MicroRNA-122-5p promotes renal fibrosis and injury in spontaneously hypertensive rats by targeting FOXO3.

Hypertensive renal injury is accompanied by tubular interstitial fibrosis leading to increased risk for renal failure. This study aimed to explore the influences of miR-122-5p in hypertension-mediated renal fibrosis and damage. 14-week-old male SHR and WKY rats were randomly assigned to treat with rAAV-miR-122-5p or rAAV-GFP for 8 weeks. There were marked increases in miR-122-5p and Kim-1 levels and decreases in FOXO3 and SIRT6 levels in hypertensive rats. Transfection with rAAV-miR-122-5p triggered exacerbation of renal fibrosis, apoptosis and inflammatory injury in SHR, associated with downregulated levels of FOXO3, SIRT6, ATG5 and BNIP3 as well as upregulated expression of Kim-1, NOX4, CTGF, and TGF-ß1. In cultured primary mouse renal tubular interstitial fibroblasts, exposure to angiotensin II resulted in obvious downregulation of FOXO3, SIRT6, ATG5, BNIP3 and nitric oxide levels as well as augmented cellular migration, oxidative stress, and inflammation, which were exacerbated by miR-122-5p mimic while rescued by miR-122-5p inhibitor and rhFOXO3, respectively. Notably, knockdown of FOXO3 strikingly blunted cellular protective effects of miR-122-5p inhibitor. In summary, miR-122-5p augments renal fibrosis, inflammatory and oxidant injury in hypertensive rats by suppressing the expression of FOXO3. Pharmacological inhibition of miR-122-5p has potential therapeutic significance for hypertensive renal injury and fibrosis-related kidney diseases.

TRIM33 protects osteoblasts from oxidative stress-induced apoptosis in osteoporosis by inhibiting FOXO3a ubiquitylation and degradation.

This study aimed to probe into the effect of TRIM33 on oxidative stress-induced apoptosis of osteoblasts in osteoporosis and to probe into the underlying mechanism. The apoptosis of osteoblasts was induced by H2 O2 treatment and tested by flow cytometry. A mouse osteoporosis model was conducted by ovariectomy (OVX). The function of TRIM33 was assessed by in vitro and in vivo experiments. The mechanism of TRIM33 was determined by immunoprecipitation, immunofluorescent staining and co-transfection experiments. Here, we found that TRIM33 expression was lessened in the osteoblasts of patients with osteoporosis and was positively correlated with the bone mineral density of these patients. FOXO3a and TRIM33 were co-localized in the osteoblasts nuclei. TRIM33 silence boosted FOXO3a degradation in normal osteoblasts, while TRIM33 overexpression restrained FOXO3a degradation in H2 O2 -treated osteoblasts. The binding of TRIM33 to CBP and its overexpression restrained CBP-mediated FOXO3a acetylation, thereby attenuating FOXO3a ubiquitylation. The H2 O2 -induced apoptosis of osteoblasts was restrained by TRIM33 overexpression, while the FOXO3a knockdown reversed this trend. The in vivo experiments corroborated that TRIM33 overexpression attenuated the OVX-driven impacts in mice. In general, our findings expounded that TRIM33 protected osteoblasts against oxidative stress-induced apoptosis in osteoporosis and that the underlying mechanism was the restraint of FOXO3a ubiquitylation and degradation.

Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library.

Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.

B Cell Receptor-Responsive miR-141 Enhances Epstein-Barr Virus Lytic Cycle via FOXO3 Inhibition.

Antigen recognition by the B cell receptor (BCR) is a physiological trigger for reactivation of Epstein-Barr virus (EBV) and can be recapitulated in vitro by cross-linking of surface immunoglobulins. Previously, we identified a subset of EBV microRNAs (miRNAs) that attenuate BCR signal transduction and subsequently dampen lytic reactivation in B cells. The roles of host miRNAs in the EBV lytic cycle are not completely understood. Here, we profiled the small RNAs in reactivated Burkitt lymphoma cells and identified several miRNAs, such as miR-141, that are induced upon BCR cross-linking. Notably, EBV encodes a viral miRNA, miR-BART9, with sequence homology to miR-141. To better understand the functions of these two miRNAs, we examined their molecular targets and experimentally validated multiple candidates commonly regulated by both miRNAs. Targets included B cell transcription factors and known regulators of EBV immediate-early genes, leading us to hypothesize that these miRNAs modulate kinetics of the lytic cascade in B cells. Through functional assays, we identified roles for miR-141 and EBV miR-BART9 and one specific target, FOXO3, in progression of the lytic cycle. Our data support a model whereby EBV exploits BCR-responsive miR-141 and further mimics activity of this miRNA family via a viral miRNA to promote productive lytic replication.IMPORTANCE EBV is a human pathogen associated with several malignancies. A key aspect of lifelong virus persistence is the ability to switch between latent and lytic replication modes. The mechanisms governing latency, reactivation, and progression of the lytic cycle are only partly understood. This study reveals that specific miRNAs can act to support the EBV lytic phase following BCR-mediated reactivation triggers. Furthermore, this study identifies a role for FOXO3, commonly suppressed by both host and viral miRNAs, in modulating progression of the EBV lytic cycle.

Tanshinone IIA Ameliorates Inflammation Response in Osteoarthritis via Inhibition of miR-155/FOXO3 Axis.

BACKGROUND: Osteoarthritis (OA) is the most common joint disorder characterized by degeneration of the articular cartilage and joint destruction with an associated risk of mobility disability in elderly people. Although a lot of achievements have been made, OA is still regarded as an incurable disease. Therefore, the pathological mechanisms and novel therapeutic strategies of OA need more investigation. METHODS: MTT assay was conducted to measure the viability of chondrocytes after LPS treatment. Cell apoptosis was analyzed by annexin V/propidium iodide labeling. ELISA was used to determine the concentrations of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the culture supernatant of chondrocytes. The expression level of miR-155, IL-1ß, FOXO3, TNF-α, IL-6, caspase-3, and caspase-9 in chondrocytes was analyzed by RT-qPCR or Western blot. RESULTS: We found that LPS led to inflammatory responses, cell apoptosis, and increased miR-155 expression in human articular chondrocytes. Tanshinone IIA could inhibit LPS-induced inflammation and cell apoptosis of chondrocytes via regulating the expression of miR-155 and FOXO3. miR-155 directly targeted the 3'-UTR of FOXO3 to regulate its expression. CONCLUSIONS: Taken together, our data suggest tanshinone IIA ameliorates inflammation response in OA via inhibition of the miR-155/FOXO3 axis, and provide some evidences that tanshinone IIA could be designed and developed as a new promising clinical therapeutic drug for OA patients.

Xiaoyaosan decoction alleviated rat liver fibrosis via the TGFß/Smad and Akt/FoxO3 signaling pathways based on network pharmacology analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver fibrosis is an outcome of many chronic liver diseases and often results in cirrhosis, liver failure, and even hepatocarcinoma. Xiaoyaosan decoction (XYS) as a classical Traditional Chinese Medicine (TCM) formula is used to liver fibrosis in clinical practice while its mechanism is unclear. AIM OF THE STUDY: The aim of this study was to investigate the anti-fibrosis effect of XYS and to explore the molecular mechanisms by combining network pharmacology and transcriptomic technologies. MATERIALS AND METHODS: The carbon tetrachloride (CCl4)-induced liver fibrosis rat were treated with three doses of XYS. The liver fibrosis and function were evaluated by histopathological examination and serum biochemical detection. The fibrosis related protein a-SMA and collagen I were assessed by Western blot. Different expressed genes (DEGs) between XYS-treated group and model group were analyzed. The herb-component-target network was constructed combined the network pharmacology. The predict targets and pathways were validated by in vitro and in vivo experiments. RESULTS: With XYS treatment, the liver function was significantly improved, and fibrotic changes were alleviated. The a-SMA and collagen I expression levels in the liver were also decreased in XYS-treated rats compared with CCl4 model rats. 108 active components and 42 targets from 8 herbs constituted herb-compound-target network by transcriptomics and network pharmacology analysis. The KEGG pathway and GO enrichment analyses showed that the FoxO, TGFß, AMPK, MAPK, PPAR, and hepatitis B and C pathways were involved in the anti-fibrosis effects of XYS. In the liver tissues, p-FoxO3a and p-Akt expression levels were significantly increased in the CCl4 model group but decreased in the XYS-treated group. The TGFß1/Smad pathway and Akt/FoxO3 pathway were verified in LX2 cells by inhibiting phosphorylation of Smad3 and Akt activity, respectively. CONCLUSIONS: Our findings suggested that XYS markedly alleviated CCl4-induced liver fibrosis in histopathological and serum liver function analyses, and this effect may occur via the TGFß1/Smad and Akt/FoxO signaling pathways.

Kcnh2 mediates FAK/AKT-FOXO3A pathway to attenuate sepsis-induced cardiac dysfunction.

OBJECTIVES: Myocardial dysfunction is a significant manifestation in sepsis, which results in high mortality. Even Kcnh2 has been hinted to associate with the pathological process, its involved signalling is still elusive. MATERIALS AND METHODS: The caecal ligation puncture (CLP) surgery or lipopolysaccharide (LPS) injection was performed to induce septic cardiac dysfunction. Western blotting was used to determine KCNH2 expression. Cardiac function was examined by echocardiography 6 hours after CLP and LPS injection in Kcnh2 knockout (Kcnh2+/- ) and NS1643 injection rats (n ≥ 6/group). Survival was monitored following CLP-induced sepsis (n ≥ 8/group). RESULTS: Sepsis could downregulate KCNH2 level in the rat heart, as well as in LPS-stimulated cardiomyocytes but not cardiac fibroblast. Defect of Kcnh2 (Kcnh2+/- ) significantly aggravated septic cardiac dysfunction, exacerbated tissue damage and increased apoptosis under LPS challenge. Fractional shortening and ejection fraction values were significantly decreased in Kcnh2+/- group than Kcnh2+/+ group. Survival outcome in Kcnh2+/- septic rats was markedly deteriorated, compared with Kcnh2+/+ rats. Activated Kcnh2 with NS1643, however, resulted in opposite effects. Lack of Kcnh2 caused inhibition of FAK/AKT signalling, reflecting in an upregulation for FOXO3A and its downstream targets, which eventually induced cardiomyocyte apoptosis and heart tissue damage. Either activation of AKT by activator or knockdown of FOXO3A with si-RNA remarkably attenuated the pathological manifestations that Kcnh2 defect mediated. CONCLUSION: Kcnh2 plays a protection role in sepsis-induced cardiac dysfunction (SCID) via regulating FAK/AKT-FOXO3A to block LPS-induced myocardium apoptosis, indicating a potential effect of the potassium channels in pathophysiology of SCID.

LncRNA SNHG12 Improves Cerebral Ischemic-reperfusion Injury by Activating SIRT1/FOXO3a Pathway through I nhibition of Autophagy and Oxidative Stress.

BACKGROUND: Ischemia/reperfusion (I/R) injury involves complex biological processes and molecular mechanisms such as autophagy. Oxidative stress plays a critical role in the pathogenesis of I/R injury. LncRNAs are the regulatory factor of cerebral I/R injury. METHODS: This study constructs cerebral I/R model to investigate role of autophagy and oxidative stress in cerebral I/R injury and the underline regulatory mechanism of SIRT1/ FOXO3a pathway. In this study, lncRNA SNHG12 and FOXO3a expression was up-regulated and SIRT1 expression was down-regulated in HT22 cells of I/R model. RESULTS: Overexpression of lncRNA SNHG12 significantly increased the cell viability and inhibited cerebral ischemicreperfusion injury induced by I/Rthrough inhibition of autophagy. In addition, the transfected p-SIRT1 significantly suppressed the release of LDH and SOD compared with cells co-transfected with SIRT1 and FOXO3a group and cells induced by I/R and transfected with p-SNHG12 group and overexpression of cells co-transfected with SIRT1 and FOXO3 further decreased the I/R induced release of ROS and MDA. CONCLUSION: In conclusion, lncRNA SNHG12 increased cell activity and inhibited oxidative stress through inhibition of SIRT1/FOXO3a signaling-mediated autophagy in HT22 cells of I/R model. This study might provide new potential therapeutic targets for further investigating the mechanisms in cerebral I/R injury and provide.

Melatonin Attenuates Anoxia/Reoxygenation Injury by Inhibiting Excessive Mitophagy Through the MT2/SIRT3/FoxO3a Signaling Pathway in H9c2 Cells.

PURPOSE: Autophagy caused by ischemia/reperfusion (I/R) increases the extent of cardiomyocyte damage. Melatonin (Mel) diminishes cardiac injury through regulating autophagy and mitochondrial dynamics. However, illustrating the specific role of mitophagy in the cardioprotective effects of melatonin remains a challenge. The aim of our research was to investigate the impact and underlying mechanisms of melatonin in connection with mitophagy during anoxia/reoxygenation (A/R) injury in H9c2 cells. METHODS: H9c2 cells were pretreated with melatonin with or without the melatonin membrane receptor 2 (MT2) antagonist 4-P-PDOT, the MT2 agonist IIK7 and the sirtuin 3 (SIRT3) inhibitor 3-TYP for 4 hours and then subjected to A/R injury. Cell viability, cellular apoptosis, necrosis levels and oxidative markers were assessed. The expression of SIRT3 and forkhead box O3a (FoxO3a), mitochondrial function and the levels of mitophagy-related proteins were also evaluated. RESULTS: A/R injury provoked enhanced mitophagy in H9c2 myocytes. In addition, increased mitophagy was correlated with decreased cellular viability, increased oxidative stress and mitochondrial dysfunction in H9c2 cells. However, melatonin pretreatment notably increased cell survival and decreased cell apoptosis and oxidative response after A/R injury, accompanied by restored mitochondrial function. The inhibition of excessive mitophagy is involved in the cardioprotective effects of melatonin, as shown by the decreased expression of the mitophagy-related molecules Parkin, Beclin1, and BCL2-interacting protein 3-like (BNIP3L, best known as NIX) and decreased light chain 3 II/light chain 3 I (LC3 II/LC3 I) ratio and upregulation of p62 expression. Moreover, the decreased expression of SIRT3 and FoxO3a in A/R-injured H9c2 cells was abrogated by melatonin, but these beneficial effects were attenuated by the MT2 antagonist 4-P-PDOT or the SIRT3 inhibitor 3-TYP and enhanced by the MT2 agonist IIK7. CONCLUSION: These results indicate that melatonin protects H9c2 cells during A/R injury through suppressing excessive mitophagy by activating the MT2/SIRT3/FoxO3a pathway. Melatonin may be a useful candidate for alleviating myocardial ischemia/reperfusion (MI/R) injury in the future, and the MT2 receptor might become a therapeutic target.

Aucubin administered by either oral or parenteral route protects against cisplatin-induced acute kidney injury in mice.

Aucubin is pharmacologically active natural compound which possesses numerous beneficial properties. This study aimed to evaluate the protective effect of aucubin against cisplatin (CP)-induced acute kidney injury in mice and the mechanism of its action. Aucubin was administrated to mice orally or intraperitoneally (ip) (1.5 and 5 mg/kg) for two consecutive days, two days after ip injection of cisplatin (CP), 11 mg/kg. Treatment with aucubin by both routes of administration ameliorated histopathological changes and reduced elevated serum markers of kidney injury. CP administration increased renal expression of heme oxygenase-1 (HO-1) and 4-hydroxynonenal (4-HNE), as well as tumor necrosis factor-alpha (TNF-α), which was dose-dependently ameliorated by aucubin. Moreover, aucubin reduced increased renal expression of cleaved caspase-3 and -9 and decreased poly (ADP-ribose) polymerase (PARP) cleavage. Mechanistically, aucubin suppressed the activation of several signaling pathways involved in inflammation and apoptosis, including nuclear factor-kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), Akt, extracellular signal-regulated kinase 1/2 (ERK1/2) and forkhead box O3a (FOXO3a). Parenteral application was marginally but statistically more effective in reducing CP-induced kidney injury than oral administration. The findings of this study suggest that aucubin acts as a protective agent against CP-induced nephrotoxicity, which should be further investigated.

Beneficial impact of epigallocatechingallate on LDL-C through PCSK9/LDLR pathway by blocking HNF1α and activating FoxO3a.

BACKGROUND: Green tea drinking has been proven to lower lipid and exert cardiovascular protection, while the potential mechanism has not been fully determined. This study was to investigate whether the beneficial impact of epigallocatechingallate (EGCG), a type of catechin in green tea on lipids is associated with proprotein convertase subtilisin/kexin type 9 (PCSK9) pathways. METHODS: We studied the effects and underlying molecular mechanism of EGCG or green tea on regulating cholesterol from human, animal and in vitro. RESULTS: In the age- and gender-matched case control observation, we found that individuals with frequent tea consumption (n = 224) had the lower plasma PCSK9 and low density lipoprotein cholesterol (LDL-C) levels compared with ones without tea consumption (n = 224, p < 0.05). In the high fat diet (HFD) fed rats, EGCG administration significantly lowered circulating PCSK9 concentration and liver PCSK9 expression, along with up-regulated LDL receptor (LDLR) expression but decreased level of LDL-C. In hepatic cell study, similar results were obtained regarding the impact of EGCG on LDLR and PCSK9 expression. The assay transposase-accessible chromatic with high-throughput sequencing (ATAC-seq) and subsequent results suggested that two transcription factors, hepatocyte nuclear factor-1α (HNF-1α) and forkhead box class O (FoxO) 3a involved in inhibitory action of EGCG on PCSK9 expression. CONCLUSIONS: The present study demonstrates that EGCG suppresses PCSK9 production by promoting nuclear FoxO3a, and reducing nuclear HNF1α, resulting in up-regulated LDLR expression and LDL uptake in hepatocytes. Thereby inhibiting liver and circulating PCSK9 levels, and ultimately lowering LDL-C levels.

HIF­1α affects trophoblastic apoptosis involved in the onset of preeclampsia by regulating FOXO3a under hypoxic conditions.

Preeclampsia (PE) is a pregnancy-specific syndrome that has severe implications on perinatal mortality and morbidity. Excessive apoptosis of trophoblasts induced by hypoxia may be associated with the development of PE, but the exact pathogenesis is unknown. Forkhead box O transcription factor 3a (FOXO3a) is activated under hypoxic conditions. Furthermore, hypoxia­inducible factor­1α (HIF­1α) is sensitive to variations in partial oxygen pressure. Thus, the aims of the present study were to investigate the expression levels of HIF­1α and FOXO3a in placental samples of early onset severe PE, and their effect on trophoblastic apoptosis under hypoxic conditions. Cobalt chloride was used to establish the hypoxic model. The present study examined the expression levels of HIF­1α and FOXO3a in the placental tissues and HTR8/SVneo cells under hypoxic conditions. It was found that HIF­1α and FOXO3a were highly expressed in placental tissues of patients with PE and in HTR8/SVneo cells under hypoxic conditions. Furthermore, knockdown of FOXO3a using a specific small interfering RNA (siRNA) decreased apoptosis in HTR8/SVneo cells. Moreover, it was found that after knockdown of HIF­1α using siRNA, FOXO3a expression and the apoptotic rate were reduced in HTR8/SVneo cells. Therefore, the present results indicated that the elevated expression of HIF­1α increased trophoblastic apoptosis by regulating FOXO3a, which may be involved in the pathogenesis of PE.

Dexmedetomidine protects against lidocaine-induced neurotoxicity through SIRT1 downregulation-mediated activation of FOXO3a.

Lidocaine, a typical local anesthetic, has been shown to directly induce neurotoxicity in clinical settings. Dexmedetomidine (DEX) is an alpha-2-adrenoreceptor agonist that has been used as anxiolytic, sedative, and analgesic agent which has recently found to protect against lidocaine-induced neurotoxicity. Nicotinamide adenine dinucleotide-dependent deacetylase sirtuin-1 (SIRT1)/forkhead box O3 (FOXO3a) signaling is critical for maintaining neuronal function and regulation of the apoptotic pathway. In the present study, we designed in vitro and in vivo models to investigate the potential effects of lidocaine and DEX on SIRT1 and FOXO3a and to verify whether SIRT1/FOXO3a-mediated regulation of apoptosis is involved in DEX-induced neuroprotective effects against lidocaine. We found that in both PC12 cells and brains of mice, lidocaine decreased SIRT1 level through promoting the degradation of SIRT1 protein. Lidocaine also increased FOXO3a protein level and increased the acetylation of SIRT1 through inhibiting SIRT1. Upregulation of SIRT1 or downregulation of FOXO3a significantly inhibited lidocaine-induced changes in both cell viability and apoptosis. DEX significantly inhibited the lidocaine-induced decrease of SIRT1 protein level and increase of FOXO3a protein level and acetylation of FOXO3a. Downregulation of SIRT1 or upregulation of FOXO3a suppressed DEX-induced neuroprotective effects against lidocaine. The data suggest that SIRT1/FOXO3a is a potential novel target for alleviating lidocaine-induced neurotoxicity and provide more theoretical support for the use of DEX as an effective adjunct to alleviate chronic neurotoxicity induced by lidocaine.

Role of FoxO3a as a negative regulator of the cardiac myofibroblast conversion induced by TGF-ß1.

Cardiac fibroblasts (CFs) are necessary to maintain extracellular matrix (ECM) homeostasis in the heart. Normally, CFs are quiescent and secrete small amounts of ECM components, whereas, in pathological conditions, they differentiate into more active cells called cardiac myofibroblasts (CMF). CMF conversion is characteristic of cardiac fibrotic diseases, such as heart failure and diabetic cardiomyopathy. TGF-ß1 is a key protein involved in CMF conversion. SMADs are nuclear factor proteins activated by TGF-ß1 that need other proteins, such as forkhead box type O (FoxO) family members, to promote CMF conversion. FoxO1, a member of this family protein, is necessary for TGF-ß1-induced CMF conversion, whereas the role of FoxO3a, another FoxO family member, is unknown. FoxO3a plays an important role in many fibrotic processes in the kidney and lung. However, the participation of FoxO3a in the conversion of CFs into CMF is not clear. In this paper, we demonstrate that TGF-ß1 decreases the activation and expression of FoxO3a in CFs. FoxO3a regulation by TGF-ß1 requires activated SMAD3, ERK1/2 and Akt. Furthermore, we show that FoxO1 is crucial in the FoxO3a regulation induced by TGF-ß1, as shown by overexpressed FoxO1 enhancing and silenced FoxO1 suppressing the effects of TGF-ß1 on FoxO3a. Finally, the regulation of TGF-ß1-induced CMF conversion was enhanced by FoxO3a silencing and suppressed by inhibited FoxO3a degradation. Considering these collective findings, we suggest that FoxO3a acts as a negative regulator of the CMF conversion that is induced by TGF-ß1.

Circ-DONSON promotes malignant progression of glioma through modulating FOXO3.

OBJECTIVE: The aim of this study was to investigate the expression level of circ-DONSON in glioma and to explore its effect on glioma metastasis and the underlying mechanism. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine circ-DONSON expression in 40 paired glioma tumor tissues and adjacent tissues. Meanwhile, the relation between circ-DONSON level and clinical parameters of glioma and the prognosis of patients was analyzed. The expression of circ-DONSON in glioma cell lines was analyzed by qRT-PCR as well. In addition, circs-DONSON silencing model was constructed in glioma cell lines. Cell counting kit-8 (CCK-8), cell scratch, and transwell migration assays were performed to investigate the effect of circ-DONSON on biological functions of glioma cells. Finally, the interplay between FOXO3 and circ-DONSON was explored. RESULTS: QRT-PCR results revealed that the expression level of circ-DONSON in glioma tumor tissues was remarkably higher than that of adjacent tissues, and the difference was statistically significant (p<0.05). Compared with patients with low expression of circ-DONSON, significantly higher prevalence of lymph node or distant metastasis and worse prognosis were observed in patients with high expression of circ-DONSON (p<0.05). The proliferation and migration abilities of glioma cells in circ-DONSON silenced group were remarkably suppressed when compared with NC group (p<0.05). Additionally, FOXO3 expression was remarkably down-regulated in glioma cell lines and tissues. FOXO3 expression was negatively correlated with circ-DONSON expression. In addition, cell reverse experiment demonstrated that circ-DONSON and FOXO3 can regulate each other, thereby together affecting the malignant progression of glioma. CONCLUSIONS: Circ-DONSON was remarkably associated with lymph node or distant metastasis, as well as poor prognosis of patients with glioma. Furthermore, it promoted the metastasis of glioma cells via regulating FOXO3.

MiRNA-96-5p contributed to the proliferation of gastric cancer cells by targeting FOXO3.

Various microRNAs (miRNAs, miRs) and the forkhead box O (FOXO) family proteins have been shown to influence gastric cancer progression and development. Here, we aimed to identify the gastric cancer related miRNAs and their relationship with the FOXO family. MiRNA profiles were generated by miRNA microarray screening from pre-operative plasma samples. Quantitative reverse transcription PCR and western bolt were used to determine the expression levels of miR-96 and FOXO family. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide assay and colony formation assay were used to test the cell viability. The miR-96-5p and FOXO3 interaction were confirmed by luciferase reporter assay. Our results demonstrated the excessive expression of miR-96-5p in gastric cancer cell lines and plasma samples from gastric cancer patients. In addition, the protein levels of FOXO3 were decreased in tissue samples from gastric cancer patients. Moreover, miR-96-5p accelerated the gastric cancer cell proliferation by directly targeting FOXO3. Therefore, we conclude that iR-96-5p might promote the progression of gastric cancer by directly targeting FOXO3 mRNA and downregulating the expression of FOXO3 protein, which provides new insights for the molecular mechanism of gastric cancer.

A drug library screen identifies Carbenoxolone as novel FOXO inhibitor that overcomes FOXO3-mediated chemoprotection in high-stage neuroblastoma.

The transcription factor FOXO3 has been associated in different tumor entities with hallmarks of cancer, including metastasis, tumor angiogenesis, maintenance of tumor-initiating stem cells, and drug resistance. In neuroblastoma (NB), we recently demonstrated that nuclear FOXO3 promotes tumor angiogenesis in vivo and chemoresistance in vitro. Hence, inhibiting the transcriptional activity of FOXO3 is a promising therapeutic strategy. However, as no FOXO3 inhibitor is clinically available to date, we used a medium-throughput fluorescence polarization assay (FPA) screening in a drug-repositioning approach to identify compounds that bind to the FOXO3-DNA-binding-domain (DBD). Carbenoxolone (CBX), a glycyrrhetinic acid derivative, was identified as a potential FOXO3-inhibitory compound that binds to the FOXO3-DBD with a binding affinity of 19 µM. Specific interaction of CBX with the FOXO3-DBD was validated by fluorescence-based electrophoretic mobility shift assay (FAM-EMSA). CBX inhibits the transcriptional activity of FOXO3 target genes, as determined by chromatin immunoprecipitation (ChIP), DEPP-, and BIM promoter reporter assays, and real-time RT-PCR analyses. In high-stage NB cells with functional TP53, FOXO3 triggers the expression of SESN3, which increases chemoprotection and cell survival. Importantly, FOXO3 inhibition by CBX treatment at pharmacologically relevant concentrations efficiently repressed FOXO3-mediated SESN3 expression and clonogenic survival and sensitized high-stage NB cells to chemotherapy in a 2D and 3D culture model. Thus, CBX might be a promising novel candidate for the treatment of therapy-resistant high-stage NB and other "FOXO-resistant" cancers.

Reduction of circular RNA Foxo3 promotes prostate cancer progression and chemoresistance to docetaxel.

Dysregulation of circular RNA Foxo3 (circFoxo3) has been reported to be involved in breast cancer and non-small lung cancer progression. However, little is known about the role of circFoxo3 in prostate cancer, which the present study seeks to investigate. CircFoxo3 expression was analyzed in 22 low-grade prostate cancer samples, 24 high-graded prostate cancer samples, and 18 normal prostate tissues, finding that its quantity was significantly decreased in high-graded compared to low-grade prostate cancer and normal prostate tissues. CircFoxo3 inhibited prostate cancer cell survival, migration, invasion and chemoresistance to docetaxel, which was related to circFoxo3's repression of Foxo3 and EMT. Silencing circFoxo3 expression promoted prostate cancer cell survival, migration, invasion and chemoresistance to docetaxel, as well as the positive effects of androgen on prostate cancer viability. Delivery of circfoxo3 enhanced chemosensitivity to docetaxel of prostate tumor-bearing mice and prolonged the life span of mice, while reduction with siRNAs promoted chemoresistance to docetaxel and shorted the life span of the tumor-bearing mice. Targeting circFoxo3/Foxo3/EMT may provide an applicable strategy for exploring potential prognostic and therapeutic approaches for prostate cancer.

Nodal pathway activation due to Akt1 suppression is a molecular switch for prostate cancer cell epithelial-to-mesenchymal transition and metastasis.

Several studies have unraveled the negative role of Akt1 in advanced cancers, including metastatic prostate cancer (mPCa). Hence, understanding the consequences of targeting Akt1 in the mPCa and identifying its downstream novel targets is essential. We studied how Akt1 deletion in PC3 and DU145 cells activates the Nodal pathway and promotes PCa epithelial-to-mesenchymal transition (EMT) and metastasis. Here we show that Akt1 loss increases Nodal expression in PCa cells accompanied by activation of FoxO1/3a, and EMT markers Snail and N-cadherin as well as loss of epithelial marker E-cadherin. Treatment with FoxO inhibitor AS1842856 abrogated the Nodal expression in Akt1 deleted PCa cells. Akt1 deficient PCa cells exhibited enhanced cell migration and invasion in vitro and lung metastasis in vivo, which were attenuated by treatment with Nodal pathway inhibitor SB505124. Interestingly, Nodal mRNA analysis from two genomic studies in cBioportal showed a positive correlation between Nodal expression and Gleason score indicating the positive role of Nodal in human mPCa. Collectively, our data demonstrate Akt1-FoxO3a-Nodal pathway as an important mediator of PCa metastasis and present Nodal as a potential target to treat mPCa patients.

Extracellular Vesicles from Apoptotic Cells Promote TGFß Production in Macrophages and Suppress Experimental Colitis.

The clearance of apoptotic cells is an essential process to maintain homeostasis of immune system, which is regulated by immunoregulatory cytokines such as TGFß. We show here that Extracellular Vesicles (EVs) were highly released from apoptotic cells, and contributed to macrophage production of TGFß in vitro and in vivo. We further elucidated mechanistically that phosphatidylserine in EVs was a key triggering-factor, and transcription factor FOXO3 was a critical mediator for apoptotic EV-induced TGFß in macrophages. Importantly, we found that macrophages pre-exposed to EVs exhibited an anti-inflammatory phenotype. More strikingly, administration of EVs in vivo promotes Tregs differentiation and suppresses Th1 cell response, and ameliorates experimental colitis. Thus, apoptotic-EV-based treatment might be a promising therapeutic approach for human autoimmune disease.