Expression Of Forkhead Box O3a Research Articles

D-galactose causes embryonic development arrest and placental development disorders in mice by increasing ROS and inhibiting SIRT1/FOXO3a axis

IntroductionDoes an elevation in d-Galactose (D-Gal) levels within the body contribute to abnormal embryonic development and placental dysfunction during pregnancy? MethodsMouse embryos were cultivated to the blastocyst stage under varying concentrations of D-Gal. The blastocyst formation rate was measured, and the levels of reactive oxygen species (ROS), sirtuin 1 (SIRT1), and forkhead box O3a (FOXO3a) in blastocysts were assessed. Mice were intraperitoneally injected with either saline or D-Gal with or without SRT1720. On the 14th day of pregnancy, the fetal absorption rate and placental weight were recorded. Placental levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were determined. The expression of senescence-related factors, such as senescence-associated β-galactosidase (SA-β-gal) in the placenta was examined, and the expression of placental SIRT1, FOXO3a and p21 was evaluated by immunohistochemistry and Western blotting. ResultsD-Gal adversely affects early embryonic development in vitro, resulting in a decreased blastocyst formation rate. Furthermore, D-Gal downregulates SIRT1 and FOXO3a while increasing ROS levels in blastocysts. Concurrently, D-Gal induces placental dysfunction, characterized by an elevated fetal absorption rate, reduced placental weight, diminished SOD activity, and increased MDA content. The senescence-related factor SA-β-gal was detected in the placenta, along with altered expression of placental SIRT1, FOXO3a, and p21. The SIRT1 agonist SRT1720 mitigated this damage by increasing SIRT1 and FOXO3a expression. DiscussionThe inhibition of early embryonic development and placental dysfunction induced by D-Gal may be attributed to the dysregulation of SIRT1. Activating SIRT1 emerges as a potentially effective strategy for alleviating the adverse effects of D-Gal exposure.

Melatonin regulates autophagy in granulosa cells from patients with premature ovarian insufficiency via activating Foxo3a.

Premature ovarian insufficiency (POI) is a diverse form of female infertility characterized by a decline in ovarian function before the age of 40. Melatonin (MT) is a potential clinical treatment for restoring or safeguarding ovarian function in POI. However, the specific therapeutic mechanism underlying this effect remains unclear. To address this, we conducted experiments using human granulosa cells (GCs) from both POI and normal patients. We examined the expression levels of autophagy-related genes and proteins in GCs through qRT-PCR and western blot analysis. Autophagy flux was monitored in GCs infected with GFP-LC3-adenovirus, and the regulatory function of MT in autophagy was investigated. Additionally, we employed pharmacological intervention of autophagy using 3-Methyladenine (3-MA) and RNA interference of Forkhead box O-3A (FOXO3A) to elucidate the mechanism of MT in the autophagy process. Compared to GCs from normal patients, GCs from POI patients exhibited irregular morphology, decreased proliferation, increased apoptosis, and elevated ROS levels. The expression of autophagy-related genes was downregulated in POI GCs, resulting in reduced autophagic activity. Furthermore, MT levels were decreased in POI GCs, but exogenous MT effectively activated autophagy. Mechanistically, melatonin treatment downregulated FOXO3A expression and induced phosphorylation in POI GCs. Importantly, silencing FOXO3A abolished the protective effect of melatonin on GCs. These findings indicate that autophagy is downregulated in POI GCs, accompanied by a deficiency in MT. Moreover, we demonstrated that supplementing MT can rescue autophagy levels and enhance GC viability through the activation of FOXO3A signaling. Thus, MT-FOXO3A may serve as a potential therapeutic target for POI treatment.

METTL14-m6A-FOXO3a axis regulates autophagy and inflammation in ankylosing spondylitis

The role of m6A in ankylosing spondylitis (AS) remains largely obscure. In this study, we found that m6A modification was decreased in T cells of AS, and the abnormal m6A modification was attributed to the downregulation of methyltransferase-like 14 (METTL14). METTL14 exerted a critical role in regulating autophagy activity and inflammation via targeting Forkhead box O3a (FOXO3a). Mechanistically, the loss of METTL14 decreased the expression of FOXO3a, leading to the damage of autophagic flux and the aggravation of inflammation. Inversely, the forced expression of METTL14 upregulated the expression of FOXO3a, thereby activating autophagy and alleviating inflammation. Furthermore, our results revealed that METTL14 targeted FOXO3a mRNA and regulated its expression and stability in a m6A-dependent manner. These findings uncovered the functional importance of m6A methylation mechanisms in the regulation of autophagy and inflammation, which expanded our understanding of this interaction and was critical for the development of therapeutic strategies for AS.

RASSF1A promotes radiosensitivity in nasopharyngeal carcinoma by promoting FoxO3a and inhibiting the Nrf2/TXNRD1 signaling pathway.

Radiotherapy is widely used as the first-line treatment for nasopharyngeal carcinoma (NPC). However, the resistance of some patients to treatment lowers its clinical effectiveness. Compared to typical epithelial cells, NPC markedly lowers the Ras-association domain family 1A (RASSF1A) protein expression. RASSF1A overexpression sensitizes NPC cells to radiotherapy. Mechanistically, RASSF1A promotes the expression of Forkhead box O3a (FoxO3a) in the nucleus and inhibits the Nuclear factor E2-related factor 2 (Nrf2) signaling pathway via binding to the Kelch-like ECH-associated protein 1 (Keap1) promoter. Through elevating intracellular ROS levels, RASSF1A overexpression inhibits the expression of thioredoxin reductase 1 (TXNRD1), a crucial Nrf2 target gene, and increases NPC sensitivity to radiation. Immunohistochemical staining of NPC tissue sections revealed that the expression of RASSF1A is negatively correlated with that of TXNRD1. The traditional Chinese medicine component andrographolide (AGP), which induces RASSF1A expression, increased the sensitivity of NPC cells to radiotherapy in vitro and in vivo. Our findings implied that RASSF1A increases the sensitivity of NPC to radiation by increasing FoxO3a expression in the nucleus, inhibiting the Nrf2/TXNRD1 signaling pathway, and elevating intracellular ROS levels. AGP targets RASSF1A and may be a promising adjuvant sensitizer for enhancing radiosensitivity in NPC.

Antitumor and radiosensitizing effects of Anagallis arvensis hydromethanolic extract on breast cancer cells through upregulating FOXO3, Let-7, and mir-421 Expression

This study aimed to observe the anticancer, as well as radiosensitizing, effect of Anagallis arvensis extract (AAE) on the growth of breast cancer cells (BCCs). This effect was evaluated through measured cell cycle arrest plus apoptosis through flowcytometry analyses associated with a protein level of estrogen receptors (ER-α and ER-β), Fork head box O3a (FOXO3), and phosphorylated histone H2AX (γ-H2AX) by western blotting analysis. Further, the gene expression of mir-18, mir-101, mir-421, and let-7 was detected via a real-time polymerase chain reaction (RTPCR). AAE and/or γ-radiation (4Gy) inhibited the mediator-induced growth activation and enhanced radiosensitivity in the BCC lines (MCF-7 and MDA-MB-231). AAE reduced both cell cycle progression and cell growth via induced apoptosis. Additionally, the protein expression of ERα and ERβ was decreased, while the protein expression of FOXO3 and γ-H2AX was increased. Moreover, the gene expression of mir-18 and mir-101 was decreased, while the gene expression of let-7 and mir-421 was stimulated in BCCs. AAE has an anticancer with a radiosensitizing effect and is recommended for BC treatment.

FOXO3a Alleviates the Inflammation and Oxidative Stress via Regulating TGF-β and HO-1 in Ankylosing Spondylitis.

This study aimed to investigate whether Forkhead box O3a (FOXO3a) modulates inflammation and oxidative stress in ankylosing spondylitis (AS). We applied bioinformatics analysis, quantitative real-time polymerase chain reaction, immunoblotting, enzyme linked immunosorbent assay, chromatin immunoprecipitation, and dual-luciferase reporter assay. Gene overexpression and knockdown of FOXO3a were conducted via lentivirus and small interfering RNA, respectively. Downregulated FOXO3a expression was first confirmed in AS patients. Interleukin-8 (IL-8) and IL-17A were highly expressed and negatively related with FOXO3a in AS. Total antioxidant capacity (T-AOC) were markedly decreased and positively associated with FOXO3a in AS. Overexpression of FOXO3a inhibited the secretion of inflammatory cytokines and promoted the production of antioxidant enzymes in Jurkat cells. Transforming growth factor-β (TGF-β) and heme oxygenase 1 (HO-1), which had binding sites to FOXO3a based on bioinformatics analysis, were abnormally expressed and positively related with FOXO3a. Accordingly, FOXO3a obviously elevated the protein and transcription levels of TGF-β and HO-1 in Jurkat cells. The above results were verified by silencing FOXO3a. Moreover, FOXO3a directly interacted with and promoted the transcription of TGF-β and HO-1. In summary, the modulation of cellular inflammation and oxidative stress via FOXO3a-mediated TGF-β and HO-1 activation is partly involved in the pathogenesis of AS.

Upregulation of METTL14 contributes to trophoblast dysfunction by elevating FOXO3a expression in an m6A-dependent manner

IntroductionPreeclampsia, a specific complication of pregnancy, is a leading cause of perinatal and maternal mortality worldwide. N6-methyladenosine (m6A) is a prevalent and reversible modification of mammalian mRNAs, and is known to play an important role in various physiological and pathological processes. However, little is known about its possible effects on trophoblasts in preeclampsia. MethodsColorimetric RNA m6A methylation quantification assay and dot blotting were used to assess the levels of global RNA m6A modification in placental tissues collected from females with normal pregnancy and preeclampsia, while the mRNA levels of major m6A methyltransferases/demethylases were investigated by quantitative real-time polymerase chain reaction. The effects of methyltransferase-like 14 (METTL14) on trophoblasts were evaluated using cell counting kit-8, transwell invasion assay, autophagic flux assay, and Annexin V/propidium iodide apoptosis assay. The molecular mechanism underlying the regulation of forkhead box O3a (FOXO3a) expression by METTL14 was determined using methylated RNA immunoprecipitation and transcription inhibition assays. ResultsGlobal RNA m6A methylation and METTL14 expression were significantly increased in placental tissues obtained from patients with preeclampsia. In vitro studies showed that overexpression of METTL14 in HTR-8/SVneo cells inhibited trophoblast proliferation and invasion, but induced trophoblast autophagy and apoptosis. We further demonstrated that METTL14 epigenetically elevated FOXO3a expression via an m6A-dependent mechanism. FOXO3a inhibition effectively prevented the impairment of trophoblast proliferation and invasion, and diminished the induction of trophoblast autophagy and apoptosis in METTL14-overexpressing HTR-8/SVneo cells. DiscussionIncreased METTL14-mediated m6A modification results in an adverse impact on trophoblast function by elevating FOXO3a expression.

Activation of forkhead box O3a by mono(2-ethylhexyl)phthalate and its role in protection against mono(2-ethylhexyl)phthalate-induced oxidative stress and apoptosis in human cardiomyocytes.

Mono(2-ethylhexyl)phthalate (MEHP), the active metabolite of di(2-ethylhexyl)phthalate (DEHP), is known to exert cardiotoxicity. The aim of the present study was to investigate the role of forkhead box O3a (FOXO3a) in MEHP-induced human AC16 cardiomyocyte injuries. MEHP reduced cell viability and mitochondrial membrane potential (ΔΨm), whereas it increased lactate dehydrogenase (LDH) leakage, production of reactive oxygen species (ROS), and apoptosis in cardiomyocytes. The expression of FOXO3a and its target genes, mitochondrial superoxide dismutase (Mn-SOD) and apoptosis repressor with caspase recruitment domain (ARC), increased after MEHP exposure, but the expression of p-FOXO3a protein was decreased. Overexpression of FOXO3a decreased the production of ROS and the apoptosis rate induced by MEHP, and the expression of Mn-SOD and ARC was further increased after MEHP exposure. In contrast, knockdown of FOXO3a resulted in increased ROS production and apoptosis and suppressed the expression of Mn-SOD and ARC in the presence of MEHP. However, overexpression or knockdown of FOXO3a did not affect MEHP-induced loss of ΔΨm. In conclusion, the loss of ΔΨm and apoptosis are involved in MEHP-induced cardiomyocyte toxicity. Activation of FOXO3a defends against MEHP-induced oxidative stress and apoptosis by upregulating the expression of Mn-SOD and ARC in AC16 cardiomyocytes.

P2Y2R contributes to the development of diabetic nephropathy by inhibiting autophagy response

ObjectiveDiabetic nephropathy (DN) is one of the most common complications of diabetes and a critical risk factor for developing end-stage renal disease. Activation of purinergic receptors, including P2Y2R has been associated with the pathogenesis of renal diseases, such as polycystic kidney and glomerulonephritis. However, the role of P2Y2R and its precise mechanisms in DN remain unknown. We hypothesised that P2Y2R deficiency may play a protective role in DN by modulating the autophagy signalling pathway. MethodsWe used a mouse model of DN by combining a treatment of high-fat diet and streptozotocin after unilateral nephrectomy in wild-type or P2Y2R knockout mice. We measured renal functional parameter in plasma, examined renal histology, and analysed expression of autophagy regulatory proteins. ResultsHyperglycaemia and ATP release were induced in wild type-DN mice and positively correlated with renal dysfunction. Conversely, P2Y2R knockout markedly attenuates albuminuria, podocyte loss, development of glomerulopathy, renal tubular injury, apoptosis and interstitial fibrosis induced by DN. These protective effects were associated with inhibition of AKT-mediated FOXO3a (forkhead box O3a) phosphorylation and induction of FOXO3a-induced autophagy gene transcription. Furthermore, inhibitory phosphorylation of ULK-1 was decreased, and the downstream Beclin-1 autophagy signalling was activated in P2Y2R deficiency. Increased SIRT-1 (sirtuin-1) and FOXO3a expression in P2Y2R deficiency also enhanced autophagy response, thereby ameliorating renal dysfunction in DN. ConclusionsP2Y2R contributes to the pathogenesis of DN by impairing autophagy and serves as a therapeutic target for treating DN.

Hepcidin deficiency causes bone loss through interfering with the canonical Wnt/β-catenin pathway via Forkhead box O3a

ObjectiveHepcidin deficiency is known to cause body iron accumulation and bone microarchitecture defects, but the exact underlying mechanisms of hepcidin deficiency-induced bone loss remain unclear. Our objective was to understand the molecular mechanism of hepcidin deficiency-induced bone loss. MethodsThe bone phenotypes of wild type (WT) and hepcidin knockout (Hepcidin-KO) mice were measured by microcomputed tomography. The osteoclastic marker of the bone was measured by tartrate-resistant acid phosphatase staining. The osteoblastic marker of the bone was measured by immunostaining of osteocalcin. Primary osteoblastic and osteoclastic differentiation was performed using bone marrow cells. The mature osteoclast was determined by tartrate-resistant acid phosphatase staining, pit formation assay and relative gene expression. The mature osteoblast was determined by alkaline phosphatase activity, alkaline phosphatase staining, Alizarin Red staining and relative gene expression. The protein expression of β-catenin, TCF4/TCF7L2 and Forkhead box O3a (FOXO3a) was measured by Western blot and their combination by co-immunoprecipitation. In vivo study was performed by tail vein administration of FOXO3a-RNAi using an adeno-associated virus in Hepcidin-KO mice. ResultsWe found that Hepcidin-KO mice exhibited iron accumulation and bone loss compared with WT mice. The osteoclastic differentiation of bone marrow-derived macrophages from Hepcidin-KO mice was not significantly different from that of bone marrow–derived macrophages from WT mice. However, the osteoblastic differentiation of bone marrow–derived mesenchymal stem cells from Hepcidin-KO mice was obviously decreased compared with that of bone marrow–derived mesenchymal stem cells from WT mice. Furthermore, it was confirmed in this study that upon hepcidin deficiency, β-catenin, TCF4/TCF7L2 and FOXO3a expression in bone tissues was not altered, but β-catenin combination with TCF4/TCF7L2 was strongly inhibited by β-catenin combination with FOXO3a, indicating that the canonical Wnt/β-catenin pathway was affected. Tail vein administration of FOXO3a-RNAi using an adeno-associated virus in Hepcidin-KO mice resulted in bone mass recovery. ConclusionThese findings suggested that hepcidin deficiency might cause bone loss by interfering with the canonical Wnt/β-catenin pathway via FOXO3a, and FOXO3a inhibition would be a possible approach to treat hepcidin deficiency-induced bone loss. The translational potential of this articleHepcidin deficiency, as well as iron accumulation, has been considered as a risk factor for osteoporosis. For this kind of osteoporosis, inhibition of FOXO3a either by neutralized antibody or AAV-mediated RNAi, represents an effective and promising method.

Antitumor activity of luteolin in human colon cancer SW620 cells is mediated by the ERK/FOXO3a signaling pathway

The aim of this study was to investigate the mechanism of the anticancer activity of luteolin in metastatic human colon cancer SW620 cells. Luteolin dose-dependently reduced the viability and proliferation of SW620 cells and increased the expression of antioxidant enzymes. The expression of antiapoptotic protein Bcl-2 decreased whereas the expression of proapoptotic proteins Bax and caspase-3 increased by luteolin treatment, resulting in increased poly (ADP-ribose) polymerase (PARP) cleavage and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity. Luteolin also increased the expression of autophagic proteins Beclin-1, autophagy-related protein 5 (Atg5) and microtubule-associated protein 1A/1B-light chain 3 beta-I/II (LC3B-I/II), while the usage of 3-methyladenine suggested a prosurvival role of autophagy. Moreover, treatment with luteolin induced reversal of the epithelial-mesenchymal transition process through the suppression of the wingless-related integration site protein (Wnt)/β-catenin pathway. The cytotoxic activity of luteolin coincided with the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and forkhead box O3a (FOXO3a). Treatment with the mitogen-activated protein kinase kinase (MEK) inhibitor PD0325901 inhibited ERK-dependent FOXO3a phosphorylation, resulting in increased FOXO3a expression and apoptosis, with the suppression of autophagy. The results of the current study suggest the antitumor activity of luteolin in SW620 cells through the ERK/FOXO3a-dependent mechanism, as well as its antimetastatic potential.

Lysine-specific demethylase 1 (LSD1) promotes ovarian cancer cell progression by Forkhead box O 3a (FOXO3a) inhibition

Recently, LSD1 is considered as a possible therapeutic mark for ovarian epithelial cancer (OEC). Though, the underlying molecular mechanism by which LSD1 endorses the oncogenesis of OEC has not been fully understood. Here, we revealed that overexpression of LSD1 downregulated Forkhead box O 3a (FOXO3a), while knockdown or pharmacological inhibition of LSD1 upregulated FOXO3a expression. Specifically, LSD1 interacted with demethylated FOXO3a. The LSD1-demethylated FOXO3a degraded via an ubiquitin-proteasome pathway. Biologically, LSD1 destabilized FOXO3a to abrogate its functions in the suppression of soft agar colony and cell proliferation formation in HO8910 ovarian cancer cells. Knockdown of FOXO3a rescued the restricted cell proliferation by LSD1 downregulation. As a whole, our study clarifies a way in ovarian cancer cell growth through the negative regulation of FOXO3a by LSD1.

Adjudin synergizes with paclitaxel and inhibits cell growth and metastasis by regulating the sirtuin 3-Forkhead box O3a axis in human small-cell lung cancer.

BackgroundSmall‐cell lung cancer (SCLC), a malignant tumor, is usually widely metastatic when diagnosed. The lack of important therapeutic clinical advances makes it difficult to treat. Previous studies showed that Adjudin had anticancer effects in many other human cancers, and it was synergetic with cisplatin in non‐small cell lung cancer. However, the mechanism on SCLC was unclear.MethodsWe investigated the potential mechanism and effect of Adjudin on SCLC both in vitro and in vivo.ResultsAn SCLC xenograft model showed that Adjudin inhibited tumor growth and was significantly synergetic with paclitaxel (in vitro as well). Cell Counting Kit‐8 assays, flow cytometric analysis and western blotting showed that Adjudin effectively suppressed SCLC cell proliferation by inducing S phase arrest and caspase‐dependent apoptosis. Moreover, Transwell and scratch assays showed that Adjudin also effectively inhibited migration and invasion. Furthermore, Adjudin activated the sirtuin 3 (SIRT3)–Forkhead box O3a (FOXO3a) pathway. Downregulating SIRT3 or FOXO3a significantly attenuated Adjudin‐induced anticancer effects. Furthermore, higher expression of SIRT3 and FOXO3a were positively correlated, and both were associated with longer survival in lung cancer patients.ConclusionOverall, the present study is the first to show that Adjudin synergizes with paclitaxel and inhibits cell growth and metastasis by regulating the SIRT3–FOXO3a axis in SCLC; thus, Adjudin has great potential to be an anticancer agent.

Butein induces lung adenocarcinoma A549 cell apoptosis via activating forkhead box O 3a/p27 kinase inhibitor protein 1 and cell oxidative stress

Objective To investigate the role of Butein on lung adenocarcinoma A549 cells and the role of forkhead box O 3a (FOXO3a)/p27 kinase inhibitor protein 1 (p27kip1) and cell oxidative stress in this process. Methods A549 cells were treaded with 25, 50 and 100 μmol/L Butein, then the cell viability, cell migration, Caspase-3 activity, NADPH oxidase activity, Reactive oxygen species (ROS) and total glutathione (GSH) were detected. Further expressions of FOXO3a, p27kip1, B-cell lymphoma-2 (bcl-2) and bcl-2 associated X protein (bax) were detected by Western blotting. In addition, small interference RNA was used to knock down FOXO3a expression in A549 cell. Then the cells were divided into four groups, which were the control group, the FOXO3a siRNA group, the Butein 100 μmol/L group and FOXO3a siRNA + Butein 100 μmol/L group. Expression of FOXO3a, p27kip1, bcl-2 and bax were detected. Results After treatment of Butein (25, 50, 100 μmol/L), cell viability were reduced from 100% to (78.3±4.6)%, (63.5±4.8)% and (40.1±4.1)% (t=10.330, P=0.000) and the distance between cell boundary were increased from 100% to (120.0±8.4)%, (150.0±10.6)% and (175.0±11.1)% (t=4.777, P=0.001). In addition, Butein treatment increased Caspase-3 activity, NADPH oxidase activity and ROS production, and reduced total GSH concentration. Furthermore, Butein treatment increased FOXO3a, p27kip1 and bax expression and reduced bcl-2 expression. FOXO3a siRNA treatment blocked FOXO3a expression and p27kip1 transcription, and reversed Butein induced A549 cell apoptosis by increasing bcl-2 and reducing bax expression. Conclusion Butein can induce A549 cell apoptosis via activating FOXO3a/p27kip1 and cell oxidative stress. Key words: Butein; Lung adenocarcinoma; Forkhead box O 3a; P27 kinase inhibitor protein 1; Oxidative Stress; Cell apoptosis

Anti-microRNA-132 causes sevoflurane‑induced neuronal apoptosis via the PI3K/AKT/FOXO3a pathway.

In the present study, the mechanisms underlying the protective effects of microRNA-132 (miRNA-132) on sevoflurane-induced neuronal apoptosis were investigated. Reverse transcription-quantitative polymerase chain reaction and gene microarray hybridization were used to analyze alterations in microRNA levels. Cell viability, apoptosis and caspase-3/9 activity were measured using MTT, flow cytometry and caspase-3/9 activity kits. Immunofluorescence staining and western blot analysis were used to measure protein expression of phosphoinositide 3-kinase (PI3K) and phosphorylated (p-)AKT, forkhead box O3a (FOXO3a). In sevoflurane-induced rats, the expression of miRNA-132 was downregulated, compared with that in negative control rats. The downregulation of miRNA-132 increased neuronal apoptosis and the upregulation of miRNA-132 inhibited neuronal apoptosis in the sevoflurane-induced in vitro model. The downregulation of miRNA-132 suppressed the protein expression of PI3K and p-AKT, and suppressed the protein expression of FOXO3a in the sevoflurane-induced in vitro model. The PI3K inhibitor increased the effects of anti-miRNA-132 on neuronal apoptosis through the AKT/FOXO3a pathway in the sevoflurane-induced in vitro model. The promotion of FOXO3a inhibited the effects of anti-miRNA-132 on neuronal apoptosis through the AKT/FOXO3a pathway in the sevoflurane-induced in vitro model. These data suggested that miRNA-132 caused sevoflurane-induced neuronal apoptosis via suppression of the PI3K/AKT/FOXO3a pathway.

FOXO3a is essential for murine endometrial decidualization through cell apoptosis during early pregnancy.

Embryo implantation is essential for normal pregnancy, and the process of decidualization is critical for embryo implantation. However, the mechanism of decidualization during early pregnancy is still unknown. Forkhead box O3a (FOXO3a) is the most important functional transcription factor of the forkhead box family and is a highly conserved transcription factor of apoptosis-related genes. In the mouse uterus, FOXO3a was found to be expressed regularly from Days 1-7 of early pregnancy. Upon further exploration, it was found that FOXO3a was expressed at significantly higher levels at the implantation site than at the interimplantation site on Days 5-7 of pregnancy. Under artificial decidualization, FOXO3a was highly expressed in the first and second decidual zones. After decidualization, the expression of FOXO3a was significantly increased both in vivo and vitro. In primary stromal cells, apoptosis was reduced by decreased expression of FOXO3a after inducing decidualization. Moreover, when FOXO3a-small interfering RNA was transfected into the uteri of mice, the expression of decidualization- and apoptosis-related factors was impaired. Thus, FOXO3a might play an important role in decidualization during early pregnancy, and cell apoptosis might be one of pathways for FOXO3a-regulated decidualization.

NOVA1 acts as an oncogene in melanoma via regulating FOXO3a expression.

Increasing studies have suggested that dysregulation of RNA‐binding proteins (RBPs) contributes to cancer progression. Neuro‐oncological ventral antigen 1 (NOVA1) is a novel RBP and plays an important role in tumour development. However, the expression and role of NOVA1 in melanoma remain unknown. In this study, we indicated that NOVA1 expression was up‐regulated in melanoma samples and cell lines. Moreover, we demonstrated that knockdown of NOVA1 suppressed melanoma cell proliferation, migration and invasion in both A375 and A875 cell lines. In addition, we showed that suppressed expression of NOVA1 enhanced forkhead box O3a (FOXO3a) expression while inhibited AKT expression in melanoma cell. Furthermore, we demonstrated that inhibited expression of FoxO3A rescued NOVA1‐mediated cell proliferation, migration and invasion in melanoma cell line A375. These results suggested that NOVA1 acted as an oncogene in the development of melanoma partly through regulating FoxO3A expression.

Expression of PIM-1 in salivary gland adenoid cystic carcinoma: Association with tumor progression and patients' prognosis.

Pim-1 proto-oncogene, serine/threonine kinase (PIM-1) phosphorylates a series of substrates to exert its oncogenic function in numerous malignancies. The present study investigated the clinical significance of the PIM-1 protein, apoptosis status and apoptosis-associated proteins, including forkhead box O3a (FOXO3a), B cell lymphoma-2 (BCL-2) and BCL-2-associted agonist of cell death (BAD), were investigated in salivary gland adenoid cystic carcinoma (ACC) tissues. PIM-1 expression levels in 4 pairs of ACC tissues and corresponding normal salivary gland tissues were determined by western blot analysis. PIM-1, FOXO3a, BAD and BCL-2 expression levels in 60 ACC tissues were evaluated by immunohistochemistry (IHC). A terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay was performed to detect the apoptosis status of ACC tissues. PIM-1 was revealed to be highly expressed in ACC tissues compared with adjacent normal tissues. IHC staining results demonstrated high expression ratios of PIM-1, FOXO3a, BCL-2 and BAD [33.33% (20/60), 51.67% (31/60), 51.67% (31/60) and 55% (33/60)], respectively, and significant correlations between the expression of PIM-1 and FOXO3a and BCL-2 (P<0.05). Apoptotic rates were significantly associated with PIM-1, FOXO3a, BCL-2 and BAD expression levels (P<0.05). PIM-1 expression levels were significantly associated with tumor size, lymph node involvement, nerve invasion, distant metastasis and weakly associated with tumor node metastasis stage. Kaplan-Meier survival curves revealed that PIM-1 expression level was significantly associated with disease-free survival of patients with ACC (P=0.009). Cox regression multivariate analysis results revealed that histotype, distant metastasis and apoptotic rate were independent prognosis factors for ACC. Assessment of PIM-1 may be useful in investigating the malignant behaviors of ACC and predicting the outcome of patients with ACC.

Effect of Sirtuin 1 inhibition on matrix metalloproteinase 2 and Forkhead box O3a expression in breast cancer cells

Breast cancer is the most common invasive cancer in women worldwide. Sirtuin 1 (SIRT1) has recently been shown to have implications in regulating cancer cell growth and apoptosis. SIRT1 regulates Forkhead box O3a (FOXO3a) by both inhibiting FOXO3-induced apoptosis and potentiating the ability of FOXO3a to resist oxidative stress. Matrix metalloproteinase 2 (MMP2) participates in tumor invasion and metastasis by degrading extracellular matrix. SIRT1 up regulates MMP2 expression by its deacetylation activity. This study aimed to investigate the expression of SIRT1, FOXO3a and MMP2 in breast tissues of women with breast cancer. In addition, the effect of SIRT1 inhibition on both FOXO3a and MMP2 expression in breast cancer (MCF-7) cells was assessed. The expression levels of SIRT1, FOXO3a and MMP2 in the breast tissues were determined by real-time PCR in 60 patients with malignant tumor and in 24 patients with benign tumors. After SIRT1 inhibition, protein levels of SIRT1 and FOXO3a were assessed by Western Blot and levels of MMP2 by ELISA in MCF-7 cells. The expression levels of SIRT1, FOXO3a and MMP2 were significantly higher in breast cancer tissues compared to in benign breast tumor and adjacent normal tissues. SIRT1, MMP2 and FOXO3a expression were associated directly with each other. SIRT1 inhibition suppresses MMP2 and FOXO3a expression compared to control MCF7. Sirtinol (SIRT1 inhibitor) effectively induced inhibition of MMP2 and FOXO3a expression in MCF-7 cells, indicating the promising therapeutic strategy of targeting SIRT1 for breast cancer.

The clinicopathological significance of forkhead box P1 and forkhead box O3a in pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinoma is a highly malignant tumor with poor prognosis, and the biomarkers for the early diagnosis, targeting therapy, and prognosis are still not clinically available. This study investigated the expression of forkhead box P1 and forkhead box O3a proteins in human pancreatic ductal adenocarcinoma tumor tissues and pancreatic tissues with and without benign lesions using immunohistochemical staining. Results showed that the positive rates of forkhead box P1 and forkhead box O3a protein expression were significantly lower in pancreatic ductal adenocarcinoma tumors compared to peritumoral tissues, benign pancreatic tissues, and normal pancreatic tissues (p < 0.01). Pancreatic tissues with negative forkhead box P1 and forkhead box O3a protein expression exhibited dysplasia or intraepithelial neoplasia. The positive rates of forkhead box P1 and forkhead box O3a expression were significantly lower in cases with tumor mass >5 cm, lymph node metastasis, invasion to surrounding tissues and organs, and tumor-node-metastasis III + IV stage disease compared to cases with tumor mass ⩽5 cm (p < 0.05), no lymph node metastasis (p < 0.001 and p = 0.001, respectively), no invasion (p = 0.003 and p = 0.004, respectively), and tumor-node-metastasis I or II stage disease (p < 0.05). Kaplan-Meier survival analysis showed that pancreatic ductal adenocarcinoma patients with negative forkhead box P1 and forkhead box O3a expression survived significantly shorter than patients with positive forkhead box P1 and forkhead box O3a expression (p = 0.000). Cox multivariate analysis revealed that negative forkhead box P1 and forkhead box O3a expression was an independent poor prognosis factor in pancreatic ductal adenocarcinoma patients. The area under the curve of a receiver operating characteristic curve was 0.642 for forkhead box P1 (95% confidence interval: 0.553-0.730) and 0.655 for forkhead box O3a (95% confidence interval: 0.6568-0.742). Loss of forkhead box P1 and forkhead box O3a protein expression is associated with carcinogenesis, progression, and poor prognosis in patients with pancreatic ductal adenocarcinomas.