Longevity factor FOXO3a: A potential therapeutic target for age-related ocular diseases.

The forkhead box protein O3 (FOXO3a) belongs to the subgroup O of the forkhead transcription factor family and plays an important role in regulating the aging process by participating in the regulation of various life processes, including cell cycle arrest, apoptosis, autophagy, oxidative stress, and DNA repair. The eye is an organ that is affected by aging earlier. However, the functional role and potential clinical applications of FOXO3a in age-related eye diseases have not received widespread attention and lacked comprehensive and clear clarification. In this review, we demonstrated the relationship between FOXO3a and visual system health, summarized the functional roles of FOXO3a in various eye diseases, and potential ocular-related therapies and drugs targeting FOXO3a in visual system diseases through a review and summary of relevant literature. This review indicates that FOXO3a is an important factor in maintaining the normal function of various tissues in the eye, and is closely related to the occurrence and development of ophthalmic-related diseases. Based on its vital role in the normal function of the visual system, FOXO3a has potential clinical application value in related ophthalmic diseases. At present, multiple molecules and drugs targeting FOXO3a have been reported to have the potential for the treatment of related ophthalmic diseases, but further clinical trials are needed. In conclusion, this review can facilitate us to grasp the role of FOXO3a in the visual system and provide new views and bases for the treatment strategy research of age-related eye diseases.

TMF suppresses chondrocyte hypertrophy in osteoarthritic cartilage by mediating the FOXO3a/BMPER pathway.

Osteoarthritis (OA) is a disease of the joints, characterized by chronic inflammation, cartilage destruction and extracellular matrix (ECM) remodeling. Aberrant chondrocyte hypertrophy promotes cartilage destruction and OA development. Collagen X, the biomarker of chondrocyte hypertrophy, is upregulated by runt-related transcription factor 2 (Runx2), which is mediated by the bone morphogenetic protein 4 (BMP4)/Smad1 signaling pathway. BMP binding endothelial regulator (BMPER), a secreted glycoprotein, acts as an agonist of BMP4. 5,7,3',4'-tetramethoxyflavone (TMF) is a natural flavonoid derived from Murraya exotica L. Results of our previous study demonstrated that TMF exhibits chondroprotective effects against OA development through the activation of Forkhead box protein O3a (FOXO3a) expression. However, whether TMF suppresses chondrocyte hypertrophy through activation of FOXO3a expression and inhibition of BMPER/BMP4/Smad1 signaling remains unknown. Results of the present study revealed that TMF inhibited collagen X and Runx2 expression, inhibited BMPER/BMP4/Smad1 signaling, and activated FOXO3a expression; thus, protecting against chondrocyte hypertrophy and OA development. However, BMPER overexpression and FOXO3a knockdown impacted the protective effects of TMF. Thus, TMF inhibited chondrocyte hypertrophy in OA cartilage through mediating the FOXO3a/BMPER signaling pathway.

Forkhead box transcription factors (FOXOs and FOXM1) in glioma: from molecular mechanisms to therapeutics.

Glioma is the most aggressive and malignant type of primary brain tumor, comprises the majority of central nervous system deaths, and is categorized into different subgroups according to its histological characteristics, including astrocytomas, oligodendrogliomas, glioblastoma multiforme (GBM), and mixed tumors. The forkhead box (FOX) transcription factors comprise a collection of proteins that play various roles in numerous complex molecular cascades and have been discovered to be differentially expressed in distinct glioma subtypes. FOXM1 and FOXOs have been recognized as crucial transcription factors in tumor cells, including glioma cells. Accumulating data indicates that FOXM1 acts as an oncogene in various types of cancers, and a significant part of studies has investigated its function in glioma. Although recent studies considered FOXO subgroups as tumor suppressors, there are pieces of evidence that they may have an oncogenic role. This review will discuss the subtle functions of FOXOs and FOXM1 in gliomas, dissecting their regulatory network with other proteins, microRNAs and their role in glioma progression, including stem cell differentiation and therapy resistance/sensitivity, alongside highlighting recent pharmacological progress for modulating their expression.

FOXO3-Activated circFGFBP1 Inhibits Extracellular Matrix Degradation and Nucleus Pulposus Cell Death via miR-9-5p/BMP2 Axis in Intervertebral Disc Degeneration In Vivo and In Vitro.

(1) Background: intervertebral disc degeneration (IVDD) defined as the degenerative changes in intervertebral disc is characterized by extracellular matrix (ECM) degradation and death in nucleus pulposus (NP) cells. (2) Methods: The model of IVDD was established in male Sprague Dawley rats using a puncture of a 21-gauge needle at the endplates located in the L4/5 intervertebral disc. Primary NP cells were stimulated by 10 ng/mL IL-1ß for 24 h to mimic IVDD impairment in vitro. (3) Results: circFGFBP1 was downregulated in the IVDD samples. circFGFBP1 upregulation inhibited apoptosis and extracellular matrix (ECM) degradation and promoted proliferation in IL-1ß-stimulated NP cells. Additionally, circFGFBP1 upregulation mitigated the loss of NP tissue and the destruction of the intervertebral disc structure in vivo during IVDD. FOXO3 could bind to the circFGFBP1 promoter to enhance its expression. circFGFBP1 upregulated BMP2 expression in NP via sponging miR-9-5p. FOXO3 enhanced the protection of circFGFBP1 in IL-1ß-stimulated NP cells, whereas a miR-9-5p increase partly reversed the protection. miR-9-5p downregulation contributed to the survival of IL-1ß-stimulated NP cells, which was partially reversed by BMP2 silence. (4) Conclusions: FOXO3 could activate the transcription of circFGFBP1 via binding to its promoter, which resulted in the enhancement of BMP2 via sponging miR-9-5p and then inhibited apoptosis and ECM degradation in NP cells during IVDD.

Contribution of microRNA-30d to the prevention of the thyroid cancer occurrence and progression: mechanism and implications.

Thyroid cancer is a major endocrine tumor and represents an emerging health problem worldwide. MicroRNAs (miRNAs) have been addressed to participate in the pathogenesis and progression of thyroid cancer. However, it remains largely unknown what functions miR-30d may exert on thyroid cancer. This study, herein, aimed to identify the functional significance and machinery of miR-30d in the progression of thyroid cancer. MiR-30b presented aberrant low expression and ubiquitin-specific protease 22 (USP22) exhibited aberrant high expression in thyroid cancer tissues and cells. The current study proposed the possible machinery that miR-30d could target and negatively regulate USP22. Additionally, USP22 could enhance the stability of SIRT1 by inducing deubiquitination which consequently contributed to FOXO3a deacetylation-induced PUMA repression. Responding to the gain- or loss-of-function of miR-30d and/or USP22, behaviors of thyroid cancer cells were altered. Accordingly, miR-30d inhibited proliferation and promoted apoptosis of thyroid cancer cells by suppressing USP22 through SIRT1/FOXO3a/PUMA axis. The effects of miR-30d and USP22-mediated SIRT1/FOXO3a/PUMA axis on thyroid tumorigenesis were finally validated in murine models. We ultimately confirmed the anti-proliferative and pro-apoptotic effect of miR-30d via suppressing USP22 through in vivo findings. Conclusively, our findings highlight that the occurrence and progression of thyroid cancer can be suppressed by miR-30d-mediated inhibition of USP22 via the SIRT1/FOXO3a/PUMA axis, which provides a attractive therapeutic target for thyroid cancer treatment.

Correlation between serum miR-122-5p and FOXO3 levels and osteoporosis in postmenopausal women with non-alcoholic fatty liver disease / 中华内分泌外科杂志

Objective:To investigate the correlation between serum miR-122-5p and FOXO3 levels and osteoporosis (OP) in postmenopausal women with non-alcoholic fatty liver disease (NAFLD) .Methods:The clinical data and serum of 30 postmenopausal women with NAFLD and 48 postmenopausal women with no-NAFLD were collected. The levels of miR-122-5p and FOXO3 in serum were detected by qRT-PCR. Triglycerides, high-density lipoproteins, and low-density lipoproteins were detected by biochemical autoanalyzer. The bone mineral density of lumbar vertebrae 1-4, Wards triangular bone, femoral neck, greater trochanter and total hip was detected by bone mineral density analyzer. The correlation between the above clinical indicators and OP was analyzed.Results:The expression of miR-122-5p in postmenopausal female NAFLD patients (0.76±0.28) was lower than that in non-NAFLD patients (1±0.31) ( t=3.43, P=0.001) . The downstream target gene FOXO3 of miR-122-5p was identified by bioinformatics website analysis. The expression of FOXO3 in postmenopausal female NAFLD patients (1.31±0.30) was higher than that in non-NAFLD patients (1±0.27) ( t=4.73, P<0.001) . Student’ s t test and Logistic regression analysis showed that triglyceride, miR-122-5p and FOXO3 levels were risk factors for NAFLD (all P<0.05) . Pearson correlation coefficient showed that miR-122-5p level was significantly positively correlated with BMD of femoral neck ( r=0.488, P=0.006) , greater trochanter ( r=0.367, P=0.046) and whole hip ( r=0.404, P=0.027) . FOXO3 level was negatively correlated with bone mineral density of femoral neck ( r=-0.445, P=0.014) and whole hip ( r=-0.507, P=0.004) , while other indexes were not significantly correlated (all P>0.05) . Conclusion:Decreased serum miR-122-5p level and increased FOXO3 level in postmenopausal women with NAFLD may increase the risk of OP.

Effects of extract of pinellia on proliferation and apoptosis of airway smooth muscle cells in asthmatic rats through AMPK/FOXO3a signaling pathway / 中国免疫学杂志

Objective:To investigate effects of extract of pinellia(EP)on proliferation and apoptosis of airway smooth muscle cells(ASMCs)in asthmatic rats and its mechanism.Methods:Asthma rat model was constructed by sensitization and challenge with ovalbumin and rat ASMCs were isolated and cultured.Immunofluorescence staining was used to identify α-actin in ASMCs.After iden-tifying as meeting characteristics of ASMCs,ASMCs were divided into control group,model group,EP group,Compound C group and EP+Compound C group.MTT was used to detect cell proliferation activity;flow cytometry was used to detect cell apoptosis;ELISA was used to detect levels of inflammatory factors IL-6 and TNF-α in cell supernatant;Western blot was used to detect expressions of CyclinD1,proliferating cell nuclear antigen(PCNA),Bcl-2 associated X protein(Bax),Caspase-3,Cleaved-Caspase-3,adenylate activated protein kinase(AMPK),p-AMPK,forkhead box protein O3a(FOXO3a),p-FOXO3a proteins.Results:Immunofluores-cence staining showed that 98%of cells showed green fluorescent filaments in cytoplasm,and α-actin was positively expressed,which proved that cultured cells were ASMCs.Compared with control group,cell OD490,CyclinD1,PCNA protein expressions and IL-6 and TNF-α levels in supernatant of model group were significantly increased,apoptosis rate,Bax,Caspase-3,Cleaved-Caspase-3 protein expressions and p-AMPK/AMPK,p-FOXO3a/FOXO3a levels were significantly reduced(P<0.05);compared with model group,cell OD490,CyclinD1,PCNA protein expressions and IL-6,TNF-α levels in supernatant of EP group were significantly reduced,apoptosis rate,Bax,Caspase-3,Cleaved-Caspase-3 protein expressions,and p-AMPK/AMPK,p-FOXO3a/FOXO3a levels were significantly increased(P<0.05),the above corresponding indicators of Compound C group showed opposite trend(P<0.05);compared with EP group,cell OD490,CyclinD1,PCNA protein expressions and IL-6,TNF-α levels in supernatant of EP+Compound C group were signifi-cantly increased,apoptosis rate,Bax,Caspase-3,Cleaved-Caspase-3 protein expressions,and p-AMPK/AMPK,p-FOXO3a/FOXO3a levels were significantly reduced(P<0.05).Conclusion:EP may inhibit proliferation of ASMCs and promote cell apoptosis in asthmatic rats by activating AMPK/FOXO3a pathway.

FOXO3 Mediates Tooth Movement by Regulating Force-Induced Osteogenesis.

The high prevalence of malocclusion and dentofacial malformations means that the demand for orthodontic treatments has been increasing rapidly. As the biological basis of orthodontic treatment, the mechanism of mechanical force-induced alveolar bone remodeling during orthodontic tooth movement (OTM) has become the key scientific issue of orthodontics. It has been demonstrated that bone mesenchymal stem cells (BMSCs) are crucial for bone remodeling and exhibit mechanical sensing properties. Mechanical force can promote osteoblastic differentiation of BMSCs and osteogenesis, but the key factor that mediates mechanical force-induced osteogenesis during OTM remains unclear. In this study, by performing reverse-phase protein arrays on BMSCs exposed to mechanical force, we found that the expression level of forkhead box O3 (FOXO3) was significantly upregulated during the mechanical force-induced osteoblastic differentiation of BMSCs. The number of FOXO3-positive cells was consistently higher on the OTM side as compared with the control side and accompanied by the enhancement of osteogenesis. Remarkably, inhibiting FOXO3 with repaglinide delayed OTM by severely impairing mechanical force-induced bone formation in vivo. Moreover, knockdown of FOXO3 effectively inhibited the mechanical force-induced osteoblastic differentiation of BMSCs, whereas the overexpression of FOXO3 enhanced this effect. Mechanistically, we revealed a novel regulatory model in which FOXO3 promoted osteocalcin transcription by activating its promoter in cooperation with runt-related transcription factor 2 (RUNX2). We collectively obtained the first evidence that FOXO3 is critical for OTM, where it responds to mechanical force and directly regulates downstream osteoblastic differentiation in an efficient manner.

The expression of FOXO3 in pancreatic cancer and its effects on pancreatic cancer cells / 中华肝胆外科杂志

Objective:To investigate the expression of forkhead box protein O3(FOXO3) in pancreatic cancer and its effect on the motility and proliferation of pancreatic cancer cells.Methods:The FOXO3 expression in pancreatic cancer and adjacent tissues was retrieved from LinkedOmics database. Western blotting and real-time quantitative polymerase chain reaction were used to detect FOXO3 expression in pancreatic cancer cells and human pancreatic stellate cells. PANC-1 and MIAPaCa-2 pancreatic cancer cells with low FOXO3 expression were selected to transfect FOXO3 overexpression plasmid and negative control plasmid, respectively. The motility and proliferation ability of pancreatic cancer cells were detected by colony formation assay, cell scratch assay, Transwell assay and flow cytometry.Results:In the LinkedOmics database, the relative expression of FOXO3 protein in the cancer tissues of 64 patients with pancreatic cancer was significantly lower than that in the adjacent tissues ( t=8.36, P<0.001). The number of clones in PANC-1 cell line was (30.0±6.6) after overexpressed FOXO3, which was lower than that in negative control cells (92.7±6.7), and the difference was statistically significant ( t=11.54, P<0.001). After overexpressed FOXO3 in PANC-1 and MIAPaCa-2 cell lines, the scratch repair rate was significantly decreased compared with the control group. In Transwell experiment, the number of cells in FOXO3 overexpressed group in PANC-1 cell lines was (21.0±6.6), which was lower than that of negative control groups (55.7±8.5), and the difference was statistically significant ( t=5.59, P=0.005). The results of MIAPaCa-2 cell line were consistent with that of PANC-1 cell line. After overexpressing of FOXO3 in PANC-1 and MIAPaCa-2 cell lines, the proportion of cells in the G0/G1 phase decreased, while the proportion in the S phase increased. Conclusion:The expression of FOXO3 was decreased in pancreatic cancer. Overexpression of FOXO3 could significantly inhibit the proliferation, migration and invasion of pancreatic cancer cells and induce cell cycle arrest, which is a potential target for the treatment of pancreatic cancer.

Correlation of macrophage-related cytokines and silent information regulator 1 and forkhead box protein O3 levels in peripheral blood mononuclear cells in patients with active pulmonary tuberculosis / 中国热带医学

@#Abstract: Objective　To explore the correlation between the levels of silent information regulator 1 (SIRT1) and forkhead box protein O3 (FOXO3) in peripheral blood mononuclear cells of patients with active pulmonary tuberculosis (APTB) and macrophage-related cytokines-inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1). Methods　A total of 64 APTB patients who were treated in Yubei Hospital, the First Affiliated Hospital of Chongqing Medical University from January 2020 to December 2021 were gathered as the APTB group, 59 people with latent tuberculosis infection (LTBI) were gathered as the LTBI group, and 62 healthy people were gathered as the control group. Quantitative real-time PCR (qPCR) method was performed to measure the levels of SIRT1 mRNA and FOXO3 mRNA in peripheral blood mononuclear cells. The enzyme-linked immunosorbent assay (ELISA) was performed to measure serum iNOS and Arg-1 levels; ROC curve was used to analyze the value of SIRT1 mRNA and FOXO3 mRNA levels in the differential diagnosis of LTBI and APTB; Pearson correlation was performed to analyze the correlation of SIRT1 mRNA and FOXO3 mRNA in peripheral blood mononuclear cells of APTB patients with serum iNOS and Arg-1 levels. Results　The levels of SIRT1 mRNA, FOXO3 mRNA and serum iNOS in peripheral blood mononuclear cells decreased in control group, LTBI group and APTB group, and the level of serum Arg-1 increased in turn (P<0.05). The AUCs of SIRT1 mRNA and FOXO3 mRNA in differential diagnosis of LTBI and APTB were 0.876 and 0.887, respectively, the sensitivity was 71.2% and 76.3%, and the specificity was 96.9% and 90.6% respectively. The levels of SIRT1 mRNA and FOXO3 mRNA in peripheral blood mononuclear cells of APTB patients were positively correlated (r=0.500, P<0.05), and they were positively correlated with serum iNOS and negatively correlated with serum Arg-1 (P<0.05). The SIRT1 mRNA, FOXO3 mRNA and serum iNOS in peripheral blood mononuclear cells of APTB patients after 6 months of treatment were higher than those before treatment, and serum Arg-1 was lower than before treatment (P<0.05). Conclusions　The levels of SIRT1 mRNA and FOXO3 mRNA in peripheral blood mononuclear cells of APTB patients are low, and they are positively correlated with macrophage-related cytokine iNOS and negatively correlated with Arg-1.

Liraglutide improves bone mineral density in osteoporotic rats by mediating O3a/Wnt signaling / 中华内分泌外科杂志

Objective:To study the effect of liraglutide on the forkhead box O3a (forkhead box O3a, FoxO3a) /Wnt signaling pathway and vertebral bone density in osteoporotic rats.Methods:Female Sprague-dawley rats were divided into sham operation group, model group, and liraglutide intervention group according to the random number table method, with 10 rats in each group. Both the model group and the intervention group used bilateral oophorectomy to establish an osteoporosis model. The intervention group was injected subcutaneously with liraglutide every day, and the sham operation group and the model group were given an equal volume of normal saline. After 12 weeks of continuous administration, the bone mineral density and bone biomechanics were measured, the serum osteoprotegerin, anti-tartrate acid phosphatase, and osteocalcin levels were detected by enzyme-linked immunosorbent assay, and the O3a/Wnt pathway was detected by Real-time PCR technology. The expression level of mRNA was detected by Western blot to detect the expression level of related proteins in the O3a/Wnt pathway.Results:The bone mineral density levels of L4,5 (0.33±0.04 vs 0.18±0.03) and left and right femurs (0.37±0.05 vs 0.23±0.04 0.35±0.04 vs 0.24±0.03) of the successfully modeled rats were significantly lower than those of the sham operation group ( P<0.05) . After 12 weeks of treatment, the differences in the maximum bone load, three-point bending stress, bone density and elastic modulus of the three groups of rats were statistically significant. Among them, the sham operation group had the highest level of each index (36.53±5.23, 154.13±6.27, 0.34±0.04, 3 102.34±160.44) , followed by the intervention group (19.37±4.32, 141.54±6.58, 0.18±0.03, 2 270.18±145.53) and the model group in turn (26.17±4.68, 147.56±5.84, 0.28±0.03, 2 804.24±140.42) ( P<0.05) . There were statistically significant differences in the levels of serum osteoprotegerin, anti-tartrate acid phosphatase and osteocalcin among the three groups. Among them, the osteoprotegerin (Sham operation group vs model group vs intervention group: 7.53±0.63 vs 4.57±0.42 vs 6.15±0.61) of the sham operation group was significantly higher than that of the intervention group and the model group. The anti-tartrate acid phosphatase (Sham operation group vs model group vs intervention group: 14.34±2.87 vs 19.53±3.52 vs 15.96±3.14) and osteocalcin levels (Sham operation group vs model group vs intervention group: 0.84±0.09 vs 1.13± 0.12 vs 0.95± 0.08) of rats The factor was significantly lower than that of the intervention group and model group ( P<0.05) . The mRNA and protein expression levels of FoxO3a, Wnt2, and β-catenin in the three groups of rats were significantly different. Among them, Wnt2 and β-catenin in the sham operation group were significantly higher than the intervention group and model group, and FoxO3a was significantly lower than the intervention group and model Group ( P<0.05) . Conclusion:Liraglutide can increase bone density and improve bone biomechanics by activating O3a/Wnt signal, thereby effectively treating osteoporosis.

TAM-derived extracellular vesicles containing microRNA-29a-3p explain the deterioration of ovarian cancer.

Extracellular vesicles (EVs) secreted from tumor-associated macrophages (TAMs) are known to generate an immune-suppressive environment conducive to the development of ovarian cancer (OC). We tried to elucidate the role of TAM-derived exosomal microRNA (miR)-29a-3p in OC. miR-29a-3p, forkhead box protein O3 (FOXO3), and programmed death ligand-1 (PD-L1) expression was determined and their interactions evaluated. EVs were isolated, followed by determination of the uptake of EVs by OC cells, after which the proliferation and immune escape facilities of the OC cells were determined. OC xenograft models were constructed with EVs in correspondence with in vivo experiments. Overexpressed miR-29a-3p was detected in OC, and miR-29a-3p promoted OC cell proliferation and immune escape. EVs derived from TAMs enhanced the proliferation of OC cells. miR-29a-3p was enriched in TAM-EVs, and TAM-EVs delivered miR-29a-3p into OC cells. Downregulated FOXO3 was identified in OC, whereas miR-29a-3p targeted FOXO3 to suppress glycogen synthase kinase 3ß (GSK3ß) activity via the serine/threonine protein kinase (AKT)/GSK3ß pathway. Inhibition of TAM-derived exosomal miR-29a-3p decreased PD-L1 to inhibit OC progression through the FOXO3-AKT/GSK3ß pathway in vitro and in vivo. Taken together, the current studies highlight the FOXO3-AKT/GSK3ß pathway and the mechanism by which TAM-derived exosomal miR-29a-3p enhances the expression of PD-L1 to facilitate OC cell proliferation and immune escape.

Forkhead box protein O3a promotes glioma cell resistance to temozolomide by regulating matrix metallopeptidase and ß-catenin.

Glioblastoma multiforme (GBM) is the most common type of malignant brain tumor. GBM is currently treated with temozolomide (TMZ), although patients often exhibit resistance to this agent. Although several mechanisms underlying the resistance of GBM to TMZ have been identified, the combination of these mechanisms is not sufficient to fully account for this phenomenon. Our previous study demonstrated that knocking down the Forkhead box protein O3a (FoxO3a) gene, a member of the FoxO subfamily of transcription factors, resulted in glioma cell sensitization to TMZ, accompanied by reduced levels of nuclear ß-catenin. The aim of the present study was to specify how FoxO3a and ß-catenin are implicated in glioma cell TMZ resistance. Using the U87 and U251 parental cell lines (also designated as sensitive cell lines) and corresponding resistant cell lines (U87-TR and U251-TR, generated by repeated TMZ treatments), coupled with a combined knockdown/overexpression strategy, it was revealed that FoxO3a or ß-catenin overexpression in TMZ-treated U87 and U251 cells markedly increased cellular proliferation; co-expression of both FoxO3a and ß-catenin resulted in the highest increase. Knockdown of either FoxO3a or ß-catenin in U87-TR and U251-TR cells led to a significant decrease in cell viability, which was rescued by the re-expression of FoxO3a in FoxO3a-knockdown cells. Subsequent experiments demonstrated that, in U87-TR and U251-TR cells, FoxO3a knockdown significantly reduced the protein levels of matrix metallopeptidase (MMP)9, while overexpression of FoxO3a in U87 and U251 cells enhanced the nuclear accumulation of ß-catenin, concomitantly with an increase in MMP9 levels. Furthermore, MMP9 knockdown markedly reduced the levels of nuclear ß-catenin. Collectively, the findings of the present study suggest that FoxO3a may regulate the nuclear accumulation of ß-catenin by modulating MMP9 expression, thereby rendering glioblastoma cells resistant to TMZ, and may provide unique molecular insights into the mechanisms underlying the development of TMZ resistance in GBM.

Circulating extracellular vesicles from patients with valvular heart disease induce neutrophil chemotaxis via FOXO3a and the inhibiting role of dexmedetomidine.

We previously demonstrated that circulating extracellular vesicles (EVs) from patients with valvular heart disease (VHD; vEVs) contain inflammatory components and inhibit endothelium-dependent vasodilation. Neutrophil chemotaxis plays a key role in renal dysfunction, and dexmedetomidine (DEX) can reduce renal dysfunction in cardiac surgery. However, the roles of vEVs in neutrophil chemotaxis and effects of DEX on vEVs are unknown. Here, we investigated the impact of vEVs on neutrophil chemotaxis in kidneys and the influence of DEX on vEVs. Circulating EVs were isolated from healthy subjects and patients with VHD. The effects of EVs on chemokine generation, forkhead box protein O3a (FOXO3a) pathway activation and neutrophil chemotaxis on cultured human umbilical vein endothelial cells (HUVECs) and kidneys in mice and the influence of DEX on EVs were detected. vEVs increased FOXO3a expression, decreased phosphorylation of Akt and FOXO3a, promoted FOXO3a nuclear translocation, and activated the FOXO3a signaling pathway in vitro. DEX pretreatment reduced vEV-induced CXCL4 and CCL5 expression and neutrophil chemotaxis in cultured HUVECs via the FOXO3a signaling pathway. vEVs were also found to suppress Akt phosphorylation and activate FOXO3a signaling to increase plasma levels of CXCL4 and CCL5 and neutrophil accumulation in kidney. The overall mechanism was inhibited in vivo with DEX pretreatment. Our data demonstrated that vEVs induced CXCL4-CCL5 to stimulate neutrophil infiltration in kidney, which can be inhibited by DEX via the FOXO3a signaling. Our findings reveal a unique mechanism involving vEVs in inducing neutrophils chemotaxis and may provide a novel basis for using DEX in reducing renal dysfunction in valvular heart surgery.

miR-155 promotes fibroblast-like synoviocyte proliferation and inflammatory cytokine secretion in rheumatoid arthritis by targeting FOXO3a.

The present study aimed to explore the expression and effects of microRNA (miR)-155 in synovial fibroblasts of patients with rheumatoid arthritis (RA). A total of 89 synovial tissues from RA patients and 49 control synovial tissues were collected, and the levels of miR-155 were measured by reverse transcription quantitative-PCR and western blotting. Fibroblast-like synoviocytes (FLS) were isolated from synovial tissues from the control group and were used to evaluate the roles of miR-155 and forkhead box protein O3a (FOXO3a). MTT assay was used to measure the proliferation of FLS. The expression of miR-155 in RA synovial tissues was significantly higher than that in the control group, but the expression of FOXO3a was significantly lower. In RA synovial tissues, miR-155 expression was negatively correlated with FOXO3a expression, but was positively correlated with the release of inflammatory cytokines interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α (TNF-α). A dual-luciferase reporter system showed that miR-155 inhibited the expression of FOXO3a in FLS cells. miR-155 also promoted secretion of the inflammatory cytokines IL-1ß, IL-6 and TNF-α by FLS and proliferation of these cells by targeting FOXO3a.

Overexpression of miR-148a inhibits viability and invasion of ovarian cancer OVCAR3 cells by targeting FOXO3.

Decreased expression of microRNA (miR)-148a is associated with poor prognosis in ovarian cancer. The aim of the present study was to investigate the impact of miR-148a on tumor cell viability and invasion via targeting forkhead box protein O3 (FOXO3). Expression of miR-148a was detected in paired tumor and adjacent normal tissues. OVCAR3 cells were transfected with miR-148a mimic and inhibitor. Cell viability, apoptosis and invasion were determined. A luciferase reporter assay was used to study the association between miR-148a and FOXO3. In addition, the influence of miR-148a on tumor cell growth was investigated by performing xenograft assays in nude mice. RT-qPCR showed that miR-148a was downregulated in ovarian cancer tissues. Overexpression of miR-148a in OVCAR3 cells inhibited cell viability, suppressed invasion and promoted cellular apoptosis. The dual-luciferase assay indicated that miR-148a directly regulated the expression of FOXO3, a transcription factor of caspase-3. Western blotting confirmed that the expression of caspase-3 was regulated by the modulation of miR-148a expression. In vivo assays revealed that miR-148a overexpression inhibited the growth of OVCAR3 ×enograft tumors in nude mice. miR-148a is a tumor suppressor in ovarian cancer OVCAR3 cells and in nude mice. The suppressive effect is due to inhibiting cell viability and invasion as well as promoting apoptosis. These results may provide theoretical basis for targeting miR-148a in the treatment of ovarian cancer.

Salidroside attenuates hypoxia/reoxygenation-induced human brain vascular smooth muscle cell injury by activating the SIRT1/FOXO3α pathway.

It has been reported that salidroside (SAL), a natural dietary isothiocyanate, exhibits neuroprotective roles in cerebral ischemia-reperfusion injury. However, to the best of our knowledge, its underlying protective mechanism remains unknown. Sirtuin 1 (SIRT1) is a class III histone deacetylase involved in a variety of cellular functions. SIRT1 has been identified as a mediator of cerebral ischemia and may induce neuroprotection by activating various intracellular downstream targets, such as forkhead box protein O3α (FOXO3α). Therefore, the present study aimed to investigate whether SAL protects human brain vascular smooth muscle cells (HBVSMC) against hypoxia/reoxygenation (H/R) injury, which is a cell model of cerebral ischemia-reperfusion injury, through regulating the SIRT1-activited signaling pathway. The present study revealed that H/R treatment significantly reduced the expression of SIRT1 protein in HBVSMCs. Additionally, pretreatment with SAL reversed the H/R-induced decrease in cellular viability, increased caspase-3 activity, the appearance of apoptotic cells and the apoptosis rate in HBVSMCs. SAL attenuated the H/R-induced decrease in the expression of SIRT1 and phosphorylated FOXO3α protein in HBVSMCs, suggesting that the protective role of SAL in H/R injury occurs via the SIRT1/FOXO3α pathway. Furthermore, sirtinol, a SIRT1-specific inhibitor, suppressed the inhibitory effects of SAL on H/R-induced cytotoxicity and apoptosis as indicated by the downregulation of cell viability and upregulation of caspase-3 activity and apoptosis rate induced by sirtinol treatment in HBVSMCs. The reversal effects of SAL on H/R-induced alternation of B-cell lymphoma (Bcl-2) and Bcl-2 associated X protein expression were also attenuated by sirtinol. These results suggest that SAL exhibits neuroprotective effects against H/R injury by activating the SIRT1/FOXO3α pathway, which may become a novel potential therapeutic target for the treatment of cerebral ischemic disease.

MicroRNA-370 suppresses the progression and proliferation of human astrocytoma and glioblastoma by negatively regulating ß-catenin and causing activation of FOXO3a.

Certain microRNAs (miRs) regulate the progression and metastasis of various cancer types. In the present study, the role of miR-370 in the progression and proliferation of human astrocytoma and glioblastoma cells was assessed and the underlying molecular mechanism was investigated. miR-370 levels in clinical specimens of human glioma and peritumoral tissues were determined by reverse-transcription quantitative PCR. Oligonucleotide mimics and inhibitors were transfected into the U-251MG human astrocytoma cell line and the and U-87MG glioblastoma cell line and the cell viability of was determined by an MTT assay. The expression of ß-catenin and forkhead box protein (FOX)O3a was determined by western blot analysis. The results revealed that the expression of miR-370 in human glioma tissues was significantly decreased compared with that in peritumoral tissues. The miR-370 levels in patients with grade III/IV gliomas were significantly decreased compared with those in grade I/II. Transfection with miR-370 mimics inhibited the proliferation of U-251MG and U-87MG cells. Furthermore, the miR-370 levels were negatively correlated with ß-catenin and positively correlated with nuclear FOXO3a. In conclusion, miR-370 inhibited the proliferation of human glioma cells by regulating the levels of ß-catenin and the activation of FOXO3a, suggesting that miR-370 was a tumor suppressor in the progression of human astrocytoma and glioblastoma cells.

Hydrogen-rich water attenuates amyloid ß-induced cytotoxicity through upregulation of Sirt1-FoxO3a by stimulation of AMP-activated protein kinase in SK-N-MC cells.

Amyloid ß (Aß) peptides are identified in cause of neurodegenerative diseases such as Alzheimer's disease (AD). Previous evidence suggests Aß-induced neurotoxicity is linked to the stimulation of reactive oxygen species (ROS) production. The accumulation of Aß-induced ROS leads to increased mitochondrial dysfunction and triggers apoptotic cell death. This suggests antioxidant therapies may be beneficial for preventing ROS-related diseases such as AD. Recently, hydrogen-rich water (HRW) has been proven effective in treating oxidative stress-induced disorders because of its ROS-scavenging abilities. However, the precise molecular mechanisms whereby HRW prevents neuronal death are still unclear. In the present study, we evaluated the putative pathways by which HRW protects against Aß-induced cytotoxicity. Our results indicated that HRW directly counteracts oxidative damage by neutralizing excessive ROS, leading to the alleviation of Aß-induced cell death. In addition, HRW also stimulated AMP-activated protein kinase (AMPK) in a sirtuin 1 (Sirt1)-dependent pathway, which upregulates forkhead box protein O3a (FoxO3a) downstream antioxidant response and diminishes Aß-induced mitochondrial potential loss and oxidative stress. Taken together, our findings suggest that HRW may have potential therapeutic value to inhibit Aß-induced neurotoxicity.