in vivo using DLBCL tumor-bearing mouse models. MiR-429 and FOXO1 expression levels were downregulated, whereas DNMT3A was upregulated in DLBCL compared to the control group. PA treatment was associated with enhanced autophagy, mediated by the upregulation of miR-429 and downregulation of DNMT3A. The luciferase reporter assay and MSP confirmed that miR-429 directly inhibits DNMT3A, thereby reducing FOXO1 methylation. Subsequent experiments demonstrated that PA promotes autophagy and inhibits DLBCL progression by upregulating miR-429 and modulating the DNMT3A/FOXO1 axis. In vivo PA significantly reduced the growth of xenografted tumors through its regulatory impact on the miR-429/DNMT3A/FOXO1 axis. Palmitic acid may modulate autophagy and inhibit DLBCL progression by targeting the miR-429/DNMT3A/FOXO1 signaling pathway, suggesting a novel therapeutic target for DLBCL management.

Silencing of forkhead box protein O-1 (FOXO-1) enhances insulin-producing cell generation from adipose mesenchymal stem cells for diabetes therapy

AimsGeneration of mature β-cells from MSCs has been a challenge in the field of stem cell therapy of diabetes. Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) have made their mark in regenerative medicine, and provide several advantages compared to other MSCs sources. Forkhead box protein O-1 (FOXO-1) is an important transcription factor for normal development of β-cells, yet its over expression in β-cells may cause glucose intolerance. In this study, we isolated, characterized Ad-MSCs from rat epididymal fat pads, differentiated these MSCs into insulin producing cells (IPCs) and studied the role of FOXO-1 in such differentiation. Materials and methodsWe examined the expression of FOXO-1 and its nuclear cytoplasmic localization in the generated IPCs. Afterwards we knocked down FOXO-1 using siRNA targeting FOXO-1 (siFOXO-1). The differentiated siFOXO-1 IPCs were compared to non-targeting siRNA (siNT) IPCs regarding expression of β-cell markers by qRT-PCR and western blotting, dithizone (DTZ) staining and glucose stimulated insulin secretion (GSIS). Key findingsIsolated Ad-MSCs exhibited all characteristics of MSCs and can generate IPCs. FOXO-1 was initially elevated during differentiation followed by a decline towards end of differentiation. FOXO-1 was dephosphorylated and localized to the nucleus upon differentiation into IPCs. Knock down of FOXO-1 improved the expression of β-cell markers in final differentiated IPCs, improved DTZ uptake and showed increased insulin secretion upon challenging with increased glucose concentration. SignificanceThese results portray FOXO-1 as a hindering factor of generation of IPCs whose down-regulation can generate more mature IPCs for MSCs therapy of diabetes mellitus.

Circ_BLNK is a Unique Molecular Marker in Non-small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) patients are characterized by distant metastasis and poor prognosis. Growing evidence has implied that circular RNAs (circRNAs) are involved in multiple tumor progression, including NSCLC. The objective of the present study was to functionally dissect the role and mechanism of circ_BLNK in NSCLC development and progression. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of circ_BLNK, miR-942-5p, and forkhead box protein O1 (FOXO1) in NSCLC tissues and cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and colony formation assay detected cell proliferation; the protein expression levels were tested by western blot assay; cell apoptosis was measured by flow cytometry, and transwell assay detected cell migration and invasion. The molecular targeting relationship was determined by dual-luciferase reporter assay. The effect of circ_BLNK overexpression on tumor growth was detected by in vivo experiments and immunohistochemistry. Circ_BLNK was dramatically decreased in NSCLC, and overexpression of circ_BLNK inhibited proliferation, migration, and invasion of NSCLC cells and promoted cell apoptosis. Circ_BLNK level was negatively correlated with miR-942-5p expression and positively correlated with FOXO1 expression. Moreover, circ_BLNK acted as a sponge for miR-942-5p, which targeted FOXO1. Rescue assays presented that miR-942-5p reversed the anticancer action of circ_BLNK in NSCLC. Besides that, miR-942-5p inhibition suppressed the oncogenic behaviors, which were attenuated by FOXO1 knockdown. Animal experiments exhibited that circ_BLNK upregulation repressed tumor growth in vivo. Our study demonstrated a novel regulatory mechanism that circ_BLNK/miR-942-5p/FOXO1 axis adjusted non-small cell lung cancer development.

O-GlcNAcylation mediates H2O2-induced apoptosis through regulation of STAT3 and FOXO1.

The O-linked-β-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) is a critical post-translational modification that couples the external stimuli to intracellular signal transduction networks. However, the critical protein targets of O-GlcNAcylation in oxidative stress-induced apoptosis remain to be elucidated. Here, we show that treatment with H2O2 inhibited O-GlcNAcylation, impaired cell viability, increased the cleaved caspase 3 and accelerated apoptosis of neuroblastoma N2a cells. The O-GlcNAc transferase (OGT) inhibitor OSMI-1 or the O-GlcNAcase (OGA) inhibitor Thiamet-G enhanced or inhibited H2O2-induced apoptosis, respectively. The total and phosphorylated protein levels, as well as the promoter activities of signal transducer and activator of transcription factor3 (STAT3) and Forkhead box protein O 1 (FOXO1) were suppressed by OSMI-1. In contrast, overexpressing OGT or treating with Thiamet-G increased the total protein levels of STAT3 and FOXO1. Overexpression of STAT3 or FOXO1 abolished OSMI-1-induced apoptosis. Whereas the anti-apoptotic effect of OGT and Thiamet-G in H2O2-treated cells was abolished by either downregulating the expression or activity of endogenous STAT3 or FOXO1. These results suggest that STAT3 or FOXO1 are the potential targets of O-GlcNAcylation involved in the H2O2-induced apoptosis of N2a cells.

Tumor-derived exosomal miR-1247-3p promotes angiogenesis in bladder cancer by targeting FOXO1

ABSTRACTTumor-derived exosomes are highly correlated with tumor progression and angiogenesis. This study was designed to probe the role of tumor-derived exosomal miR-1247-3p in mediating the angiogenesis in bladder cancer. Exosomes isolation from the culture medium of normal or bladder cancer cell lines was performed using a differential centrifugation method. miR-1247-3p expression in exosomes and cells was detected by quantitative real-time PCR (qRT-PCR). The effect of exosomes on the angiogenesis of human umbilical vein endothelial cells (HUVECs) was assessed using cell counting kit-8 (CCK-8), transwell and tube formation assays. The interaction between miR-1247-3p and forkhead box protein O1 (FOXO1) was studied using luciferase reporter and RNA pull down assays. Exosomes were successfully isolated from T24, UM-UC-3, and SV-HUC-1 cells, as confirmed by corresponding identifications. Functional experiments revealed that exosomes derived from T24 and UM-UC-3 cells significantly enhanced the abilities of proliferation, migration, angiogenesis, and vascular endothelial-derived growth factor (VEGF) secretion in HUVECs. miR-1247-3p was highly expressed in exosomes derived from T24 and UM-UC-3 cells, and exosomes derived from miR-1247-3p inhibitor-transfected cells reduced HUVEC viability, migration, tube formation, and VEGF level. FOXO1 was confirmed as a direct target of miR-1247-3p. Rescue assays suggested that the effect of miR-1247-3p inhibition on the viability, migration, and angiogenesis of HUVECs was partly abrogated by the knockdown of FOXO1. Our data suggest that miR-1247-3p is up-regulated in tumor-derived exosomes, thereby inhibiting FOXO1 expression and facilitating angiogenesis in bladder cancer.

The effect of heat-killed Lactobacillus brevis SBL88 on improving selective hepatic insulin resistance in non-alcoholic fatty liver disease mice without altering the gut microbiota.

There have been several reports that some probiotics improve non-alcoholic fatty liver disease (NAFLD); however, many studies have involved cocktail therapies. We evaluated whether heat-killed Lactobacillus brevis SBL88 (L.brevis SBL88) monotherapy improves the clinical features of NAFLD. The NAFLD model was induced in mice fed a high-fat diet (HFD) (HFD mice) or HFD+1% heat-killed L.brevis SBL88 (SBL mice) for 16weeks. Histopathological liver findings were analyzed. To evaluate the gut microbiota, a modified terminal restriction fragment length polymorphism analysis of the feces was performed. RNA sequencing in the liver was performed with Ion Proton™. To investigate the direct effects of heat-killed L.brevis SBL88, an in vitro study was performed. Histopathological findings revealed that fat droplets in the liver were significantly reduced in SBL mice; however, terminal restriction fragment length polymorphism did not show alterations in the gut microbiota between HFD mice and SBL mice. RNA sequencing and pathway analysis revealed that the regulation of lipid and insulin metabolism was affected. The mRNA expression of insulin receptor substrate 2 (IRS-2) was significantly higher in SBL mice, whereas the expression of IRS-1 was not significantly different. Phospho-IRS-2 expression was also significantly increased in SBL mice. In addition, an in vitro study revealed significant alterations in IRS-2 and forkhead box protein O1 expression levels. SBL mice exhibited partially improved selective hepatic insulin resistance. Our data suggest that heat-killed L.brevis SBL88 could attenuate the clinical features of NAFLD that are not mediated by alterations in the gut microbiota.

MiR-183-5p promotes the progression of non-small cell lung cancer through targeted regulation of FOXO1

Abstract Objective To investigate miR-183-5p targeting to forkhead box protein O1 (FOXO1) and its corresponding effect on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) cells. Methods NSCLC tissues and adjacent normal tissues from 60 patients with NSCLC adenocarcinoma were obtained via pathological biopsy or intraoperative resection. Several cell lines were cultured in vitro, including the human normal lung epithelial cell line BEAS-2B and human NSCLC cell lines A549, SPCA-1, PC-9, and 95-D. miR-183-5p and FOXO1 mRNA expression in tissues and cells were detected by qRT-PCR; the corresponding correlations in NSCLC tissues were analyzed using the Pearson test, and the relationship between miR-183-5p expression and clinicopathological parameters was analyzed. The miR-183-5p-mediated regulation of FOXO1 was verified by bioinformatics prediction alongside double luciferase, RNA-binding protein immunoprecipitation (RIP) assay, and pull-down experiments. A549 cells were divided into control, anti-miR-NC, anti-miR-183-5p, miR-NC, miR-183-5p, miR-183-5p+pcDNA3.1, and miR-183-5p+pcDNA3.1-FOXO1 groups. Cell proliferation, invasion, migration, apoptosis, and cell cycle distribution were detected using an MTT assay, clone formation assay, Transwell assay, scratch test, and flow cytometry, respectively. The expression of EMT-related proteins in the cells was analyzed by western blotting. The effect of miR-185-3p silencing on the development of transplanted tumors was detected by analyzing tumor formation in nude mice. Results miR-183-5p expression was significantly higher in NSCLC tissues and cells than in adjacent normal tissues, whereas FOXO1 mRNA expression was significantly down-regulated. There was a significant negative correlation between miR-183-5p and FOXO1 mRNA in NSCLC tissues (P < 0.05). Additionally, the expression of miR-183-5p was significantly correlated with tumor size, tumor differentiation, and tumor-node-metastasis stage in patients with NSCLC (P < 0.05). miR-183-5p targeted and inhibited FOXO1 expression. Compared to the anti-miR-NC group, the cell proliferation, scratch healing rate, N-cadherin and vimentin protein expression, and the proportion of S phase cells were significantly lower in the anti-miR-183-5p group, whereas the protein expression of E-cadherin and α-catenin and the proportion of G0/G1 phase cells were significantly higher; additionally, the frequency of colony formation and invasion were significantly lower in the anti-miR-183-5p group (P < 0.05). Compared to the miR-NC group, the cell proliferation, scratch healing rate, N-cadherin and vimentin protein expression, and the proportion of S phase cells in the miR-183-5p group were significantly higher, whereas the E-cadherin and α-catenin protein expression and the proportion of G0/G1 phase cells were significantly lower; furthermore, the frequency of colony formation and invasion were significantly higher in the miR-183-5p group (P < 0.05). Compared with the miR-183-5p+pcDNA3.1 group, the OD value, scratch healing rate, N-cadherin and vimentin protein expression, and the proportion of S phase cells were significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group, whereas E-cadherin and α-catenin protein expression and the proportion of G0/G1 phase cells were significantly higher; additionally, the frequency of colony formation and invasion was significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group (P < 0.05). Overall, silencing miR-185-3p inhibited the growth of transplanted tumors and promoted FOXO1 expression. Conclusion Overexpression of miR-183-5p can inhibit apoptosis and promote the proliferation, migration, invasion, and EMT, of NSCLC cells by down-regulating FOXO1 expression.

SIRT3 improves alveolar epithelial cell damage caused by bronchopulmonary dysplasia through deacetylation of FOXO1.

Bronchopulmonary dysplasia (BPD) is a serious and long-term lung condition commonly observed in premature babies. Sirtuin 3 (SIRT3) has been reported to reduce pulmonary injury and pulmonary fibrosis. The present study investigated the specific role of SIRT3 in BPD by establishing hyperoxia-induced BPD rat and cell models. Hematoxylin and eosin staining was used to observe pathological changes in lung tissues. The expression levels of SIRT3 and forkhead box protein O1 (FOXO1), as well as its acetylation levels, were detected in hyperoxia-induced lung tissues and cells by Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Levels of reactive oxygen species, superoxide dismutase, and malondialdehyde were assessed by using biochemical kits. Following SIRT3 overexpression, the levels of inflammatory cytokines were assessed by RT-qPCR. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) and Western blot analysis. Upon FOXO1 knockout, cell inflammation, oxidative stress and apoptosis were evaluated again. Compared to the control group, the SIRT3 and FOXO1 expression levels were decreased and the FOXO1 acetylation levels were increased in hyperoxia-induced lung tissues and cells. In addition, SIRT3 reduced hyperoxia-induced inflammation, oxidative stress, and apoptosis in A549 cells, and inhibited FOXO1 acetylation to activate FOXO1. However, FOXO1 knockdown reversed the effects of SIRT3 overexpression in hyperoxia-induced A549 cells. SIRT3 relieved alveolar epithelial cell damage caused by BPD via deacetylation of FOXO1, suggesting that SIRT3 could be a therapeutic target in BPD.

Simiao Wan and its ingredients alleviate type 2 diabetes mellitus via IRS1/AKT2/FOXO1/GLUT2 signaling.

Type 2 diabetes mellitus (T2DM) is a metabolic disease. Simiao Wan (SMW) is a commonly used clinical drug for hyperuricemia treatment. SMW has been confirmed to improve insulin resistance and is expected to be a novel hypoglycemic agent. However, the hypoglycemic bioactive ingredients and mechanisms of action of SMW are unclear. To explore the hypoglycemic effects and reveal the mechanisms of SMW and bioactive ingredients (SMW-BI). The hypoglycemic effects of SMW and SMW-BI were verified in a mouse model of T2DM induced by streptozotocin (STZ) and a high-fat and high-sugar diet (HFSD). Network pharmacology was used to predict the mechanisms of SMW and SMW-BI. Histological analysis and real-time quantitative polymerase chain reaction (RT-qPCR) verified network pharmacology results. RT-qPCR results were further verified by immunofluorescence (IFC) and molecular docking. The correlation between proteins and biochemical indicators was analyzed by Spearman's correlation. Chlorogenic acid, phellodendrine, magnoflorine, jateorhizine, palmatine, berberine, and atractydin were identified as SMW-BI. After 8 weeks of treatment, SMW and SMW-BI decreased the levels of fasting blood glucose (FBG), total cholesterol (TC), triacylglycerols (TG) and low-density lipoprotein cholesterol (LDL-C), increased the level of high-density lipoprotein cholesterol (HDL-C), alleviated weight loss, and increased serum insulin levels in T2DM mice. In addition, SMW and SMW-BI improved hepatocyte morphology in T2DM mice, decreased the number of adipocytes, and increased liver glycogen. Network pharmacological analysis indicated that SMW and SMW-BI may exert hypoglycemic by regulating insulin receptor substrate 1 (IRS1)/RAC-beta serine/threonine-protein kinase (AKT2)/forkhead box protein O1 (FOXO1)/glucose transporter type 2 (GLUT2) signaling. Moreover, correlation analysis showed that SMW and SMW-BI were associated with activation of IRS1, AKT2, and GLUT2, and inhibiting FOXO1. RT-qPCR revealed that SMW and SMW-BI could increase levels of IRS1, AKT2, and GLUT2 in the livers of T2DM mice and lower the level of FOXO1. Furthermore, immunofluorescence analysis showed that FOXO1 expression in the livers of T2DM mice decreased after oral administration of SMW and SMW-BI. Furthermore, molecular docking showed that SMW-BI could bind directly to IRS1 and AKT2. SMW and SMW-BI are potential hypoglycemic drugs that alleviate T2DM by regulating IRS1/AKT2/FOXO1 signaling. Our study provides a research idea for screening the bioactive ingredients in traditional Chinese medicine (TCM).

The hepatoprotective effects of n3-polyunsaturated fatty acids against non-alcoholic fatty liver disease in diabetic rats through the FOXO1/PPARα/GABARAPL1 signalling pathway

AimWe compared the efficacy of n3-polyunsaturated fatty acids (n3-PUFAs) and metformin in halting the progression of non-alcoholic fatty liver disease (NAFLD) developed in the milieu of insulin deficiency. Main methodsNAFLD was induced by a chronic high-fat diet (HFD) in male Sprague Dawley rats, rendered diabetic by a low dose streptozotocin (STZ). Diabetic rats were treated with n3-PUFAs (300 mg/kg/d) or metformin (150 mg/kg/d) for 8 weeks. Improvements in the NAFLD score and hepatic insulin resistance (IR) were addressed and correlated to changes in the hepatic expression of Forkhead box protein O1 (FOXO-1), microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) and gamma-aminobutyric acid receptor-associated protein-like 1 (GABARAPL1) genes. Hepatic peroxisome proliferator-activated receptor alpha (PPAR-α), and B-cell lymphoma 2 (Bcl-2) protein expression was also assessed. Key findingsDriven by insulin deficiency and HFD, the FOXO-1 gene along with its downstream targets, MAP1LC3B and GABARAPL1, were highly expressed in the liver tissue of the HFD/STZ group. Meanwhile, hepatic expression of PPAR-α and Bcl-2 was markedly decreased. These abnormalities coincided with a marked increase in the hepatic IR and NAFLD activity. Comparable to metformin, n3-PUFAs were able to rearrange hepatic PPAR-α and FOXO-1 expression in HFD/STZ rats, resulting in improved diabetic/steatotic liver phenotype. SignificanceAlong with the enhancement of PPAR-α expression, inhibition of FoxO1/GABARAPL1/MAP1LC3B transcription is suggested as a core mechanism for the protective effects of n3-PUFAs on hepatic IR and NAFLD. Under conditions of insulin deficiency, n3-PUFAs retain their potential as a safe and promising approach for the control of NAFLD.

Fighting Fire with Fire: Exosomes and Acute Pancreatitis-Associated Acute Lung Injury.

Acute pancreatitis (AP) is a prevalent clinical condition of the digestive system, with a growing frequency each year. Approximately 20% of patients suffer from severe acute pancreatitis (SAP) with local consequences and multi-organ failure, putting a significant strain on patients' health insurance. According to reports, the lungs are particularly susceptible to SAP. Acute respiratory distress syndrome, a severe type of acute lung injury (ALI), is the primary cause of mortality among AP patients. Controlling the mortality associated with SAP requires an understanding of the etiology of AP-associated ALI, the discovery of biomarkers for the early detection of ALI, and the identification of potentially effective drug treatments. Exosomes are a class of extracellular vesicles with a diameter of 30-150 nm that are actively released into tissue fluids to mediate biological functions. Exosomes are laden with bioactive cargo, such as lipids, proteins, DNA, and RNA. During the initial stages of AP, acinar cell-derived exosomes suppress forkhead box protein O1 expression, resulting in M1 macrophage polarization. Similarly, macrophage-derived exosomes activate inflammatory pathways within endothelium or epithelial cells, promoting an inflammatory cascade response. On the other hand, a part of exosome cargo performs tissue repair and anti-inflammatory actions and inhibits the cytokine storm during AP. Other reviews have detailed the function of exosomes in the development of AP, chronic pancreatitis, and autoimmune pancreatitis. The discoveries involving exosomes at the intersection of AP and acute lung injury (ALI) are reviewed here. Furthermore, we discuss the therapeutic potential of exosomes in AP and associated ALI. With the continuous improvement of technological tools, the research on exosomes has gradually shifted from basic to clinical applications. Several exosome-specific non-coding RNAs and proteins can be used as novel molecular markers to assist in the diagnosis and prognosis of AP and associated ALI.

Exosomal MicroRNA-325 Enhances Trophoblast Migration and Invasion Through Downregulation of Lethal-7b and Upregulation of Forkhead Box Protein O1 Expression in Preeclampsia

We aimed to explore the mechanism by how microRNA (miRNA)-325 derived from marrow mesenchymal stem cell exosomes (MSC-exos) affects the trophoblast progression in preeclampsia (PE). RT-qPCR detected the level of miRNA let-7b and FOXO1 in the placenta tissue of PE patients. Functional experiment was performed to analyze the effect of FOXO1 inhibitor and let-7b mimics on cell migration, invasion and apoptosis through Transwell assay and TUNEL staining. The trophoblast cell was co-cultured with overexpressed-miR-325 MSC-exos to measure gene expression and cell progression. let-7b was highly and FOXO1 was lowly expressed in PE placenta tissue. let-7b directly targeted and inhibited FOXO1 expression. Importantly, as miR-325 was internalized by trophoblast cells through MSC-exos, MSC-exos overexpressing miR-325 inhibited let-7b expression in trophoblasts, up-regulated FOXO1 and activated AKT signaling pathway. Further, MSC-exos treatment promoted invasion and migration of trophoblast cell and inhibited apoptosis. In conclusion, miR-325 derived from MSC-exos promotes the invasion and migration of trophoblast cells in PE through inhibition of let7b and upregulation of FOXO1.

Effect of miR-183-5p on Cholestatic Liver Fibrosis by Regulating Fork Head Box Protein O1 Expression.

Liver fibrosis is a common pathological feature of end-stage liver disease and has no effective treatment. MicroRNAs (miRNAs) have been found to modulate gene expression in liver disease. But the potential role of miRNA in hepatic fibrosis is still unclear. The objective of this research is to study the potential mechanism and biological function of miR-183-5p in liver fibrosis. In this study, we used high-throughput sequencing to find that miR-183-5p is upregulated in human fibrotic liver tissues. In addition, miR-183-5p was upregulated both in rat liver fibrosis tissue induced by bile-duct ligation (BDL) and activated LX-2 cells (human hepatic stellate cell line) according to the result of quantitative real-time PCR (RT-qPCR). Moreover, the inhibition of miR-183-5p alleviated liver fibrosis, decreased the fibrotic biomarker levels in vitro and in vivo, and led toLX-2 cell proliferation inhibition and, apoptosis induction. The result of dual-luciferase assay revealed that miR-183-5p suppressed fork head box protein O1 (FOXO1) expression by binding to its 3′UTR directly. Next, we used lentivirus to overexpress FOXO1 in LX-2 cells, and we found that overexpression of FOXO1 reversed the promotion of miR-183-5p on liver fibrosis, reducing the fibrotic biomarker levels inLX-2 cells, inhibitingLX-2 cell proliferation, and promoting apoptosis. Furthermore, overexpression of FOXO1 prevented the activation of the transforming growth factor (TGF)-β signaling pathway in TGF-β1-induced LX-2 cells according to the result of western blotting. In conclusion, the findings showed thatmiR-183-5p might act as a key regulator of liver fibrosis, and miR-183-5p could promote cholestatic liver fibrosis by inhibiting FOXO1 expression through the TGF-β signaling pathway. Thus, inhibition of miR-183-5pmay be a new way to prevent and improve liver fibrosis.

Genetic and Functional Evaluation of the Role of FOXO1 in Antituberculosis Drug-Induced Hepatotoxicity.

Background The accumulation of the hepatotoxic substance protoporphyrin IX (PPIX) induced by aminolevulinate synthase 1 (ALAS1) activation is one of the important mechanisms of antituberculosis drug-induced hepatotoxicity (ATDH). Forkhead box protein O1 (FOXO1) may activate ALAS1 transcription. However, little is known about their roles in ATDH; we performed a study to determine the association between polymorphisms in the two genes and ATDH susceptibility. Then, we verified this possible association by cellular functional experiments. Materials and Methods Tag single-nucleotide polymorphisms (TagSNPs) in the two genes were genotyped in 746 tuberculosis patients. The frequencies of the alleles, genotypes, genetic models, and haplotype distribution of the variants were compared between the case and control groups. L-02 cells and HepG2 cells were incubated with the indicated concentration of isoniazid (INH) and rifampicin (RIF) for the desired times, and then the expression levels of ALAS1 and FOXO1 mRNAs and proteins were detected. HepG2 cells were transiently transfected with FOXO1 siRNA to observe the effect of changes in the FOXO1 expression on the cell survival rate and ALAS1 expression. Results The C allele at rs2755237 and the T allele at rs4435111 in the FOXO1 gene were associated with a decreased risk of ATDH. The expression of ALAS1 in both L-02 cells and HepG2 cells was increased by the coadministration of INH/RIF (600/200 μM) for 24 h. Although FOXO1 expression was reduced slightly by the same treatment, its content in the nucleus was significantly increased. However, the cell survival rate and ALAS1 expression level were not significantly altered by the downregulation of FOXO1 in HepG2 cells. Conclusions Variants of the rs4435111 and rs2755237 loci in the FOXO1 gene were associated with susceptibility to ATDH. Coadministration of INH/RIF promoted the transfer of FOXO1 from the cytoplasm to the nucleus, but the functional significance of its nuclear translocation requires further verification.

Circ_GRN Promotes the Proliferation, Migration, and Inflammation of Vascular Smooth Muscle Cells in Atherosclerosis Through miR-214-3p/FOXO1 Axis.

Dysfunction of vascular smooth muscle cells (VSMCs) assumes a fundamental part in the pathogenesis of atherosclerosis (AS). Circular RNA granulin precursor (circ_GRN) was identified to promote the proliferation and invasion of human VSMCs (HVSMCs) in an in vitro AS model. However, the underlying mechanisms remain unclear. Levels of circ_GRN, microRNA (miR)-214-3p, and forkhead box protein O1 (FOXO1) were detected using quantitative real-time polymerase chain reaction and Western blot assays. The proliferation, migration, and inflammatory response of HVSMCs were evaluated by using flow cytometry, colony formation, Cell Counting Kit-8, Western blot, transwell assays, and enzyme-linked immunosorbent assay, respectively. The binding interaction between miR-214-3p and circ_GRN or FOXO1 was detected by dual-luciferase reporter assay. In this study, we found that circ_GRN was elevated in the serum of AS and oxidized low-density lipoprotein (ox-LDL)-induced HVSMCs. The in vitro AS model was established by exposing HVSMCs to ox-LDL, and we found circ_GRN knockdown reversed ox-LDL-evoked cell proliferation, migration, and inflammation. In a mechanical study, miR-214-3p directly bound to circ_GRN or FOXO1, and circ_GRN could regulate FOXO1 expression by competitively binding to miR-214-3p. Importantly, we demonstrated that miR-214-3p inhibition attenuated the protective effects of circ_GRN knockdown on ox-LDL-induced HVSMCs; besides that, miR-214-3p overexpression abolished ox-LDL-triggered HVSMC proliferation, migration, and inflammation, which were counteracted by FOXO1 upregulation. In conclusion, circ_GRN promoted the proliferation, migration, and inflammation of HVSMCs through miR-214-3p/FOXO1 axis in ox-LDL-induced AS model in vitro, suggesting the potential involvement in an AS process, which provided a potential candidate for future clinic intervention in AS.

Therapeutic effects of Chlorella vulgaris on carbon tetrachloride induced liver fibrosis by targeting Hippo signaling pathway and AMPK/FOXO1 axis.

This study was conducted to present the mechanism of the therapeutic effects of Chlorella vulgaris extract (CV) on the carbon tetrachloride (CCl4) induced liver fibrosis model. Primarily, the mechanism of antioxidant effects of CV were investigated via measuring the expression of forkhead box protein O1 (FOXO1) and phosphorylated 5' adenosine monophosphate-activated protein kinase (p-AMPK) as upstream regulators of superoxide dismutase (SOD) and catalase (CAT). Subsequently, we investigated the regulatory effect of CV treatment on the yes-associated protein (YAP) and transcriptional coactivators with a PDZ-binding motif (TAZ) as fibrogenic factors. Male Wistar rats received CCl4 and olive oil solution 1ml/kg intraperitoneally for 12weeks, twice weekly. CV 50 and 100mg/kg were administered on a daily basis by gavage in the last 4weeks. Ultimately, liver marker enzymes and hepatic hydroxyproline content were measured. The activity of SOD and CAT and the expression of YAP, TAZ, FOXO1, SOD, and CAT were analyzed. Finally, the protein levels of YAP, TAZ, and p-AMPK were detected. CV administration decreased liver marker enzymes and hydroxyproline content significantly. The expression and protein levels of YAP and TAZ reduced by CV treatment. Furthermore, the augmentation of expression and function of CAT and SOD by CV treatment was followed by an increase in the expression of FOXO1 and protein level of p-AMPK. Our data revealed that the stimulation of expression and function of SOD and CAT by CV treatment could be mediated by FOXO1/p-AMPK axis. Moreover, anti-fibrotic effect of CV might be associated with its inhibitory effect on the hepatic expression of YAP and TAZ. Chlorella vulgaris treatment ameliorates liver fibrosis via two cellular mechanisms. A) Likely, Chlorella vulgaris treatment increases gene expression of enzymatic antioxidants superoxide dismutase (SOD) and catalase (CAT) via upregulating its upstream regulatory elements i.e. phosphorylated 5' adenosine monophosphate-activated protein kinase (p-AMPK) and forkhead box protein O1 (FOXO1). These possible regulatory effects maybe lead to reduce reactive oxygen species level (ROS). B) Chlorella vulgaris treatment decreases hepatic protein level and gene expression of key elements of Hippo signaling pathway i.e. Yes-associated protein (YAP) and Transcriptional coactivators with a PDZ-binding motif (TAZ). Figure created with BioRender ( https://biorender.com ). ROS: Reactive oxygen species, YAP: Yes-associated protein, TAZ: Transcriptional coactivators with a PDZ-binding motif, FOXO1: Fork head Box O1, AMPK: 5' adenosine monophosphate activated protein kinase, SOD: Superoxide dismutase, CAT: Catalase, P: Phosphate group.

Prognostic assessment of forkhead box protein O1 (FOXO1) and paired box 3 (PAX3) in epithelial ovarian cancer

Objective: The transcription factor Forkhead box protein O1 (FOXO1) has been reported to play an important role in various human cancers. However, its role in epithelial ovarian cancer (EOC) has not yet been clarified. Here, we evaluated the expression and clinical significance of FOXO1 in EOC.

FOXO1 suppresses PGC-1β gene expression in skeletal muscles.

Peroxisome proliferator‐activated receptor‐gamma coactivator‐1β (PGC‐1β) is a transcriptional regulator whose increased expression activates energy expenditure‐related genes in skeletal muscles. However, how PGC‐1β is regulated remains largely unclear. Here, we show that PGC‐1β gene expression is negatively correlated with the expression of a transcription factor, forkhead box protein O1 (FOXO1), whose expression is increased during muscle atrophy. In the skeletal muscles of FOXO1‐overexpressing transgenic mice, PGC‐1β gene expression is decreased. Denervation or plaster cast‐based unloading, as well as fasting, increases endogenous FOXO1 expression in skeletal muscles, with decreased PGC‐1β expression. In the skeletal muscles of FOXO1‐knockout mice, the decrease in PGC‐1β expression caused by fasting was attenuated. Tamoxifen‐inducible FOXO1 activation in C2C12 myoblasts causes a marked decrease of PGC‐1β expression. These findings together reveal that FOXO1 activation suppresses PGC‐1β expression. During atrophy with FOXO1 activation, decreased PGC‐1β may decrease energy expenditure and avoid wasting energy.

Prognostic implications of forkhead box protein O1 (FOXO1) and paired box 3 (PAX3) in epithelial ovarian cancer

BackgroundTranscription factors forkhead box protein O1 (FOXO1) and paired box 3 (PAX3) have been reported to play important roles in various cancers. However, their role in epithelial ovarian cancer (EOC) has not been elucidated yet. Therefore, we evaluated the expression and clinical significance of FOXO1 and PAX3 in EOC.MethodsImmunohistochemical analyses of FOXO1 and PAX3 in 212 EOCs, 57 borderline ovarian tumors, 153 benign epithelial ovarian tumors, and 79 nonadjacent normal epithelial tissues were performed using tissue microarray. Various clinicopathological variables, including the survival of EOC patients, were compared. In addition, the effect of FOXO1 on cell growth was assessed in EOC cell lines.ResultsFOXO1 and PAX3 protein expression levels were significantly higher in EOC tissues than in nonadjacent normal epithelial tissues, benign tissues, and borderline tumors (all p < 0.001). In EOC tissues, FOXO1 expression was positively correlated with PAX3 expression (Spearman’s rho = 0.118, p = 0.149). Multivariate survival analysis revealed that high FOXO1 expression (hazard ratio = 2.77 [95% CI, 1.48–5.18], p = 0.001) could be an independent prognostic factor for overall survival. Most importantly, high expression of both FOXO1 and PAX3 showed a high hazard ratio (4.60 [95% CI, 2.00–10.55], p < 0.001) for overall survival. Also in vitro results demonstrated that knockdown of FOXO1 was associated with decreased cell viability, migration, and colony formation.ConclusionsThis study revealed that high expression of FOXO1/PAX3 is an indicator of poor prognosis in EOC. Our results suggest the promising potential of FOXO1 and PAX3 as prognostic and therapeutic markers. The possible link between biological functions of FOXO1 and PAX3 in EOC warrants further studies.

Prognostic implication of forkhead box protein O1 (FOXO1) and paired box gene 3 (PAX3) in epithelial ovarian cancer.

61 Background: Transcriptional factor, Forkhead box protein O1 (FOXO1) has been reported to play an imported role in human cancer, but the role in epithelial ovarian cancer (EOC) has not yet been clarified. Here, we evaluatedthe expression and clinical significance of FOXO1 in EOC. Methods: Immunohistochemical analyses of FOXO1 and PAX3 in 212 in EOCs, 57 borderline ovarian tumors and 153 benign epithelial ovarian tumors and 79 nonadjacent normal epithelial tissues were performed using tissue microarray analysis. The data were compared with clinicopathological variables including the survival of EOC patients. Also, the effect of FOXO1 on cell growth were assessed in EOC cell lines. Results: The expressions of FOXO1 and PAX3 protein were significantly higher in EOC tissues than in nonadjacent normal epithelial tissues, benign tissues and borderline tumors respectively (all p&lt; 0.001). Overexpression of FOXO1 was significantly associated with poor grade ( p = 0.004). FOXO1 expression showed trend of positive correlation with that of PAX3 in EOC tissues ( Spearman’s rho0.118, p= 0.149). Multivariate survival analysis revealed that the high expression of FOXO1 (hazard ratio = 2.74 [95% CI, 1.22–13.10], p = 0.001) could be an independent prognostic factor for overall survival. Most importantly, high expression of both FOXO1 and PAX3 showed high hazard ratio (hazard ratio = 5.53 [95% CI, 2.47–12.40], p&lt; 0.001) for overall survival. In vitro result revealed that knockdown of FOXO1 was associated decreased cell viability and migration. Conclusions: This study reveals that high expression of FOXO1/PAX3 is an indicator of poor prognosis in EOC. Our results not only suggest the promising potential of FOXO1 and PAX3 as a prognostic and survival marker, but also warrant further studies on a possible link between the biological function of FOXO1 and PAX3 of EOC.