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Cardiovascular disease including diabetic cardiomyopathy (DbCM) represents the leading cause of death in people with diabetes. DbCM is defined as ventricular dysfunction in the absence of underlying vascular diseases and/or hypertension. The known molecular mediators of DbCM are multifactorial, including but not limited to insulin resistance, altered energy metabolism, lipotoxicity, endothelial dysfunction, oxidative stress, apoptosis, and autophagy. FoxO1, a prominent member of forkhead box O transcription factors, is involved in regulating various cellular processes in different tissues. Altered FoxO1 expression and activity have been associated with cardiovascular diseases in diabetic subjects. Herein we provide an overview of the role of FoxO1 in various molecular mediators related to DbCM, such as altered energy metabolism, lipotoxicity, oxidative stress, and cell death. Furthermore, we provide valuable insights into its therapeutic potential by targeting these perturbations to alleviate cardiomyopathy in settings of type 1 and type 2 diabetes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Shafaati and Gopal.)

An increasing body of research suggests that promoting microglial autophagy hinders the neuroinflammation initiated though the NLRP3 inflammasome activation in Alzheimer's disease (AD). The function of FoxG1, a crucial transcription factor involved in cell survival by regulating mitochondrial function, remains unknown during the AD process and neuroinflammation occurs. In the present study, we firstly found that Aβ peptides induced AD-like neuroinflammation upregulation and downregulated the level of autophagy. Following low-dose Aβ25-35 stimulation, FoxG1 expression and autophagy exhibited a gradual increase. Nevertheless, with high-concentration Aβ25-35 treatment, progressive decrease in FoxG1 expression and autophagy levels as the concentration of Aβ25-35 escalated. In addition, FoxG1 has a positive effect on cell viability and autophagy in the nervous system. In parallel with the Aβ25-35 stimulation, we employed siRNA to decrease the expression of FoxG1 in N2A cells. A substantial reduction in autophagy level (Beclin1, LC3II, SQSTM1/P62) and a notable growth in inflammatory response (NLRP3, TNF-α, and IL-6) were observed. In addition, we found FoxG1 overexpression owned the effect on the activation of AMPK/mTOR autophagy pathway and siRNA-FoxG1 successfully abolished this effect. Lastly, FoxG1 suppressed the NLRP3 inflammasome and enhanced the cognitive function in AD-like mouse model induced by Aβ25-35. Confirmed by cellular and animal experiments, FoxG1 suppressed NLRP3-mediated neuroinflammation, which was strongly linked to autophagy regulated by AMPK/mTOR. Taken together, FoxG1 may be a critical node in the pathologic progression of AD and has the potential to serve as therapeutic target., (© 2024. The Author(s).)

Epstein-Barr virus (EBV) is reportedly the first identified human tumor virus, and is closely related to the occurrence and development of nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and several lymphomas. PD-L1 expression is elevated in EBV-positive NPC and GC tissues; however, the specific mechanisms underlying the EBV-dependent promotion of PD-L1 expression to induce immune escape warrant clarification. EBV encodes 44 mature miRNAs. In this study, we find that EBV-miR-BART11 and EBV-miR-BART17-3p upregulate the expression of PD-L1 in EBV-associated NPC and GC. Furthermore, EBV-miR-BART11 targets FOXP1, EBV-miR-BART17-3p targets PBRM1, and FOXP1 and PBRM1 bind to the enhancer region of PD-L1 to inhibit its expression. Therefore, EBV-miR-BART11 and EBV-miR-BART17-3p inhibit FOXP1 and PBRM1, respectively, and enhance the transcription of PD-L1 (CD274, http://www.ncbi.nlm.nih.gov/gene/29126 ), resulting in the promotion of tumor immune escape, which provides insights into potential targets for EBV-related tumor immunotherapy., (© 2022. The Author(s).)

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. After a thorough investigation, the Editor has concluded that the acceptance of this article was partly based upon the positive advice of one illegitimate reviewer report. The report was submitted from an email account which was provided to the journal as a suggested reviewer during the submission of the article. Although purportedly a real reviewer account, the Editor has concluded that this was not of an appropriate, independent reviewer. This manipulation of the peer-review process represents a clear violation of the fundamentals of peer review, our publishing policies, and publishing ethics standards. Apologies are offered to the reviewer whose identity was assumed and to the readers of the journal that this deception was not detected during the submission process. Also, a section of the ‘circFOXP1/Merge’ panel from Fig. 1B appears similar to a section of the ‘18s/Merge’ panel from Figure 1B of the article., (Copyright © 2021. Published by Elsevier B.V.)

Aims: Phenotypic switching of vascular smooth muscle cells (VSMCs) is essential for the formation of abdominal aortic aneurysms (AAAs). MicroRNA-23b (miR-23b) has recently been shown to play a vital role in maintaining the VSMC contractile phenotype; however, little is known about the role of miR-23b in the formation of AAAs. Here, we investigated whether miR-23b prevents AAA formation by inhibiting VSMC phenotypic switching., Materials and Methods: We administered angiotensin II (Ang II, 1000 ng/kg/min) or vehicle to 10-12-week-old male apolipoprotein E knockout (ApoE -/- ) or C57BL/6J mice via subcutaneous osmotic minipumps for 4 weeks., Key Findings: The expression of miR-23b was significantly reduced in the aorta during the early onset of AAA in angiotensin II-treated ApoE -/- mice and in human AAA samples. In vitro experiments showed that the suppression of SMC contractile marker gene expression induced by Ang II was accelerated by miR-23b inhibitors but inhibited by mimics. In vivo studies revealed that miR-23b deficiency in Ang II-treated C57BL/6J mice aggravated the formation of AAAs in these mice compared with control mice; the opposite results were observed in miR-23b-overexpressing mice. Mechanistically, miR-23b knockdown significantly increased the expression of the transcription factor forkhead box O4 (FoxO4) during VSMC phenotypic switching induced by Ang II. In addition, a luciferase reporter assay showed that FoxO4 is a target of miR-23b in VSMCs., Significance: Our study revealed a pivotal role for miR-23b in protecting against aortic aneurysm formation by maintaining the VSMC contractile phenotype., (Copyright © 2021. Published by Elsevier Inc.)

Background & Aims: Regulatory T-cells (Tregs) impair cancer immunosurveillance by creating an immunosuppressive environment that fosters tumor cell survival. Our previous findings demonstrated that neutrophil extracellular traps (NETs), which are involved both in innate and adaptive immunity, are abundant in livers affected by non-alcoholic steatohepatitis (NASH). However, how NETs interact with Tregs in the development of NASH-associated hepatocellular carcinoma (NASH-HCC) is not known., Methods: A choline-deficient, high-fat diet+diethylnitrosamine mouse model and the stelic animal model were utilized for NASH-HCC and a western diet mouse model was used for NASH development. Treg depletion was achieved using FoxP3-DTR mice. RNA sequencing was used to explore the mechanism by which NETs could regulate Treg differentiation. Bioenergetic analyses of naïve CD4 + T-cells were assessed by Seahorse., Results: Although the absolute number of CD4 + T-cells is lower in NASH livers, the Treg subpopulation is selectively increased. Depleting Tregs dramatically inhibits HCC initiation and progression in NASH. There is a positive correlation between increased NET and hepatic Treg levels. RNA sequencing data reveals that NETs impact gene expression profiles in naïve CD4 + T-cells, with the most differentially expressed genes being those involved in mitochondrial oxidative phosphorylation. By facilitating mitochondrial respiration, NETs can promote Treg differentiation. Metabolic reprogramming of naïve CD4 + T-cells by NETs requires toll-like receptor 4. Blockade of NETs in vivo using Pad4 -/- mice or DNase I treatment reduces the activity of Tregs., Conclusions: Tregs can suppress immunosurveillance in the premalignant stages of NASH. NETs facilitate the crosstalk between innate and adaptive immunity in NASH by promoting Treg activity through metabolic reprogramming. Therapies targeting NETs and Treg interactions could offer a potential strategy for preventing HCC in patients with NASH., Lay Summary: Regulatory T-cells (Tregs) can promote tumor development by suppressing cancer immunosurveillance, but their role in carcinogenesis during non-alcoholic steatohepatitis (NASH) progression is unknown. Herein, we discovered that selectively increased intrahepatic Tregs can promote an immunosuppressive environment in NASH livers. Neutrophil extracellular traps (NETs) link innate and adaptive immunity by promoting Treg differentiation via metabolic reprogramming of naïve CD4 + T-cells. This mechanism could be targeted to prevent liver cancer in patients with NASH., Competing Interests: Conflict of interest The authors declare that they have no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021. Published by Elsevier B.V.)

Despite absent expression in normal hematopoiesis, the Forkhead factor FOXC1, a critical mesenchymal differentiation regulator, is highly expressed in ∼30% of HOXA high acute myeloid leukemia (AML) cases to confer blocked monocyte/macrophage differentiation. Through integrated proteomics and bioinformatics, we find that FOXC1 and RUNX1 interact through Forkhead and Runt domains, respectively, and co-occupy primed and active enhancers distributed close to differentiation genes. FOXC1 stabilizes association of RUNX1, HDAC1, and Groucho repressor TLE3 to limit enhancer activity: FOXC1 knockdown induces loss of repressor proteins, gain of CEBPA binding, enhancer acetylation, and upregulation of nearby genes, including KLF2. Furthermore, it triggers genome-wide redistribution of RUNX1, TLE3, and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes, including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping as a feature of leukemia cell differentiation and reveal that FOXC1 prevents this by stabilizing enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex to oncogenic effect., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Emerging evidence indicates that the incidence of nasopharyngeal carcinoma (NPC) remains high in endemic regions despite changing environmental factors, suggesting that genetic traits contribute to its development. Recently, long non-coding RNA-microRNA-messenger RNA (lncRNA-miRNA-mRNA) axis has been reported to be implicated in the pathophysiological processes of malignancies. Moreover, initial bioinformatic analysis revealed a highly expressed lncRNA Forkhead box D3 antisense RNA1 (FOXD3-AS1) for mechanistic network underlying NPC in this present study. Therefore, this study aims to delineate the ability of lncRNA FOXD3-AS1 to influence the NPC progression. The relationship among lncRNA FOXD3-AS1, miR-185-3p, and FOXD3 was identified with bioinformatics prediction, dual-luciferase reporter gene assays, RNA-binding protein immunoprecipitation, and RNA pull-down assays. Furthermore, effects of lncRNA FOXD3-AS1 on malignant phenotypes in vitro, alongside tumor formation in vivo, of transfected NPC stem-like cells were examined with gain- and loss-of-function experiments. Our findings revealed that lncRNA FOXD3-AS1 and FOXD3 exhibited increased expression levels, while miR-185-3p exhibited diminished levels in NPC. The levels of lncRNA FOXD3-AS1 and FOXD3 were further correlated with tumor node metastasis stage and pathological type of patients with NPC. LncRNA FOXD3-AS1 was also confirmed to negatively regulate the miR-185-3p expression, which further targeted the downstream gene FOXD3. In addition, lncRNA FOXD3-AS1 knockdown repressed cell stemness, colony formation, viability, invasion, migration, and in vivo tumor growth, and accelerated cell apoptosis. Moreover, FOXD3 silencing or miR-185-3p overexpression reversed the effects of lncRNA FOXD3-AS1. Our findings provide evidence indicating that lncRNA FOXD3-AS1 could bind to miR-185-3p to upregulate the FOXD3 expression, thereby promoting the development of NPC.

FOXO proteins are transcription factors that are involved in numerous physiological processes and in various pathological conditions, including cardiovascular disease, cancer, diabetes and chronic neurological diseases. For example, FOXO proteins are context-dependent tumour suppressors that are frequently inactivated in human cancers, and FOXO3 is the second most replicated gene associated with extreme human longevity. Therefore, pharmacological manipulation of FOXO proteins is a promising approach to developing therapeutics for cancer and for healthy ageing. In this Review, we overview the role of FOXO proteins in health and disease and discuss the pharmacological approaches to modulate FOXO function.

Genetically modified tumor cells harboring immunomodulators may be used as therapeutic vaccines to stimulate antitumor immunity. The therapeutic benefit of these tumor vaccines is extensively investigated and mechanisms by which they boost antitumor response may be further explored. Tumor cells are large secretors of extracellular vesicles (EVs). These EVs are able to vehiculate RNA and proteins to target cells, and engineered EVs also vehiculate recombinant proteins. In this study, we explore immunomodulatory properties of EVs derived from antitumor vaccines expressing the TNFSF ligands 4-1BBL and OX40L, modulating immune response mediated by immune cells and eliminating tumors. Our results suggest that the EVs secreted by genetically modified tumor cells harboring TNFSF ligands can induce T cell proliferation, inhibit the transcription factor FoxP3, associated with the maintenance of Treg phenotype, and enhance antitumor activity mediated by immune cells. The immunomodulatory extracellular vesicles have potential to be further engineered for developing new approaches for cancer therapy.

The pathogenesis of hepatocellular carcinoma (HCC) is a multistep process, involving the progressive accumulation of molecular alterations and transcriptomic alterations. The Forkhead-box (FOX) transcription factor family is characterized by its unique DNA binding domain (FKH or winged-helix domain). Human FOX family consists of about 17 subfamilies, at least 43 members. Some of them are liver-enriched transcription factors, suggesting that they may play a crucial role in the development or/and functions of the liver. Dysregulation of FOX transcription factors may contribute to the pathogenesis of HCC because they can activate or suppress the expression of various tumor-related molecules, and pinpoint different molecular and cellular events. Here we summarized, analyzed and discussed the status and the functions of the human FOX family of transcription factors in HCC, aiming to help the further development of them as potential therapeutic targets or/and diagnostic/prognostic markers for HCC., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Single-cell RNA-seq (scRNA-seq) is emerging as a powerful tool to dissect cell-specific effects of drug treatment in complex tissues. This application requires high levels of precision, robustness, and quantitative accuracy-beyond those achievable with existing methods for mainly qualitative single-cell analysis. Here, we establish the use of standardized reference cells as spike-in controls for accurate and robust dissection of single-cell drug responses., Results: We find that contamination by cell-free RNA can constitute up to 20% of reads in human primary tissue samples, and we show that the ensuing biases can be removed effectively using a novel bioinformatics algorithm. Applying our method to both human and mouse pancreatic islets treated ex vivo, we obtain an accurate and quantitative assessment of cell-specific drug effects on the transcriptome. We observe that FOXO inhibition induces dedifferentiation of both alpha and beta cells, while artemether treatment upregulates insulin and other beta cell marker genes in a subset of alpha cells. In beta cells, dedifferentiation and insulin repression upon artemether treatment occurs predominantly in mouse but not in human samples., Conclusions: This new method for quantitative, error-correcting, scRNA-seq data normalization using spike-in reference cells helps clarify complex cell-specific effects of pharmacological perturbations with single-cell resolution and high quantitative accuracy.

Background: Forkhead box K1 (FOXK1) is a transcription factor belonging to the Forkhead box (FOX) family and is closely related to the development of various cancers, but the functional mechanism through which FOXK1 regulates autophagy and epithelial-mesenchymal transition (EMT) in the acidic microenvironment of gastric cancer (GC) remains unclear., Results: Our results indicated that the inhibition of FOXK1 induced autophagy and thus exerted antimetastatic effects in an acidic microenvironment. The dual inhibition of mammalian target of rapamycin (mTOR) and FOXK1 enhanced autophagy and reversed EMT of acidic GC cells. In addition, FOXK1 activated transcription in conjunction with the MAZ promoter., Conclusion: Together, our results suggest that FOXK1 can be used as an independent prognostic indicator for GC patients. We also revealed a new strategy involving the cotargeting of FOXK1 and autophagy to reverse the effects of EMT. MAZ is involved in the development and progression of GC as a downstream target of FOXK1., Methods: Here, the cellular responses to the inhibition of FOXK1 in GC were studied in vivo and in vitro through wound healing assays, transwell assays, Western blotting, laser confocal microscopy and transmission electron microscopy. The molecular mechanisms of FOXK1 and Myc-associated zinc finger protein (MAZ) were studied via chromatin immunoprecipitation sequencing (ChIP-seq), bioinformatics, Western blotting, and quantitative real-time PCR (q-PCR).

Transcriptional factors (TFs) play a central role in governing gene expression under physiological conditions including the processes of embryonic development, metabolic homeostasis and response to extracellular stimuli. Conceivably, the aberrant dysregulations of TFs would dominantly result in various human disorders including tumorigenesis, diabetes and neurodegenerative diseases. Serving as the most evolutionarily reserved TFs, Fox family TFs have been explored to exert distinct biological functions in neoplastic development, by manipulating diverse gene expression. Recently, among the Fox family members, the pilot roles of FoxAs attract more attention due to their functions as both pioneer factor and transcriptional factor in human tumorigenesis, particularly in the sex-dimorphism tumors. Therefore, the pathological roles of FoxAs in tumorigenesis have been well-explored in modulating inflammation, immune response and metabolic homeostasis. In this review, we comprehensively summarize the impressive progression of FoxA functional annotation, clinical relevance, upstream regulators and downstream effectors, as well as valuable animal models, and highlight the potential strategies to target FoxAs for cancer therapies., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Getting a grip on how we may age healthily is a central interest of biogerontological research. To this end, a number of academic teams developed platforms for life- and healthspan assessment in Caenorhabditis elegans. These are very appealing for medium- to high throughput screens, but a broader implementation is lacking due to many systems relying on custom scripts for data analysis that others struggle to adopt. Hence, user-friendly recommendations would help to translate raw data into interpretable results. The aim of this communication is to streamline the analysis of data obtained by the WorMotel, an economically and practically appealing screening platform, in order to facilitate the use of this system by interested researchers. We here detail recommendations for the stepwise conversion of raw image data into activity values and explain criteria for assessment of health in C. elegans based on locomotion. Our analysis protocol can easily be adopted by researchers, and all needed scripts and a tutorial are available in S1 and S2 Files., Competing Interests: The authors have declared that no competing interests exist.

The use of autoantigen-specific regulatory T cells (Tregs) as a cellular therapy for autoimmune diseases is appealing. However, it is challenging to isolate and expand large quantity of Tregs expressing disease-relevant T-cell receptors (TCR). To overcome this problem, we used an approach aiming at redirecting the specificity of polyclonal Tregs through autoreactive TCR gene transfer technology. In this study, we examined whether Tregs engineered through retroviral transduction to express a TCR cross-reactive to two CNS autoantigens, myelin oligodendrocyte glycoprotein (MOG) and neurofilament-medium (NF-M), had a superior protective efficacy compared with Tregs expressing a MOG mono-specific TCR. We observed that engineered Tregs (engTregs) exhibited in vitro regulatory effects related to the antigenic specificity of the introduced TCR, and commensurate in potency with the avidity of the transduced TCR. In experimental autoimmune encephalomyelitis (EAE), adoptively transferred engTregs proliferated, and migrated to the CNS, while retaining FoxP3 expression. EngTregs expressing MOG/NF-M cross-reactive TCR had superior protective properties over engTregs expressing MOG-specific TCR in MOG-induced EAE. Remarkably, MOG/NF-M bi-specific TCR-engTregs also improved recovery from EAE induced by an unrelated CNS autoantigen, proteolipid protein (PLP). This study underlines the benefit of using TCRs cross-reacting towards multiple autoantigens, compared with mono-reactive TCR, for the generation of engTregs affording protection from autoimmune disease in adoptive cell therapy., Competing Interests: Declaration of competing interest The authors have declared that no conflict of interest exists., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Previous studies have shown the carcinogenic role of long-chain non-coding RNAs (lncRNA) TRERNA1. However, the role of TRERNA1 in non-small cell lung cancer (NSCLC) has not been reported. This research aims to explore the regulatory effect of TRERNA1/FOXL1 axis on the malignant progression of NSCLC., Patients and Methods: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the expression levels of TRERNA1 and FOXL1 in 39 pairs of tumor tissues and paracancerous ones collected from NSCLC patients. The potential relation between TRERNA1 expression and clinical indicators of NSCLC patients was analyzed. Meanwhile, expression levels of TRERNA1 and FOXL1 in NSCLC cell lines were also detected by qRT-PCR. In addition, TRERNA1 knockdown model was constructed in H358 and SPC-A1 cells. Cell counting kit-8 (CCK-8), cell colony formation assay, and flow cytometry were applied to analyze the influence of TRERNA1 on NSCLC cell biological functions. Finally, Dual-Luciferase reporter gene assay and cell reverse recovery experiments were performed to figure out the underlying mechanisms of TRERNA1 in regulating NSCLC progression., Results: QRT-PCR results indicated that the expression level of lncRNA TRERNA1 in tumor tissue samples of NSCLC patients was remarkably higher than that in adjacent tissues. Compared with NSCLC patients with low expression of TRERNA1, patients with high TRERNA1 expression had a worse pathological stage and overall survival. Similarly, compared with cells in sh-NC group, the proliferation ability of cells in sh-TRERNA1 group was remarkably attenuated. In addition, cell ratio in the G1 phase increased after knockdown of TRERNA1, suggesting the arrested G1/S cell cycle. Subsequently, FOXL1 was downregulated in NSCLC cell lines and tumor tissues. Meanwhile, FOXL1 level was verified to be negatively correlated with TRERNA1 level. Additionally, the binding between TRERNA1 and FOXL1 was confirmed by Dual-Luciferase reporter gene assay. Cell reverse investigation indicated the involvement of FOXL1 in TRERNA1-regulated malignant progression of NSCLC., Conclusions: LncRNA TRERNA1 was up-regulated both in NSCLC tissues and cell lines. Its level was associated with pathological stage and poor prognosis in NSCLC. In addition, lncRNA TRERNA1 could promote the malignant progression of NSCLC via modulating FOXL1.

Programmed cell death 1 (PD-1) is critical for T regulatory cells (Tregs) to maintain peripheral tolerance to self-antigens. In the tumor microenvironment, interaction between PD-1 and its ligands supports tumor immune evasion. Pembrolizumab blocks interactions of PD-1 with its ligands, enhancing antitumor and clinical responses. We and others have reported that pembrolizumab does not affect function or phenotype of thymic-derived Tregs; however, little is known about its effect on extrathymic differentiation of peripheral Tregs. In this study, we investigated the effect of pembrolizumab on in vitro-induced Tregs (iTregs). Our work showed that PD-1 blockade interferes with iTreg differentiation and has no potential effect on the stability of FOXP3 after differentiation. Additionally, we found that both nontreated and pembrolizumab-treated iTregs were suppressive. However, pembrolizumab-treated iTregs were relatively less suppressive in higher Treg ratios and failed to produce IL-10 compared with their nontreated counterparts. Different methods including transcriptomic analyses confirmed that the downregulation of FOXP3 was mediated by activating mTOR and STAT1 and inhibiting MAPK pathways, shifting the iTreg polarization in favor of Th1 and Th17 subsets. To confirm the role of mTOR activation, we found that rapamycin diminished the effect of pembrolizumab-mediated downregulation of FOXP3. Ingenuity pathway analysis revealed that pembrolizumab-treated iTregs showed upregulation of genes promoting DNA repair and immune cell trafficking, in addition to downregulation of genes supporting cellular assembly and organization. To our knowledge, this is the first study to show that pembrolizumab interferes with differentiation of human FOXP3 + iTregs and to disclose some of the molecular pathways involved., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Background/aim: Cytokine-induced killer (CIK) cells are ex vivo expanded major histocompatibility complex (MHC)-unrestricted cytotoxic cells with promising effects against a variety of cancer types. Regulatory T-cells (T-reg) have been shown to reduce the effectiveness of CIK cells against tumor cells. Peptide P60 has been shown to inhibit the immunosuppressive functions of T-regs. This study aimed at examining the effect of p60 on CIK cells efficacy against renal and pancreatic cancer cells., Materials and Methods: The effect of P60 on CIK cytotoxicity was examined using flow cytometry, WST-8-based cell viability assay and interferon γ (IFNγ) ELISA., Results: P60 treatment resulted in a significant decrease in the viability of renal and pancreatic cancer cell lines co-cultured with CIK cells. No increase in IFNγ secretion from CIK cells was detected following treatment with P60. P60 caused no changes in the distribution of major effector cell populations in CIK cell cultures., Conclusion: P60 may potentiate CIK cell cytotoxicity against tumor cells., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

In nematode Caenorhabditis elegans, some microRNAs (miRNAs) could be dysregulated by multi-walled carbon nanotubes (MWCNTs), suggesting their involvement in regulating the response of nematodes to MWCNTs. Among these dysregulated miRNAs induced by MWCNT exposure, prolonged exposure to MWCNTs increased mir-35 expression. mir-35 further acted in the intestine to regulate the response to MWCNTs. In the intestine, a transcription factor MAB-3 was identified as its target in regulating the response to MWCNTs. Moreover, during the control of response to MWCNTs, MAB-3 acted upstream of DAF-16, a fork head transcriptional factor in insulin signaling pathway. Therefore, MWCNTs exposure potentially dysregulates intestinal mir-35 and its direct target MAB-3, which may activate a protective intestinal response of nematodes against the MWCNTs toxicity.

Phosphatidylcholine is one of the major phospholipids comprising cellular membrane and is known to have several health-promoting activities, including the improvement of brain function and liver repair. In this paper, we examine the in vivo effect of dietary supplementation with phosphatidylcholine on the response to environmental stressors and aging in C. elegans . Treatment with phosphatidylcholine significantly increased the survival of worms under oxidative stress conditions. However, there was no significant difference in response to stresses caused by heat shock or ultraviolet irradiation. Oxidative stress is believed to be one of the major causal factors of aging. Then, we examined the effect of phosphatidylcholine on lifespan and age-related physiological changes. Phosphatidylcholine showed a lifespan-extending effect and a reduction in fertility, possibly as a tradeoff for long lifespan. Age-related decline of motility was also significantly delayed by supplementation with phosphatidylcholine. Interestingly, the expressions of well-known longevity-assuring genes, hsp-16.2 and sod-3 , were significantly upregulated by dietary intervention with phosphatidylcholine. DAF-16, a transcription factor modulating stress response genes, was accumulated in the nucleus by phosphatidylcholine treatment. Increase of the ROS level with phosphatidylcholine suggests that the antioxidant and lifespan-extending effects are due to the hormetic effect of phosphatidylcholine. Phosphatidylcholine also showed a protective effect against amyloid beta-induced toxicity in Alzheimer's disease model animals. Experiments with long-lived mutants revealed that the lifespan-extending effect of phosphatidylcholine specifically overlapped with that of reduced insulin/IGF-1-like signaling and required DAF-16. These findings showed the antioxidant and antiaging activities of phosphatidylcholine for the first time in vivo . Further studies focusing on the identification of underlying cellular mechanisms involved in the antiaging effect will increase the possibility of using phosphatidylcholine for the development of antiaging therapeutics.

Nitric oxide (NO) stimulates soluble guanylyl cyclase (sGC) activity, leading to elevated intracellular cyclic guanosine 3',5'-monophosphate (cGMP) and subsequent vascular smooth muscle relaxation. It is known that downregulation of sGC expression attenuates vascular dilation and contributes to the pathogenesis of cardiovascular disease. However, it is not well understood how sGC transcription is regulated. Here, we demonstrate that pharmacological inhibition of Forkhead box subclass O (FoxO) transcription factors using the small-molecule inhibitor AS1842856 significantly blunts sGC α and β mRNA expression by more than 90%. These effects are concentration-dependent and concomitant with greater than 90% reduced expression of the known FoxO transcriptional targets, glucose-6-phosphatase and growth arrest and DNA damage protein 45 α (Gadd45 α ). Similarly, sGC α and sGC β protein expression showed a concentration-dependent downregulation. Consistent with the loss of sGC α and β mRNA and protein expression, pretreatment of vascular smooth muscle cells with the FoxO inhibitor decreased sGC activity measured by cGMP production following stimulation with an NO donor. To determine if FoxO inhibition resulted in a functional impairment in vascular relaxation, we cultured mouse thoracic aortas with the FoxO inhibitor and conducted ex vivo two-pin myography studies. Results showed that aortas have significantly blunted sodium nitroprusside-induced (NO-dependent) vasorelaxation and a 42% decrease in sGC expression after 48-hour FoxO inhibitor treatment. Taken together, these data are the first to identify that FoxO transcription factor activity is necessary for sGC expression and NO-dependent relaxation., (Copyright © 2019 by The Author(s).)

Forkhead box class O6 (FOXO6) is an important member of FOXO family, which has been demonstrated to be implicated in tumor development. However, the role of FOXO6 in colorectal cancer (CRC) is still unclear. The study aimed to investigate the potential roles of FOXO6 in the development of CRC. Our results showed that FOXO6 was overexpressed in CRC tissues and cell lines. FOXO6 knockdown inhibited cell proliferation, as well as repressed the migration and invasion of CRC cells. Additionally, we found that FOXO6 knockdown altered cellular metabolism by inhibiting glycolysis and promoting mitochondrial respiration. Furthermore, FOXO6 knockdown inhibited the activation of PI3K/Akt/mTOR pathway in CRC cells. The results herein indicated that FOXO6 knockdown inhibited cell proliferation, migration, invasion, and glycolysis in CRC cells. PI3K/Akt/mTOR pathway was involved in the effects of FOXO6 on CRC cells. These findings suggested that FOXO6 might be a potential target for the CRC therapy., (© 2018 Wiley Periodicals, Inc.)

Zebrafish gata4/5/6 genes encode transcription factors that lie on the apex of the regulatory hierarchy in primitive myelopoiesis. However, little is known about the roles of microRNAs in gata4/5/6- regulated processes. Performing RNA-Seq deep sequencing analysis of the expression changes of microRNAs in gata4/5/6- knockdown embryos, we identified miR-210-5p as a regulator of zebrafish primitive myelopoiesis. Knocking down gata4/5/6 (generating gata5/6 morphants) significantly increased miR-210-5p expression, whereas gata4/5/6 overexpression greatly reduced its expression. Consistent with inhibited primitive myelopoiesis in the gata5/6 morphants, miR-210-5p overexpression repressed primitive myelopoiesis, indicated by reduced numbers of granulocytes and macrophages. Moreover, knocking out miR-210 partially rescued the defective primitive myelopoiesis in zebrafish gata4/5/6 -knockdown embryos. Furthermore, we show that the restrictive role of miR-210-5p in zebrafish primitive myelopoiesis is due to impaired differentiation of hemangioblast into myeloid progenitor cells. By comparing the set of genes with reduced expression levels in the gata5/6 morphants to the predicted target genes of miR-210-5p, we found that foxj1b and slc3a2a , encoding a forkhead box transcription factor and a solute carrier family 3 protein, respectively, are two direct downstream targets of miR-210-5p that mediate its inhibitory roles in zebrafish primitive myelopoiesis. In summary, our results reveal that miR-210-5p has an important role in the genetic network controlling zebrafish primitive myelopoiesis., (© 2019 Jia et al.)

Genome editing technology greatly facilitates the genetic modification of various cells and animals. The common marmoset (Callithrix jacchus), a small non-human primate which exhibits high reproductive efficiency, is a widely used animal model in biomedical research. Developing genome editing techniques in the common marmoset will further enhance its utility. Here, we report the successful establishment of a knock-in (KI) method for marmoset embryonic stem cells (ESCs), which is based on the CRISPR-Cas9 system. The use of CRISPR-Cas9, mediated by homologous recombination (HR), enhanced the KI efficiency in marmoset ESCs. Furthermore, we succeeded in performing KI in early-stage marmoset embryos. In the course of the experiments, we found that HR in the marmoset ESCs is innately highly efficient. This suggested that the marmoset possesses a repair mechanism for DNA double-strand breaks. The current study will facilitate the generation of genetically modified marmosets and gene function analysis in the marmoset.

Lung cancer is one of the most common cancers in the world, which accounts for about 27% of all cancer deaths. However, the mechanisms underlying the pathogenesis of lung cancer cells remain largely elusive. In this study, we examined the role of the Forkhead box protein P1 (FOXP1) in lung cancer development. Our Oncomine analysis shows that FOXP1 is downregulated in lung adenocarcinoma compared with normal lung tissue. Knockdown of FOXP1 promotes the growth and invasion of PC9 and A549 cells by regulating genes of chemokine signaling molecules, including CCR1, ADCY5, GNG7, VAV3, and PLCB1. Simultaneous knockdown of CCR1 and FOXP1 attenuated FOXP1 knockdown-induced increase of lung cancer cell growth. Finally, knockdown of FOXP1 in PC9 cells promotes the tumorigenesis via CCR1 signaling in xenograft mouse model. Taken together, our data suggest that FOXP1 plays important roles in preventing lung adenocarcinoma development via suppressing chemokine signaling pathways.

Glioma is a type of malignant brain tumor. Forkhead box C1 (FOXC1) is a conserved transcription factor that is involved in tumorigenesis; however, the function of FOXC1 in glioma remains unclear. The present study aimed to investigate the effects of FOXC1 silencing on the epithelial‑to‑mesenchymal transition (EMT) of glioma cells. FOXC1‑specific small interfering RNAs were employed to downregulate the expression levels of FOXC1 in glioma cells. The proliferation, migration and invasion of glioma cells were assessed by MTT assay, wound healing assay and Transwell assay. Western blot analysis was performed to reveal the effects of FOXC1 on EMT‑associated proteins and β‑catenin signaling. The results revealed that, following FOXC1 silencing, the proliferation, migration and invasion of glioma cells were decreased. The expression levels of EMT‑associated proteins were also affected. Further examination demonstrated that β‑catenin signaling was involved in the effects of FOXC1 on glioma cells. Previous results suggested that overexpression of β‑catenin reversed the effects of FOXC1 silencing on glioma cells. The present study demonstrated that FOXC1 may regulate the EMT of glioma cells, potentially via β‑catenin signaling. Therefore, FOXC1 may be a potential therapeutic target for the treatment of glioma.

Aberrant expression of Forkhead box (FOX) transcription factors plays vital roles in carcinogenesis. However, the function of the FOX family member FOXC1 in maintenance of colorectal cancer (CRC) malignancy is unknown. Herein, FOXC1 expression in CRC specimens in The Cancer Genome Atlas (TCGA) cohort was analyzed and validated using immunohistochemistry with a tissue microarray. The effect of FOXC1 expression on proliferation of and glycolysis in CRC cells was assessed by altering its expression in vitro and in vivo. Mechanistic investigation was carried out using cell and molecular biological approaches. Our results showed that FOXC1 expression was higher in CRC specimens than in adjacent benign tissue specimens. Univariate survival analyses of the patients from whom the study specimens were obtained, and validated cohorts indicated that ectopic FOXC1 expression was significantly correlated with shortened survival. Silencing FOXC1 expression in CRC cells inhibited their proliferation and colony formation and decreased their glucose consumption and lactate production. In contrast, FOXC1 overexpression had the opposite effect. Furthermore, increased expression of FOXC1 downregulated that of a key glycolytic enzyme, fructose-1,6-bisphosphatase 1 (FBP1). Mechanistically, FOXC1 bound directly to the promoter regions of the FBP1 gene and negatively regulated its transcriptional activity. Collectively, aberrant FBP1 expression contributed to CRC tumorigenicity, and decreased FBP1 expression coupled with increased FOXC1 expression provided better prognostic information than did FOXC1 expression alone. Therefore, the FOXC1/FBP1 axis induces CRC cell proliferation, reprograms metabolism in CRCs, and constitutes potential prognostic predictors and therapeutic targets for CRC.

Background/aims: Aberrantly expressed miRNAs play a vital role in the development of some cancers, such as human osteosarcoma (OS). However, the detailed molecular mechanisms underlying miR-186-5p-involved osteosarcoma are unclear., Methods: qRT-PCR and western blot analysis were employed to measure the expressions of miR-186-5p and forkhead box k1 (FOXK1). CCK-8 assay evaluated the effect of miR-186-5p and FOXK1 on cell proliferation. Transwell assay confirmed cell migration and invasion. Eventually, the dual-luciferase reporter assay validated 3'-untranslated region (3'-UTR) of FOXK1 as a direct target of miR-186-5p., Results: Down-regulation of miR-186-5p was identified in OS tissues and cell lines, and negatively correlated with distant metastasis, Enneking stage and poor 5-year prognosis as well as the expression of forkhead box k1 (FOXK1) protein. Further assays demonstrated that miR-186-5p overexpression had inhibitory effects on in-vitro cell proliferation, cell cycle, and in-vivo tumor growth. miR-186-5p overexpression also inhibited the epithelial-tomesenchymal transition (EMT), migration and invasion of OS cells. Importantly, miR-186-5p directly targeted FOXK1 3'-UTR and negatively regulated its expression. Silencing of FOXK1 expression enhanced the inhibitory effects of miR-186-5p on OS cell proliferation, migration and invasion., Conclusion: These findings highlighted miR-186-5p as a tumor suppressor in the regulation of progression and metastatic potential of OS, and may benefit the development of therapies targeting miR-186-5p in patients with OS., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Insulin and insulin-like signaling regulates a broad spectrum of growth and metabolic responses to a variety of internal and environmental stimuli. For example, the inhibition of insulin-like signaling in C. elegans mediates its response to both osmotic stress and starvation. We report that in response to osmotic stress the cytosolic sulfotransferase SSU-1 antagonizes insulin-like signaling and promotes developmental arrest. Both SSU-1 and the DAF-16 FOXO transcription factor, which is activated when insulin signaling is low, are needed to drive specific responses to reduced insulin-like signaling. We demonstrate that SSU-1 functions in a single pair of sensory neurons to control intercellular signaling via the nuclear hormone receptor NHR-1 and promote both the specific transcriptional response to osmotic stress and altered lysophosphatidylcholine metabolism. Our results show the requirement of a sulfotransferase-nuclear hormone receptor neurohormonal signaling pathway for some but not all consequences of reduced insulin-like signaling.

Arsenic trioxide (ATO) has been reported to inhibit the activity of Ten-eleven translocation methylcytosine dioxygenase (TET). TET modulates FOXP3 expression, while dysregulation of FOXP3 expression promotes the malignant progression of leukemia cells. We examined the role of TET-FOXP3 axis in the cytotoxic effects of ATO on the human acute myeloid leukemia cell line, U937. ATO-induced apoptosis in U937 cells was characterized by activation of caspase-3/-9, mitochondrial depolarization, and MCL1 downregulation. In addition, ATO-treated U937 cells showed ROS-mediated inhibition of TET2 transcription, leading to downregulation of FOXP3 expression and in turn, suppression of FOXP3-mediated activation of Lyn and Akt. Overexpression of FOXP3 or Lyn minimized the suppressive effect of ATO on Akt activation and MCL1 expression. Promoter luciferase activity and chromatin immunoprecipitation assays revealed the crucial role of Akt-mediated CREB phosphorylation in MCL1 transcription. Further, ATO-induced Akt inactivation promoted GSK3β-mediated degradation of MCL1. Transfection of constitutively active Akt expression abrogated ATO-induced MCL1 downregulation. MCL1 overexpression lessened the ATO-induced depolarization of mitochondrial membrane and increased the viability of ATO-treated cells. Thus, our data suggest that ATO induces mitochondria-mediated apoptosis in U937 cells through its suppressive effect on TET2-FOXP3-Lyn-Akt axis-modulated MCL1 transcription and protein stabilization. Our findings also indicate that the same pathway underlies ATO-induced death in human leukemia HL-60 cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: Cytokine-induced killer cells (CIK) is a type of immune cell with antitumor activity induced by a variety of cytokines. Regulatory T cells (Treg) is a T cell subgroup featured as immunosuppressive function. Existing CIK cultivation system may inevitably induce Treg. Forkhead box protein 3 (Foxp3) is an essential transcription factor for Treg function. This study aimed to investigate the effects of CIK on the leukemia cell HL-60., Materials and Methods: This work silenced Foxp3 expression on the basis of CIK induction, aiming to investigate its killing effect on HL-60 cells. Peripheral blood mononuclear cells were separated and differentiated to CIK in vitro. CD3+CD56+ and CD4+CD25+Foxp3+ Treg cells were detected by flow cytometry. CIK cells were co-cultured with HL-60 cells under the effector-target ratio at 20:1, 10:1, and 5:1, respectively. The killing activity of CIK on HL-60 cells was determined by CCK-8 assay., Results: The ratio of CD3+, CD3+CD8+, and CD3+CD56+ cells gradually increased during CIK induction. Foxp3 interference significantly reduced Treg cell ratio on the 7th day (p < 0.05). Treg cell ratio was significantly lower in Foxp3 interference group at 1.62% ± 0.07% compared with control (p < 0.05). The killing activity of CIK on HL-60 cells enhanced following the increase of effector-target ratio. Interference of Foxp3 significantly elevated the killing activity of CIK on HL-60 cells with effector-target ratio dependence (p < 0.05). CIK can effectively suppress HL-60 cell growth. Treg significantly inhibited the anti-tumor effect of CIK., Conclusions: Interference of Foxp3 expression significantly declined Treg level and attenuated its suppression impact on CIK, thus enhancing the killing effect of CIK on HL-60 cells.

Objectives: Hepatic insulin resistance is a hallmark of type 2 diabetes and obesity. Insulin receptor signaling through AKT and FOXO has important metabolic effects that have traditionally been ascribed to regulation of gene expression. However, whether all the metabolic effects of FOXO arise from its regulation of protein-encoding mRNAs is unknown., Methods: To address this question, we obtained expression profiles of FOXO-regulated murine hepatic microRNAs (miRNAs) during fasting and refeeding using mice lacking Foxo1, 3a, and 4 in liver (L-Foxo1,3a, 4)., Results: Out of 439 miRNA analyzed, 175 were differentially expressed in Foxo knockouts. Their functions were associated with insulin, Wnt, Mapk signaling, and aging. Among them, we report a striking increase of miR-205-5p expression in L-Foxo1,3a,4 knockouts, as well as in obese mice. We show that miR-205-5p gain-of-function increases AKT phosphorylation and decreases SHIP2 in primary hepatocytes, resulting in FOXO inhibition. This results in decreased hepatocyte glucose production. Consistent with these observations, miR-205-5p gain-of-function in mice lowered glucose levels and improved pyruvate tolerance., Conclusions: These findings reveal a homeostatic miRNA loop regulating insulin signaling, with potential implications for in vivo glucose metabolism., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Diabetes is caused by combined abnormalities in insulin production and action. The pathophysiology of these defects has been studied extensively and is reasonably well understood. Their causes are elusive and their manifestations pleiotropic, likely reflecting the triple threat of genes, environment, and lifestyle. Treatment, once restricted to monotherapy with secretagogues or insulin, now involves complex combinations of expensive regimens that stem the progression but do not fundamentally alter the underlying causes of the disease. As advances in our understanding of insulin action and β-cell failure reach a critical stage, here I draw on lessons learned from our research on insulin regulation of gene expression and pancreatic β-cell dedifferentiation to address the question of how we can translate this exciting biology into mechanism-based interventions to reverse the course of diabetes., (© 2018 by the American Diabetes Association.)

Aberrant expression of FOXN2, a member of the Forkhead box transcription factors, has been found in several types of cancer. However, the underlying mechanisms of FOXN2 deregulation in tumorigenesis remain largely unknown. Here, we find that FOXN2 binds to and is ubiquitinated by β-Trcp ubiquitin ligase and RSK2 kinase for degradation. Furthermore, we demonstrate that the Ser365 and Ser369 sites in a conserved DSGYAS motif are critical for the degradation of FOXN2 by β-Trcp and RSK2. Moreover, gain-of-function and loss-of-function studies show that FOXN2 impairs cell proliferation in vitro and in vivo and enhances the radiosensitivity of lung cancer. Importantly, β-Trcp-mediated and RSK2-mediated degradation of FOXN2 promotes tumorigenesis and radioresistance in lung cancer cells. Collectively, our study reveals a novel post-translational modification of FOXN2 and suggests that FOXN2 may be a potential therapeutic and radiosensitization target for lung cancer.

The epithelial-mesenchymal transition (EMT) has been suggested as a new target for therapeutic intervention of metastatic cancer. Forkhead box protein C2 (FOXC2) is known to be necessary for initiating and maintaining EMT, and therefore bestows on cancer cells metastatic and cancer stem cell (CSC)-like phenotypes, allowing cells to acquire higher motility, invasiveness, self-renewal, and therapy resistance. Here, we describe the first inhibitor of FOXC2, MC-1-F2. MC-1-F2 was able to induce cadherin switching and reverse EMT through the degradation of FOXC2 and blocking of its nuclear localization. In addition, MC-1-F2 was very effective in inhibiting cancer cell migration and invasion. As the first small-molecule inhibitor of FOXC2 and the first compound targeting EMT-associated transcription factor, MC-1-F2 will pave the way for a new anticancer therapeutic agent targeting metastatic cancer and help to elucidate the network of EMT signaling pathways., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Increasing evidence has demonstrated that aberrant forkhead box protein C1 (FOXC1) expression contributes to tumorigenesis in multiple types of malignant tumor. However, the clinical significance and biological roles of FOXC1 in cervical cancer remain unknown. The expression levels of FOXC1 were examined in human cervical cancer tissues and cells using reverse transcription‑quantitative polymerase chain reaction, immunohistochemistry and western blotting. Furthermore, high FOXC1 expression was significantly associated with advanced clinical stages, a high degree of malignancy and a poor outcome. FOXC1 silencing inhibited cell growth and enhanced cell apoptosis. Knockdown of FOXC1 markedly suppressed cell migration and invasion in vitro, and resulted in downregulation of phosphorylated‑RAC‑α serine/threonine‑protein kinase, proto‑oncogene c‑Myc and B‑cell lymphoma 2. In conclusion, these data indicated that upregulation of FOXC1 contributed to the development of cervical cancer by increasing the growth and motility of the cervical cancer cells, thereby worsening the disease progression in these patients.

Objective: This study aims to identify serum microRNAs (miRNAs) related to ovarian cancer., Study Design: MiRNA profiling data (GSE79943) were generated from the Gene Expression Omnibus, including 3 serum samples from healthy individuals and 4/3/16/6 serum samples from patients with ovarian cancer stage I/II/III/IV. Differentially expressed miRNAs (DEmiRNAs) were identified between controls and ovarian cancer stage I/II/III/IV by using limma package (p-value <0.05 and |log 2 fold change| ≥0.5). miRWALK2.0 database was used to find experiment-validated targets of DEmiRNAs, and CTD database was utilized to screen known genes related to ovarian cancer. clusterProfiler package was used to perform pathway enrichment analysis of DEmiRNAs. Targets of DEmiRNAs were validated by using GSE40595, involving 8 normal ovarian stroma, 31 ovarian cancer stroma, 6 human ovarian surface epthelium, and 32 ovarian tumor epthelial component., Results: Between stage I/II/III/IV and control, 39/143/29/39 DEmiRNAs were identified, which were regarded as key miRNAs. Between 4 DEmiRNA sets, 15 common DEmiRNAs were identified (e.g. up-regulated hsa-miR-1181 and hsa-miR-4314). Hsa-miR-1181 participated in "Jak-STAT signaling pathway" and "miRNAs in cancer"; hsa-miR-4314 took part in cancer-related pathways. STAT3 and KRAS, known marker genes of ovarian cancer, were targeted by hsa-miR-1181 and hsa-miR-4314, respectively. Besides, FOXP1 was targeted by hsa-miR-1181; FOXP1-AS1 and FOXP1-IT1 were down-regulated in ovarian cancer. GRWD1, IP6K1, and NEGR1 were targeted by hsa-miR-4314; GRWD1, IP6K1, and NEGR1 were down-regulated in ovarian tumor., Conclusion: MiR-1181 and miR-4314 might promote ovarian tumorigenesis via down-regulating FOXP1 and GRWD1/IP6K1/NEGR1, respectively. In addition, the 15 common DEmiRNAs might provide directions for ovarian cancer diagnosis., (Copyright © 2018. Published by Elsevier B.V.)

Objective: To identify the functioning mode of miR-378 on non-small cell lung cancer (NSCLC) and provide therapeutic targets for NSCLC., Patients and Methods: Expression levels of miR-378 in human NSCLC tissue samples and NSCLC-derived cell lines were measured by using quantitative Real-time polymerase chain reaction (PCR). Cell proliferation capacity was assessed by methyl thiazolyl tetrazolium (MTT) assay and colony formation assay. Cell apoptosis and cell cycle distribution were identified by flow cytometry. Downstream target gene was confirmed by using luciferase and Western blotting assays., Results: MiR-378 was significantly elevated in NSCLC tissues when compared with para-carcinoma tissues (n=42). Decreased-miR-378 could attenuate cell proliferation capacity, as well as promoted cell apoptosis and induced cell cycle arrest at G0/G1 phase. FOXG1 was chosen as the target gene of miR-378 by bioinformatics analysis and luciferase reporter assay. Moreover, restoration of miR-378 could impair the tumor suppression role of downregulated-miR-378 on NSCLC growth., Conclusions: Decreased-miR-378 exerted tumor-suppressive effects on NSCLC growth via targeting FOXG1 in vitro, which provided an innovative and candidate target for diagnosis and treatment of NSCLC.

Background/aims: The etiology of asthma, which is a complicated disorder with various symptoms, including wheezing, coughing, and airflow obstruction, remains poorly understood. In addition, the effects of microRNAs (miRs) have not been explored. This study explored the effect of microRNA-200a (miR-200a) on airway smooth muscle cells (ASMCs) and airway remodeling in asthmatic mice. Furthermore, we speculated that miR-200a achieves its effect by targeting FOXC1 via the PI3K/AKT signaling pathway based on differentially expressed gene screening, target miR predictions and a bioinformatics analysis., Methods: Eighty mice were assigned to a saline group or an ovalbumin (OVA) group, and the OVA group was transfected with a series of inhibitors, activators, and siRNAs to test the established mouse model. Airway reactivity and the ratio of eosinophils (EOSs) to leukocytes were detected. An ELISA was adopted to measure the levels of interleukin (IL)-4, IL-6, IL-8, tumor necrosis factor (TNF)-α, and immunoglobulin E (IgE). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to determine the expression of FOXC1, PI3K, AKT, NF-κB, cyclin D1, TGF-β1 and p-AKT in ASMCs. Finally, CCK-8 assays were performed to detect cell proliferation and flow cytometry to detect apoptosis and cell cycle entry., Results: The bioinformatics analysis indicated that miR-200a mediated the PI3K/AKT signaling pathway by targeting FOXC1. In addition, mouse models of asthma were established. An elevated expression of miR-200a, a decreased mRNA and protein expression of FOXC1, PI3K, AKT, NF-κB, cyclin D1 and TGF-β1, a decreased expression of p-AKT, suppressed cell proliferation, accelerated apoptosis, and an increased number of cells at the G0/G1 phase were observed following the upregulation of miR-200a and downregulation of FOXC1., Conclusion: The overexpression of miR-200a may downregulate FOXC1, thereby inhibiting the activation of the PI3K/AKT signaling pathway and ultimately suppressing ASMC proliferation and airway remodeling in asthmatic mice. This evidence supports the potential that miR-200a represents a new approach to treating asthma., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The FOXO (Forkhead box O) transcription factors are implicated in several signaling pathways and play a vital role in various cellular and physiological processes include for instance, ROS (reactive oxygen species) response, cell proliferation, regulation of programmed cell death, longevity, metabolism and cancer and regulation of cell cycle. In humans, the four FOXO family members are responsible for resemblance in their structure, regulation and functions. FOXO1 gene is highly expressed in adipose tissues and it affects the regulation of glycogenolysis and gluconeogenesis through insulin signaling. The gene of FOXO3 is highly expressed in the kidney, heart, spleen and brain and is characterized as diverse forkhead DNA-binding domain of transcription factors. The FOXO3 is a tumor suppressor gene and found to interact with p53, the trigger for apoptosis through BCl2 family genes and a regulator of Notch signaling pathway for the self-renewal of stem cells. Therefore, FOXOs remains to be a fascinating and potential target to acquire novel therapeutic approaches to cure cancer. This review will provide a comprehensive overview about the biology of FOXO proteins, which can be utilized for developing current therapeutic approaches to treat cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background/aims: Metastasis is the primary cause of colorectal cancer (CRC)-related death. However, the molecular mechanisms underlying metastasis in CRC remain unclear., Methods: We evaluated mRNA and protein expression levels by quantitative real-time reverse transcription PCR, western blotting, immunofluorescence, tissue microarrays, and immunohistochemistry assays. We also assessed the migration and invasion abilities of CRC cells in vitro by wound healing assays, invasion and migration assays, western blot analysis, and immunofluorescence. Tumor metastasis was evaluated in nude mice in vivo., Results: A positive correlation was observed between the expression patterns of Forkhead box k1 (FOXK1) and Snail in CRC. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that Snail directly bound to and activated the human FOXK1 gene promoter. Moreover, the Snail-FOXK1 axis promote epithelial mesenchymal transition (EMT)-mediated CRC cell invasion and metastasis. FOXK1 and Snail expression levels were correlated with tumor progression and served as significant predictors of overall survival in patients with CRC. Furthermore, overexpression of FOXK1 induced the EMT by upregulating the expression of cysteine-rich angiogenic inducer 61 (Cyr61). Luciferase assays showed that Cyr61 was a direct transcriptional target of FOXK1. Down regulation of Cyr61 decreased FOXK1-enhanced "CRC cell" migration, invasion, and metastasis. Additionally, FOXK1 expression was positively correlated with Cyr61 expression and was associated with poor prognosis., Conclusions: The Snail/FOXK1/Cyr61 signaling axis regulates the EMT and metastasis of CRC., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Forkhead box k1 (FOXK1) is a member of the FOX class of transcription factors and it is dysregulated in many solid tumors including hepatocellular carcinoma, gastric cancer, colorectal cancer and prostate cancer. However, the expression status of FOXK1 and its clinical significance in esophageal cancer (EC) is still uncertain. Our study aimed at investigating the significance of FOXK1 expression in human EC and its biological function in the development of EC. We found that FOXK1 was overexpressed in EC tissues compared with corresponding non-tumor tissues using immunohistochemistry. And high FOXK1 expression was related to poor differentiation of EC. The Kaplan-Meier curve indicated that high FOXK1 expression may result in poor prognosis of EC patients. Furthermore, overexpression of FOXK1 in EC9706 cell inhibited cell apoptosis and promoted cell proliferation and migration, and suppression of FOXK1 in EC109 cell obtained reverse results. Our data suggest that FOXK1 plays an oncogenic role in EC pathogenesis and can serve as a therapeutic target for patients with EC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: Regulatory T cells (Tregs) impair the clinical benefit of cancer immunotherapy. To optimize the antitumor efficacy of therapeutic dendritic cell (DC) vaccines, we aimed to inhibit Foxp3, a transcription factor required for Treg function., Methods: Mice bearing established syngeneic LM3 and 4T1 breast tumors were treated with antitumor DC vaccines and a synthetic peptide (P60) that has been shown to inhibit Foxp3., Results: Treatment with P60 improved the therapeutic efficacy of DC vaccines in these experimental models. In addition, monotherapy with P60 inhibited tumor growth in immunocompetent as well as in immuno-compromised animals bearing established tumors. We found expression of Foxp3 in human and murine breast tumor cells. P60 inhibited IL-10 secretion in breast cancer cells that expressed Foxp3., Conclusions: Our results suggest that Foxp3 blockade improves the therapeutic efficacy of DC vaccines by inhibition of Tregs and through a direct antitumor effect. This strategy could prove useful to neutralize the immunosuppressive microenvironment and to boost antitumor immunity in breast cancer.

Acute onset of organ failure in heatstroke is triggered by rhabdomyolysis of skeletal muscle. Here, we showed that elevated temperature increases free cytosolic Ca 2+ [Ca 2+ ]f from RYR (ryanodine receptor)/UNC-68 in vivo in the muscles of an experimental model animal, the nematode Caenorhabditis elegans This subsequently leads to mitochondrial fragmentation and dysfunction, and breakdown of myofilaments similar to rhabdomyolysis. In addition, treatment with an inhibitor of RYR (dantrolene) or activation of FoxO (Forkhead box O)/DAF-16 is effective against heat-induced muscle damage. Acute onset of organ failure in heatstroke is triggered by rhabdomyolysis of skeletal muscle. To gain insight into heat-induced muscle breakdown, we investigated alterations of Ca 2+ homeostasis and mitochondrial morphology in vivo in body-wall muscles of C. elegans exposed to elevated temperature. Heat stress for 3 hr at 35° increased the concentration of [Ca 2+ ]f, and led to mitochondrial fragmentation and subsequent dysfunction in the muscle cells. A similar mitochondrial fragmentation phenotype is induced in the absence of heat stress by treatment with a calcium ionophore, ionomycin. Mutation of the unc-68 gene, which encodes the ryanodine receptor that is linked to Ca 2+ release from the sarcoplasmic reticulum, could suppress the mitochondrial dysfunction, muscle degeneration, and reduced mobility and life span induced by heat stress. In addition, in a daf-2 mutant, in which the DAF-16/FoxO transcription factor is activated, resistance to calcium overload, mitochondrial fragmentation, and dysfunction was observed. These findings reveal that heat-induced Ca 2+ accumulation causes mitochondrial damage and consequently induces muscle breakdown., (Copyright © 2017 Momma et al.)

Background: The histone deacetylase (HDAC) inhibitor, which has potential effects on epigenetic modifications, had been reported to attenuate renal fibrosis. CD4 + forkhead box P3 (FOXP3) + T regulatory (Treg) cells may be converted to inflammation-associated T helper 17 cells (Th17) with tissue fibrosis properties. The association between FOXP3 + IL-17 + T cells and the attenuation of renal fibrosis by the HDAC inhibitor is not clear., Methods: This study evaluated the roles of the HDAC inhibitor, Treg cells and their differentiation into Th17 cells, which aggravate chronic inflammation and renal fibrosis in a unilateral ureteral obstruction (UUO) mouse model. The study groups included control and UUO mice that were monitored for 7, 14 or 21 days., Results: Juxtaglomerular (JG) hyperplasia, angiotensin II type 1 receptor (AT1R) expression and lymphocyte infiltration were observed in renal tissues after UUO but were decreased after trichostatin A (TSA) treatment, a HDAC inhibitor. The number of CD4 + FOXP3 + T cells increased progressively, along with the number of FOXP3 + interleukin (IL)-17 + T cells, after 14 days, and their numbers then progressively decreased with increasing CD4 + IL-17 + T cell numbers, as demonstrated by double immunohistochemistry. Progressive renal fibrosis was associated with the loss of CD4 + FOXP3 + IL-17 + T cells in splenic single-cell suspensions. FOXP3 + IL-17 + T cells expressed TGF-β1 both in vitro and in vivo, and TGF-β1 expression was significantly knockdown by IL-17 siRNA in vitro. These cells were found to play a role in converting Tregs into IL-17- and TGF-β1-producing cells., Conclusions: TSA treatment decreased JG hyperplasia, the percentage of FOXP3 + IL-17 + cells and the degree of fibrosis, suggesting that therapeutic benefits may result from epigenetic modifications.

In ruminants, Interferon tau (IFNT) is the pregnancy recognition protein produced by the mononuclear trophectoderm of the conceptus, and is secreted into the uterine lumen during the peri-attachment period. In our previous study, the high-throughput RNA sequencing (RNA-seq) data obtained from bovine conceptuses during the peri-attachment period identified two IFNT mRNAs, IFNT2 and IFNTc1. However, how each of these IFNT variants regulates endometrial gene expression has not been characterized. Using RNA-seq analysis, we evaluated how IFNT2 and IFNTc1 affected transcript expression in primary bovine endometrial epithelial cells (EECs). IFNT treatment induced 348 differentially expressed genes (DEGs); however, there are few DEGs in IFNT2 or IFNTc1 treated EECs, indicating that IFNT2-induced DEGs were similar to those induced by IFNTc1 treatment. In in silico analysis, we identified four IFNT2- and IFNTc1-induced pathways: 1) type II interferon signaling, 2) proteasome degradation, 3) type III interferon signaling, and 4) DNA damage response. We further demonstrated that IFNT2 and IFNTc1 up-regulated several transcription factors, among which forkhead box S1 (FOXS1) was identified as the most highly expressed gene. Furthermore, the knockdown of FOXS1 in IFNT2- or IFNTc1-treated EECs similarly down-regulated 9 genes including IRF3 and IRF9, and up-regulated 9 genes including STAT1, STAT2, and IRF8. These represent the first demonstration that effects of each IFNT on EECs were studied, and suggest that endometrial response as well as signaling mechanisms were similar between two IFNT variants existed in utero.

Background and Objectives: MicroRNAs are small non-coding RNAs involved in pathogenesis and progression of human malignancies. MicroRNA-491-5p (miR-491-5p) is down-regulated in many human cancers where it would serve as a tumour suppressor. However, the role played by miR-491-5p in pathogenesis of human osteosarcoma has remained largely unknown. This study has been conducted to examine effects of miR-491-5p on migration and proliferation of cells of the SAOS-2 and MG63 osteosarcoma lines, and mechanisms of those effects., Materials and Methods: Levels of miR-491-5p expression in osteosarcoma tissues and in human osteosarcoma cell lines were studied using qualitative real-time polymerase chain reaction (qRT-PCR) methods. Cell viability was detected using the CCK-8 and EdU assays, while the transwell assay was used to evaluate migration and invasion. Apoptosis was analysed uing flow cytometry and the Hoechst 33342 nuclear staining method. A dual-luciferase reporter system was used to confirm the target gene of miR-491-5p. The electrophoretic mobility shift assay (EMSA) with DIG-labelled double-stranded FOXP4 oligonucleotides was used to confirm whether or not miR-491-5p suppressed FOXP4 activation., Results: Cells of osteosarcoma tissues and cell lines had low levels of miR-491-5p expression, but high levels of forkhead-box P4 (FOXP4) expression. Transfection of SAOS-2 and MG63 cells with miR-491-5p mimics inhibited expression of FOXP4 protein, which suppressed cell growth and migration, but induced apoptosis. Dual-luciferase reporter assays confirmed FOXP4 as the target gene for miR-491-5p. Overexpression of miR-491-5p suppressed FOXP4 activity in SAOS-2 and MG63 cells. Knockdown of FOXP4 in SAOS-2 and MG63 cells using an RNAi strategy resulted in reduced levels of cell proliferation and migration, but increased levels of apoptosis., Conclusion: Our in vitro studies showed that up-regulation of miR-491-5p suppressed proliferation of the human osteosarcoma cells and induced apoptosis by targeting FOXP4. These findings suggest that miR-491-5p could be further studied as a potential clinical diagnostic or predictive biomarker for human osteosarcoma., (© 2016 John Wiley & Sons Ltd.)

A locus on chromosome 9q22 harbors a SNP (rs965513) firmly associated with risk of papillary thyroid carcinoma (PTC). The locus also comprises the forkhead box E1 (FOXE1) gene, which is implicated in thyroid development, and a long noncoding RNA (lncRNA) gene, papillary thyroid cancer susceptibility candidate 2 (PTCSC2). How these might interact is not known. Here we report that PTCSC2 binds myosin-9 (MYH9). In a bidirectional promoter shared by FOXE1 and PTCSC2, MYH9 inhibits the promoter activity in both directions. This inhibition can be reversed by PTCSC2, which acts as a suppressor. RNA knockdown of FOXE1 in primary thyroid cells profoundly interferes with the p53 pathway. We propose that the interaction between the lncRNA, its binding protein MYH9, and the coding gene FOXE1 underlies the predisposition to PTC triggered by rs965513., Competing Interests: The authors declare no conflict of interest.

A disruption of the crucial balance between regulatory T-cells (Tregs) and Th17-cells was recently implicated in various autoimmune disorders. Tregs are responsible for the maintenance of self-tolerance, thus inhibiting autoimmunity, whereas pro-inflammatory Th17-cells contribute to the induction and propagation of inflammation. Distortion of the Th17/Treg balance favoring the  pro-inflammatory Th17 side is hence suspected to contribute to exacerbation of autoimmune disorders. This review aims to summarize recent data and advances in targeted therapeutic modification of the Th17/Treg-balance, as well as information on the efficacy of candidate therapeutics with respect to the treatment of autoimmune diseases.