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Objective To explore the role and mechanism of RNA demethylase fat mass and obesity-associated protein (FTO) in the ferroptosis in testicular interstitial cells induced by di (2-ethylhexyl) phthalate (DEHP). Methods Forty 3-week-old C57BL/6 male mice were randomly divided into a control group (corn oil) and 3 dosed DEHP treatment groups (5, 250 and 500 mg/kg), and received an intragastric infusion of corresponding agents for 35 d, respectively. After mouse testicular interstitial TM3 cells was treated with 0, 100, 200 and 400 μmol/L mono-2-ethylhexyl phthalate (MEHP) for 24 h, corresponding plasmids were transfected to construct Fto overexpressing TM3 cells. Serum testosterone level was detected by ELISA, expression of testicular proteins was detected with immunohistochemical assay, and contents of Fe2+, malondialdehyde (MDA) and lipid peroxides in the testicle were detected by colorimetry. Methylated RNA immunoprecipitation, RT-PCR, and Western blotting were used to detect the level of N6-methyladenosine (m6A) modification. Results In the mice exposed to 250 and 500 mg/kg DEHP, the serum testosterone level was significantly reduced (P < 0.01), contents of Fe2+, MAD and lipid peroxides in testicular tissue were obviously increased (P < 0.01), and protein levels of RNA demethylase FTO, and ferroptosis related molecules ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) were significantly down-regulated (P < 0.05), while those of transferrin receptor (TFRC), ferroportin (FPN), cyclooxygenase-2 (COX-2), and acyl-CoA synthetase long-chain family member 4 (ACSL4) were notably up-regulated (P < 0.05). MEHP treatment for 24 h resulted in remarkably decreased cell viability in the TM3 cells, increased production of intracellular reactive oxygen species (ROS), reduced mitochondrial membrane potential (MMP) (P < 0.01), down-regulated mRNA and protein levels of Fto (P < 0.01), and the changes in other ferroptosis related proteins were consistent with the trend in testicular tissue, indicating ferroptosis in testicular interstitial cells. Intervention with ferroptosis inhibitor Fer-1 or overexpression of Fto significantly inhibited MEHP-induced toxicity and ferroptosis in TM3 cells (P < 0.05), and overexpression of Fto reduced the m6A modification of Gpx4 and Fth1 mRNA (P < 0.05). Conclusion Abnormal m6A modification of Gpx4 and Fth1 caused by inhibiting FTO expression may be the mechanism of ferroptosis in testicular interstitial cells induced by DEHP.

Odontoblastic differentiation of human stem cells from the apical papilla (hSCAPs) is crucial for continued root development and dentin formation in immature teeth with apical periodontitis (AP). Fat mass and obesity-associated protein (FTO) has been reported to regulate bone regeneration and osteogenic differentiation profoundly. However, the effect of FTO on hSCAPs remains unknown. This study aimed to identify the potential function of FTO in hSCAPs' odontoblastic differentiation under normal and inflammatory conditions and to investigate its underlying mechanism preliminarily. Histological staining and micro-computed tomography were used to evaluate root development and FTO expression in SD rats with induced AP. The odontoblastic differentiation ability of hSCAPs was assessed via alkaline phosphatase and alizarin red S staining, qRT-PCR, and Western blotting. Gain- and loss-of-function assays and online bioinformatics tools were conducted to explore the function of FTO and its potential mechanism in modulating hSCAPs differentiation. Significantly downregulated FTO expression and root developmental defects were observed in rats with AP. FTO expression notably increased during in vitro odontoblastic differentiation of hSCAPs, while lipopolysaccharide (LPS) inhibited FTO expression and odontoblastic differentiation. Knockdown of FTO impaired odontoblastic differentiation, whereas FTO overexpression alleviated the inhibitory effects of LPS on differentiation. Furthermore, FTO promoted the expression of secreted modular calcium-binding protein 2 (SMOC2), and the knockdown of SMOC2 in hSCAPs partially attenuated the promotion of odontoblastic differentiation mediated by FTO overexpression under LPS-induced inflammation. This study revealed that FTO positively regulates the odontoblastic differentiation ability of hSCAPs by promoting SMOC2 expression. Furthermore, LPS-induced inflammation compromises the odontoblastic differentiation of hSCAPs by downregulating FTO, highlighting the promising role of FTO in regulating hSCAPs differentiation under the inflammatory microenvironment. [ABSTRACT FROM AUTHOR]

Aim: Rheumatoid arthritis (RA) inflicts chronic joint inflammation, leading to cartilage damage and potential disability. This study delves into the impact of fat mass and obesity‐associated protein (FTO) on the invasion and migration of fibroblast‐like synoviocytes (FLSs) in RA, aiming to identify a novel target for RA treatment. Methods: RA‐FLSs and control‐FLSs from RA patients and healthy controls were isolated and cultured. An FTO overexpression vector was constructed and transfected into RA‐FLSs, with subsequent measurement of FTO, miR‐126‐5p, and FOXO3 expression levels using quantitative real‐time polymerase chain reaction or western blot assays. The proliferation, invasion, and migration capabilities of RA‐FLSs were assessed. The total m6A modification of cellular mRNA was quantified. The interaction between m6A and DiGeorge syndrome critical region 8 (DGCR8) on pri‐miR‐126‐5p was evaluated using methylated RNA immunoprecipitation. The binding relationship between miR‐126‐5p and FOXO3 3′UTR sequence was verified. Results: FTO was downregulated in RA‐FLSs. FTO overexpression inhibited the invasion and migration of RA‐FLSs. FTO reduced the m6A modification level of pri‐miR‐126‐5p in RA‐FLSs, inhibiting miR‐126‐5p expression by reducing the binding of DGCR8 to pri‐miR‐126‐5p. miR‐126‐5p targeted and inhibited FOXO3 expression. Overexpression of miR‐126‐5p or FOXO3 knockdown could partially reverse FTO overexpression's inhibitory effect on the invasion and migration of RA‐FLSs. Conclusion: FTO overexpression curtailed miR‐126‐5p by diminishing the m6A modification level of pri‐miR‐126‐5p, subsequently enhancing FOXO3 expression and restraining the invasion and migration of RA‐FLSs. [ABSTRACT FROM AUTHOR]

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Acute kidney injury (AKI) is a common disorder without effective therapy yet. Renal ischemia/reperfusion (I/R) injury is a common cause of AKI. MicroRNA miR-192-5p has been previously reported to be upregulated in AKI models. However, its functional role in renal I/R injury is not fully understood. This study aimed to investigate the effects and the underlying mechanism of miR-192-5p in renal I/R progression. Hypoxia/reoxygenation (H/R)-induced cell injury model in HK-2 cells and I/R-induced renal injury model in mice were established in this study. Cell counting kit-8 assay was performed to determine cell viability. Quantitative real-time PCR and western blot analysis were performed to detect gene expressions. Hematoxylin-eosin and periodic acid-Schiff staining were performed to observe the histopathological changes. Enzyme-linked immunosorbent assay was performed to detect the kidney markers' expression. In vivo and in vitro results showed that miR-192-5p was up-regulated in the I/R-induced mice model and H/R-induced cell model, and miR-192-5p overexpression exacerbated I/R-induced renal damage. Then, the downstream target of miR-192-5p was analyzed by combining the differentially expressed mRNAs and the predicted genes and confirmed using a dual-luciferase reporter assay. It was found that miR-192-5p was found to regulate fat mass and obesity-associated (FTO) protein expression by directly targeting the 3' untranslated region of FTO mRNA. Moreover, in vivo and in vitro studies unveiled that FTO overexpression alleviated renal I/R injury and promoted HK-2 cell viability via stimulating autophagy flux. In conclusion, miR-192-5p aggravated I/R-induced renal injury by blocking autophagy flux via down-regulating FTO. [ABSTRACT FROM AUTHOR]

Uveitis, a vision-threatening inflammatory disease worldwide, is closely related to resident microglia. Retinal microglia are the main immune effector cells with strong plasticity, but their role in uveitis remains unclear. N6-methyladenosine (m6A) modification has been proven to be involved in the immune response. Therefore, we in this work aimed to identify the potentially crucial m6A regulators of microglia in uveitis. Through the single-cell sequencing (scRNA-seq) analysis and experimental verification, we found a significant decrease in the expression of fat mass and obesity-associated protein (FTO) in retinal microglia of uveitis mice and human microglia clone 3 (HMC3) cells with inflammation. Additionally, FTO knockdown was found to aggravate the secretion of inflammatory factors and the mobility/chemotaxis of microglia. Mechanistically, the RNA-seq data and rescue experiments showed that glypican 4 (GPC4) was the target of FTO, which regulated microglial inflammation mediated by the TLR4/NF-κB pathway. Moreover, RNA stability assays indicated that GPC4 upregulation was mainly regulated by the downregulation of the m6A “reader” YTH domain family protein 3 (YTHDF3). Finally, the FTO inhibitor FB23-2 further exacerbated experimental autoimmune uveitis (EAU) inflammation by promoting the GPC4/TLR4/NF-κB signaling axis, and this could be attenuated by the TLR4 inhibitor TAK-242. Collectively, a decreased FTO could facilitate microglial inflammation in EAU, suggesting that the restoration or activation of FTO function may be a potential therapeutic strategy for uveitis.

BACKGROUND: Glycogen synthase kinase-3ß (GSK3β), fat mass and obesity-associated protein (FTO), and toll-like receptors 4 (TLR4) take on critical significance in different biological processes, whereas their interactions remain unclear. The objective was the investigation of the interaction effect in cerebral ischemia-reperfusion (I/R) injury. METHODS: The function of the cerebral cortex in the mouse middle cerebral artery occlusion (MCAO) model (each group n = 6) and P12 cells oxygen-glucose deprivation/reoxygenation (OGD/R) model was analyzed using short hairpin GSK3β lentivirus and overexpression of FTO lentivirus (in vitro), TLR4 inhibitor (TAK242), and LiCl to regulate GSK3β, FTO, TLR4 expression, and GSK3β activity, respectively. RESULTS: After GSK3β knockdown in the OGD/R model of PC12 cells, the levels of TLR4 and p-p65 were lower than in the control, and the level of FTO was higher than in the control. Knockdown GSK3β reversed the OGD/R-induced nuclear factor kappa-B transfer to the intranuclear nuclei. As indicated by the result, TLR4 expression was down-regulated by overexpressed FTO, and TLR4 expression was up-regulated notably after inhibition of FTO with the use of R-2HG. After the inhibition of the activity of GSK3β in vivo, the reduction of FTO in mice suffering from MCAO was reversed. CONCLUSIONS: Our research shows that GSK3β/FTO/TLR4 pathway contributes to cerebral I/R injury. [ABSTRACT FROM AUTHOR]

Benzo[a]pyrene (B[a]P) is neurotoxic; however, the mechanism and prevention are still unclear. In this study, we assessed the intervention effect of metformin (MET) on cognitive dysfunction in mice induced by B[a]P from the perspective of glucolipid metabolism. Forty-two male healthy ICR mice were randomly categorized into 6 groups and were gavaged with B[a]P (0, 2.5, 5, or 10 mg/kg), 45 times for 90 days. The controls were gavaged with edible peanut oil, and the intervention groups were co-treated with B[a]P (10 mg/kg) and MET (200 or 300 mg/kg). We assessed the cognitive function of mice, observed the pathomorphological and ultrastructural changes, and detected neuronal apoptosis and glucolipid metabolism. Results showed that B[a]P dose-dependently induced cognitive impairment, neuronal damage, glucolipid metabolism disorder in mice, and enhanced proteins of fat mass and obesity-associated protein (FTO) and forkhead box protein O6 (FoxO6) in the cerebral cortex and liver, which were alleviated by the MET intervention. The findings indicated the critical role of glucolipid metabolism disorder in the cognitive impairment in mice caused by B[a]P and the prevention of MET against B[a]P neurotoxicity by regulating glucolipid metabolism via restraining FTO/FoxO6 pathway. The finding provides a scientific basis for the neurotoxicity and prevention strategies of B[a]P. [ABSTRACT FROM AUTHOR]

Amyloid‐β (Aβ) is thought to be a critical pathologic factor of retinal pigment epithelium (RPE) degeneration in age‐related macular degeneration (AMD). Aβ induces inflammatory responses in RPE cells and recent studies demonstrate the N6‐methyladenosine (m6A) regulatory role in RPE cell inflammation. m6A is a reversible epigenetic posttranslational modification, but its relationship with Aβ‐induced RPE degeneration is yet to be thoroughly investigated. The present study explored the role and mechanism of m6A in Aβ‐induced RPE degeneration model. This model was induced via intravitreally injecting oligomeric Aβ and the morphology of its retina was analyzed. One of m6A demethylases, the fat mass and obesity‐associated (FTO) gene expression, was assessed. An m6A‐messenger RNA (mRNA) epitranscriptomic microarray was employed for further bioinformatic analyses. It was confirmed that Aβ induced FTO upregulation within the RPE. Hypopigmentation alterations and structural disorganization were observed in Aβ‐treated eyes, and inhibition of FTO exacerbated retinal degeneration and RPE impairment. Moreover, the m6A‐mRNA epitranscriptomic microarray suggested that protein kinase A (PKA) was a target of FTO, and the PKA/cyclic AMP‐responsive element binding (CREB) signaling pathway was involved in Aβ‐induced RPE degeneration. m6A‐RNA binding protein immunoprecipitation confirmed that FTO demethylated PKA within the RPE cells of Aβ‐treated eyes. Altered expression of PKA and its downstream targets (CREB and brain‐derived neurotrophic factor) was confirmed by quantitative reverse‐transcription polymerase chain reaction and Western blot analyses. Hence, this study's findings shed light on FTO‐mediated m6A modification in Aβ‐induced RPE degeneration and indicate potential therapeutic targets for AMD. [ABSTRACT FROM AUTHOR]

BackgroundChemical modification of RNA is a recent hotspot in the field of epigenetics, but the specific mechanism of chemical modification of RNA in aluminum neurotoxicity has not been fully reported.ObjectiveTo investigate the alterations of fat mass and obesity-associated protein (FTO), that demethylates N6-methyladenosine (m6A), and brain-derived neurotrophic factor (BDNF) in different brain regions of rats and rat adrenal pheochromocytoma differentiated cells (PC12 cells) following aluminum exposure.MethodsAnimal experiment: Twenty-four healthy male SD rats were randomly divided into a control group (normal saline) and 10, 20, and 40 μmol·kg−1 exposure groups according to body weight, with 6 rats in each group. Maltol aluminum [Al(mal)3] was injected intraperitoneally every other day for 3 months. Cell experiment: PC12 cells were divided into a control group and 100, 200, and 400 μmol·L−1 exposure groups exposed to Al(mal)3 for 24 h. After exposure, the learning and memory ability of rats was measured by water maze experiment, and the protein expression levels of FTO and BDNF in rat cortex (n=6) and hippocampus (n=6) samples as well as in PC12 cells (n=5) were determined by Western blotting.ResultsThe results of water maze test showed that the escape latency of the 40 μmol·kg−1Al(mal)3 group was higher than those of the control group, the 10 μmol·kg−1Al(mal)3 group, and the 20 μmol·kg−1Al(mal)3 group on day 3, 4, and 5 of training (P

Background Over the past three decades, with substantial changes in lifestyle, the tendency to gain weight has increased, which is resulting in significant consequences affecting an individual’s well-being. The fat mass and obesity-associated (FTO) gene is involved in food intake and energy expenditure and plays a crucial role in regulating homeostasis and controlling energy expenditure by hindering signals that generate from the brain. Edible seaweeds have been shown to enhance satiety owing to their health benefits. Methods Extensive screening of plant-derived anti-obesity compounds and seaweed compounds was conducted and validated for ADME properties and toxicity prediction. Further, the top ranked compounds were docked against the FTO protein to identify potential inhibitors and were subjected to molecular dynamic simulation studies to understand the binding stability of ligand protein complex. Finally, MM/PBSA studies were performed to calculate the binding free energy of the protein-ligand complexes. Results Through the virtual screening of 1,210 compounds, 443 compounds showed good docking scores less than −7.00 kcal/mol. Drug likeness screenings of 443 compounds showed that only 369 compounds were in accordance with these properties. Further toxicity prediction resulted in 30 non-toxic compounds. Molecular docking studies revealed four top ranked marine compounds. Finally, RL074 (2-hydroxyluzofuranone B) and RL442 (10-acetoxyangasiol) from marine red alga Laurencia sp showed good stability from molecular dynamic simulation studies. MM/PBSA results revealed that BT012 (24ε-hydroperoxy-6β-hydroxy-24-ethylcholesta-4,-28(29)-dien-3-one), an oxygenated fucosterol from brown alga Turbinaria conoides, possessed higher binding energy. Hence, with all the data obtained it could be concluded that three seaweed compounds, BT012, RL074 and RL442, may act as a potential anti-obesity lead compound in targeting FTO.

Non-small cell lung cancer (NSCLC) represents one of the primary causes of cancer-related mortality all over the world. Following our initial finding of the upregulated expression of E2F transcription factor-1 (E2F1) in the NSCLC-related microarray, this study aimed to explore the regulatory role of E2F1 and underlying mechanism in NSCLC development. NSCLC cell viability, migration, and invasion were evaluated utilizing Cell Counting Kit 8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), wound-healing, and Transwell assays. Loss- and gain-function assays were performed to determine the effects of the fat mass and obesity-associated protein (FTO)/E2F1/neural epidermal growth factor-like 2 (NELL2) axis on NSCLC cell behaviors in vitro and NSCLC tumor growth in vivo. E2F1 was highly expressed in both NSCLC tissues and cells. E2F1 augmented the viability, migration, and invasion of NSCLC cells, which was attributable to E2F1 transcriptionally activating NELL2. FTO upregulated the expression of E2F1 by inhibiting the m6A modification of E2F1. The FTO/E2F1/NELL2 axis modulated NSCLC cell viability, migration, and invasion in vitro as well as affected NSCLC tumor growth and metastasis in vivo. The FTO/E2F1/NELL2 axis may impart pro-tumorigenic effects on the cell behavior of NSCLC cells and thus accelerate NSCLC progression.

The fat mass and obesity-associated protein (FTO) is vital for modulating dynamic reversible process of N6-methyladenosine (m6A) level in the nucleus and the abnormal expression of FTO correlates with generation and development of Alzheimer's disease, obesity and several cancers. The sensitive detection of FTO is therefore of significance for diagnosis of FTO-related diseases. In this work, on the basis of the demethylation enzyme capability of FTO and the dual Mg2+-dependent DNAzyme recycled signal magnification strategy, establishment of a highly sensitive electrochemical FTO biosensor is described. The target FTO molecules catalyze the demethylation of m6A-modified sequences to form effective cleavage substrates for the Mbo I endonuclease, leading to the release of active DNAzyme sequences to trigger subsequent dual DNAzyme recycling cycles. Many methylene blue-tagged ssDNA sequences can thus be yielded via the DNAzyme amplifications and captured on the sensor electrode to generate highly amplified currents for detecting FTO in range of 0.04 pM to 10 nM with 6.11 fM detection limit. Additionally, the sensor demonstrates high selectivity and is able to be used for detecting cell lysate FTO, showing its potential for the diagnosis and study of FTO-related diseases for various application purposes. • A highly sensitive electrochemical FTO biosensor is developed. • Target activation of dual DNAzyme recycling leads to significant signal amplification. • Such sensor can achieve sensitive detection of low levels of FTO in cell lysate. [ABSTRACT FROM AUTHOR]

Hyperglycemia-induced endothelial cell injury is one of the main causes of diabetic vasculopathy. Fat mass and obesity-associated protein (FTO) was the first RNA N6-methyladenosine (m6A) demethylase identified; it participates in the pathogenesis of diabetes. However, the role of FTO in hyperglycemia-induced vascular endothelial cell injury remains unclear. The effects of FTO on cellular m6A, autophagy, oxidative stress, proliferation, and cytotoxicity were explored in human umbilical vein endothelial cells (HUVECs) treated with high glucose (33.3 mmol/mL) after overexpression or pharmacological inhibition of FTO. MeRIP-qPCR and RNA stability assays were used to explore the molecular mechanisms by which FTO regulates autophagy. High glucose treatment increased m6A levels and reduced FTO protein expression in HUVECs. Wild-type overexpression of FTO markedly inhibited reactive oxygen species generation by promoting autophagy, increasing endothelial cell proliferation, and decreasing the cytotoxicity of high glucose concentrations. The pharmacological inhibition of FTO showed the opposite results. Mechanistically, we identified Unc-51-like kinase 1 (ULK1), a gene responsible for autophagosome formation, as a downstream target of FTO-mediated m6A modification. FTO overexpression demethylated ULK1 mRNA and inhibited its degradation in an m6A-YTHDF2-dependent manner, leading to autophagy activation. Our study demonstrates the functional importance of FTO-mediated m6A modification in alleviating endothelial cell injury under high glucose conditions and indicates that FTO may be a novel therapeutic target for diabetic vascular complications. • FTO overexpression suppresses high glucose-induced endothelial cell injury. • FTO overexpression inhibit ROS formation via promotion of autophagy. • FTO selectively demethylates ULK1 (Unc-51-like kinase 1) in an m6A-YTHDF2 dependent manner. • FTO increases half-life of ULK1 transcripts and expression of proteins involved in autophagy activation. • FTO-mediated demethylation crucial for cellular homeostasis under high glucose [ABSTRACT FROM AUTHOR]

The role of fat mass and obesity-associated protein (FTO)-mediated N6-methyladenosine (m6A)-demethylation has been investigated in various types of cancers, but it is still unclear whether FTO participates in the progression of diffuse large B-cell lymphoma (DLBCL). Here, by conducting Real-Time qPCR and Western Blot analysis, we verified that FTO was especially enriched in the DLBCL cells (RCK-8, LY-3, DHL-6 and U2932) compared to normal WIL2S cells. Then, the overexpression and silencing vectors for FTO were delivered into the LY-3 and U2932 cells, and our functional experiments confirmed that silencing of FTO suppressed cell viability and division, and induced apoptotic cell death in the DLBCL cells, whereas FTO-overexpression exerted opposite effects. Further mechanical experiments showed that FTO demethylated m6A modifications in flotillin-2 (FLOT2) mRNA to sustain its stability for FLOT2 upregulation, and elevated FLOT2 subsequently increased the expression levels of phosphorylated PI3K (p-PI3K), p -Akt and p -mTOR to activate the tumor-initiating PI3K/Akt/mTOR signal pathway. Of note, FLOT2 also serve as an oncogene to enhance cancer malignancy in DLBCL, and the rescuing experiments showed that FTO-ablation induced suppressing effects on the malignant phenotypes in DLBCL were all abrogated by overexpressing FLOT2. Taken together, those data hinted that FTO-mediated m6A-demethylation upregulated FLOT2 to activate the downstream PI3K/Akt/mTOR signal pathway, leading to the aggressiveness of DLBCL, which potentially provided diagnostic, therapeutic and prognostic biomarkers for DLBCL. [Display omitted] • FTO and FLOT2 served as oncogenes to enhance cancer aggressiveness in DLBCL. • FTO positively regulated FLOT2 in DLBCL cells via inducing m6A-demethylation. • Overexpression of FTO activated the tumor-initiating PI3K/Akt/mTOR pathway through FLOT2. • FTO upregulated FLOT2 to promote cancer progression in DLBCL. [ABSTRACT FROM AUTHOR]

Colorectal carcinoma (CRC) poses heavy burden to human health and has an increasing incidence. Currently, the existing biomarkers for CRC bring about restrained clinical benefits. GSK3β is reported to be a novel therapeutic target for this disease but with undefined molecular mechanisms. Thus, we aimed to investigate the regulatory effect of GSK3β on CRC progression via FTO/MZF1/c‐Myc axis. Firstly, the expression patterns of GSK3β, FTO, MZF1 and c‐Myc were determined after sample collection. Lowly expressed GSK3β but highly expressed FTO, MZF1 and c‐Myc were found in CRC. After transfection of different overexpressed and interference plasmids, the underlying mechanisms concerning GSK3β in CRC cell functions were analysed. Additionally, the effect of GSK3β on FTO protein stability was assessed followed by detection of MZF1 m6A modification and MZF1‐FTO interaction. Mechanistically, GSK3β mediated ubiquitination of demethylase FTO to reduce FTO expression. Besides, GSK3β inhibited MZF1 expression by mediating FTO‐regulated m6A modification of MZF1 and then decreased the proto‐oncogene c‐Myc expression, thus hampering CRC cell proliferation. We also carried out in vivo experiment to verify the regulatory effect of GSK3β on CRC via FTO‐mediated MZF1/c‐Myc axis. It was found that GSK3β inhibited CRC growth in vivo which was reversed by overexpressing c‐Myc. Taken together, our findings indicate that GSK3β suppresses the progression of CRC through FTO‐regulated MZF1/c‐Myc axis, shedding light onto a new possible pathway by which GSK3β regulates CRC. [ABSTRACT FROM AUTHOR]

Background: Through experimental research on the biological function of GATA6-AS1, it was confirmed that GATA6-AS1 can inhibit the proliferation, invasion, and migration of gastric cancer cells, suggesting that GATA6-AS1 plays a role as an anti-oncogene in the occurrence and development of gastric cancer. Further experiments confirmed that the overexpression of fat mass and obesity-associated protein (FTO) inhibited the expression of GATA6-AS1, thereby promoting the occurrence and development of gastric cancer., Aim: To investigate the effects of GATA6-AS1 on the proliferation, invasion and migration of gastric cancer cells and its mechanism of action., Methods: We used bioinformatics methods to analyze the Cancer Genome Atlas (https://portal.gdc.cancer.gov/. The Cancer Genome Atlas) and download expression data for GATA6-AS1 in gastric cancer tissue and normal tissue. We also constructed a GATA6-AS1 lentivirus overexpression vector which was transfected into gastric cancer cells to investigate its effects on proliferation, migration and invasion, and thereby clarify the expression of GATA6-AS1 in gastric cancer and its biological role in the genesis and development of gastric cancer. Next, we used a database (http://starbase.sysu.edu.cn/starbase2/) to analysis GATA6-AS1 whether by m6A methylation modify regulation and predict the methyltransferases that may methylate GATA6-AS1. Furthermore, RNA immunoprecipitation experiments confirmed that GATA6-AS1 was able to bind to the m6A methylation modification enzyme. These data allowed us to clarify the ability of m6A methylase to influence the action of GATA6-AS1 and its role in the occurrence and development of gastric cancer., Results: Low expression levels of GATA6-AS1 were detected in gastric cancer. We also determined the effects of GATA6-AS1 overexpression on the biological function of gastric cancer cells. GATA6-AS1 had strong binding ability with the m6A demethylase FTO, which was expressed at high levels in gastric cancer and negatively correlated with the expression of GATA6-AS1. Following transfection with siRNA to knock down the expression of FTO, the expression levels of GATA6-AS1 were up-regulated. Finally, the proliferation, migration and invasion of gastric cancer cells were all inhibited following the knockdown of FTO expression., Conclusion: During the occurrence and development of gastric cancer, the overexpression of FTO may inhibit the expression of GATA6-AS1, thus promoting the proliferation and metastasis of gastric cancer., Competing Interests: Conflict-of-interest statement: All authors have nothing to disclose., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Fat mass and obesity-associated protein (FTO) is an enzyme that demethylates N6-methyladenosine (m6A), the most abundant RNA modifications in a cell. The upregulated expression of FTO promotes the progression of various types of cancer by modulating cell-intrinsic genes which relate to malignant potential. However, the impact of FTO on the expression of immune-checkpoint molecules in the tumor cells, which are important for immune escape, has not been well understood. We examined the relevance of FTO to programmed cell death-ligand 1 (PD-L1) expression in colon cancer cells. HCT-116 cells showed high expression of both FTO and PD-L1 proteins. The knockdown of FTO by small interfering RNA decreased mRNA and protein levels of PD-L1 in HCT-116 cells. To elucidate the underlying mechanism by which FTO regulates the expression of PD-L1, we depleted FTO in HCT-116 in the presence of IFN-γ, which is a major stimulus to upregulate PD-L1 expression. Depletion of FTO reduced PD-L1 expression in an IFN-γ signaling-independent manner. RNA immunoprecipitation assay revealed the m6A modification of the PD-L1 mRNA and the binding of FTO to the PD-L1 mRNA in HCT-116. Taken together, our results indicated that FTO could regulate PD-L1 expression in colon cancer cells and provides new insights into the regulation of PD-L1 expression by RNA modification. • The role of RNA modification on immune system in cancer is not well known. • PD-L1 is an immune-checkpoint molecule expressed on cancer cells. • RNA demethylase, FTO, regulates PD-L1 expression in colon cancer cells. • PD-L1 mRNA possesses m6A modifications, and FTO binds to the PD-L1 mRNA. [ABSTRACT FROM AUTHOR]

Liver cancer is the fourth leading cause of cancer-associated mortality worldwide. Statistics indicate that the incidence of liver cancer has been increasing and that its prognosis remains poor. Fat mass and obesity-associated protein (FTO) is a demethylase that is involved in N6-methyladenosine (m6a) RNA modification; however, to the best of our knowledge, its role in tumorigenesis and development of liver cancer remains unknown. In the present study, cell proliferation, colony formation, apoptosis, Transwell and wound healing assays of small interfering (si)RNA-FTO HepG2 cells were performed, and the levels of m6A RNA methylation were assessed. Additionally, the prognostic value of FTO in liver cancer was analyzed using immunohistochemistry analysis. The results from the EpiQuik m6A RNA methylation quantitative assay revealed that knockdown of FTO increased the total m6A methylation level. Notably, FTO promoted the proliferation and migration of liver cancer cells. Additionally, FTO expression was upregulated in patients with liver cancer and was associated with a high Edmondson Grade, which served as an independent prognostic factor for liver cancer. Results from the Kaplan-Meier survival analysis revealed that low expression levels of FTO predicted a good prognosis. The 5-year overall survival of the low FTO expression group was 68% compared with 48% in the high FTO expression group (P=0.077). In conclusion, the present study suggested that FTO regulates the tumorigenesis and development of liver cancer. [ABSTRACT FROM AUTHOR]

Fat mass and obesity-associated (FTO) protein has been identified as a critical demethylase in regulating cellular mRNA stability by removing N6-methyladenosine (m6A) residues in mRNA. Even though the role of FTO in body energy metabolism has been well established, its role in cancer cell homeostasis remains unclear. In the present study, by using RNA interference, it was indicated that FTO is required for pancreatic cancer cell proliferation. Knockdown of FTO resulted in compromised proliferation of pancreatic cancer cells. Furthermore, DNA synthesis was compromised, followed by an increase in apoptosis in FTO small interfering RNA (siRNA)-treated cells. In terms of its underlying mechanism, FTO has been indicated to interact with MYC proto-oncogene, bHLH transcription factor and to enhance its stability by decreasing its m6A level. Therefore, the aforementioned observations indicate a novel mechanism for the regulation of pancreatic cancer cells by FTO, which may provide insight on pancreatic cancer treatment strategies. [ABSTRACT FROM AUTHOR]

Abstract Nafamostat mesilate is a serine protease inhibitor and used for the treatment of pancreatitis and cancers. The fat mass and obesity-associated protein (FTO) was identified to be a demethylase and played an important role in physiological processes. The aim of this study was to examine the inhibition of Nafamostat mesilate on FTO demethylase activity. A combination of thermodynamic and enzymatic activity studies provides an insight into the recognition of Nafamostat mesilate by FTO. The binding of Nafamostat mesilate to FTO was driven by higher positive entropy changes and smaller negative enthalpy changes. The structural and thermodynamic information provides the basis for the design of more effective inhibitors for FTO. Our results might provide a novel target protein for Nafamostat mesilate. Graphical abstract The characteristics of the binding between Nafamostat mesilate and the fat mass and obesity-associated protein were investigated. Image 1 Highlights • Nafamostat mesilate is an inhibitor of FTO demethylase activity. • The binding of Nafamostat mesilate to FTO is driven by higher positive entropy changes and smaller negative enthalpy changes. • Results provide a novel target protein for Nafamostat mesilate. [ABSTRACT FROM AUTHOR]

Microglia-mediated inflammation is a major contributor to the brain damage in cerebral ischemia and reperfusion (I/R) injury, and N6-Methyladenosine (m6A) has been implicated in cerebral I/R injury. Here, we explored whether m6A modification is associated with microglia-mediated inflammation in cerebral I/R injury and its underlying regulatory mechanism using an in vivo mice model of intraluminal middle cerebral artery occlusion/reperfusion (MCAO/R) and in vitro models of primary isolated microglia and BV2 microglial cells subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) were used. We found microglial m6A modification increased and microglial fat mass and obesity-associated protein (FTO) expression decreased in cerebral I/R injury in vivo and in vitro. Inhibition of m6A modification by intraperitoneal injection of Cycloleucine (Cyc) in vivo or transfection of FTO plasmid in vitro significantly alleviated brain injury and microglia-mediated inflammatory response. Through Methylated RNA immunoprecipitation sequencing (MeRIP-Seq), RNA sequencing (RNA-Seq) and western blotting, we found that m6A modification promoted cerebral I/R-induced microglial inflammation via increasing cGAS mRNA stability to aggravate Sting/NF-κB signaling. In conclusion, this study deepens our understanding on the relationship of m6A modification and microglia-mediated inflammation in cerebral I/R injury, and insights a novel m6A-based therapeutic for inhibiting inflammatory response against ischemic stroke. [ABSTRACT FROM AUTHOR]

This research discussed the specific mechanism by which PIAS1 affects acute pancreatitis (AP). PIAS1, Foxa2, and FTO expression was assessed in Cerulein-induced AR42J cells and mice. Loss- and gain-of-function assays and Cerulein induction were conducted in AR42J cells and mice for analysis. The relationship among PIAS1, Foxa2, and FTO was tested. Cell experiments run in triplicate, and eight mice for each animal group. Cerulein-induced AP cells and mice had low PIAS1 and Foxa2 and high FTO. Cerulein induced pancreatic injury in mice and inflammation and oxidative stress in pancreatic tissues, which could be reversed by PIAS1 or Foxa2 upregulation or FTO downregulation. PIAS1 elevated SUMO modification of Foxa2 to repress FTO transcription. FTO upregulation neutralized the ameliorative effects of PIAS1 or Foxa2 upregulation on Cerulein-induced AR42J cell injury, inflammation, and oxidative stress. PIAS1 upregulation diminished FTO transcription by increasing Foxa2 SUMO modification, thereby ameliorating Cerulein-induced AP. • Cerulein-induced mice and cells have low PIAS1 and Foxa2 but high FTO expression. • PIAS1 or Foxa2 upregulation or FTO downregulation relieves AP progression in mice. • PIAS1 mediates sumoylation of Foxa2 in AP cells. • Foxa2 curbs FTO transcription to mitigate Cerulein-induced cell injury. • PIAS1 upregulation ameliorates Cerulein-induced cell injury via Foxa2/FTO axis. [ABSTRACT FROM AUTHOR]

Background: Glycogen synthase kinase-3β (GSK3β), fat mass and obesity-associated protein (FTO), and toll-like receptors 4 (TLR4) take on critical significance in different biological processes, whereas their interactions remain unclear. The objective was the investigation of the interaction effect in cerebral ischemia-reperfusion (I/R) injury., Methods: The function of the cerebral cortex in the mouse middle cerebral artery occlusion (MCAO) model (each group n = 6) and P12 cells oxygen-glucose deprivation/reoxygenation (OGD/R) model was analyzed using short hairpin GSK3β lentivirus and overexpression of FTO lentivirus ( in vitro ), TLR4 inhibitor (TAK242), and LiCl to regulate GSK3β, FTO, TLR4 expression, and GSK3β activity, respectively., Results: After GSK3β knockdown in the OGD/R model of PC12 cells, the levels of TLR4 and p-p65 were lower than in the control, and the level of FTO was higher than in the control. Knockdown GSK3β reversed the OGD/R-induced nuclear factor kappa-B transfer to the intranuclear nuclei. As indicated by the result, TLR4 expression was down-regulated by overexpressed FTO, and TLR4 expression was up-regulated notably after inhibition of FTO with the use of R-2HG. After the inhibition of the activity of GSK3β in vivo , the reduction of FTO in mice suffering from MCAO was reversed., Conclusions: Our research shows that GSK3β/FTO/TLR4 pathway contributes to cerebral I/R injury., Competing Interests: There are no conflicts of interest., (Copyright: © 2023 Brain Circulation.)

In this work, the interactions between fat mass and obesity-associated (FTO) protein and three fluoronucleoside analogues (three-member-ring compound ( 1a ), five-member-ring compound ( 1b ) and six-member-ring compound ( 1c )) have been investigated by fluorescence, ultraviolet-visible absorption spectroscopy, isothermal titration calorimetric (ITC) and molecular modeling. Analysis of fluorescence data showed that the binding between three analogues and FTO occurred via a static quenching mechanism. Both ITC and fluorescence results indicated that 1b is the strongest quencher. In contrast to spectroscopy techniques, ITC results suggested that there is no binding for 1c . ITC results showed that the binding between FTO and 1a (or 1b ) were exothermic. Fluorescence results showed that the binding between three analogues and FTO were endothermic. Results of thermodynamic analysis and molecular modeling suggested that it was entropy driven event between FTO and 1a (or 1b ). [ABSTRACT FROM AUTHOR]

As one of the most important demethylases for RNA N6-methyladenosine (m6A) modifications, fat mass and obesity-associated protein (FTO) plays anti-cancer role during prostate cancer (PC), but it is still unclear the detailed molecular mechanisms. Here, this study verified that FTO inactivated the tumor-accelerating PI3K/Akt/mTOR pathway to hamper PC development through regulating the downstream miR-139–5p/zinc finger protein 217 (ZNF217) axis. Through performing clinical analysis, it was revealed that FTO was apparently ablated in the cancerous tissues compared to the normal tissues collected from PC patients, and patients with high-expressed FTO predicted a favorable prognosis. Functional experiments confirmed that overexpression of FTO suppressed cell proliferation, mitosis, epithelial-mesenchymal transition (EMT), tumorigenesis and lung metastasis both in vitro and in vivo. The following mechanical experiments verified that FTO stabilized miR-139–5p to increase its expression levels in a m6A-dependent manner, and elevated miR-139–5p induced degradation of ZNF217 through binding to ZNF217 mRNA, resulting in the inactivation of the PI3K/Akt/mTOR signal pathway. Finally, our rescuing experiments confirmed that overexpressed FTO-induced tumor-suppressing effects on PC cells were abrogated by miR-139–5p ablation and ZNF217 overexpression. Collectively, this study firstly validated that FTO exerted its anti-tumor effects in PC through regulating the miR-139–5p/ZNF217 axis in a m6A-dependent manner, providing novel biomarkers for the advancement of anti-cancer agents for PC treatment. [Display omitted] • FTO suppressed the malignant phenotypes in prostate cancer. • FTO-mediated m6A-demethylation increased the expression levels and stability of miR-139–5p. • miR-139–5p degraded ZNF217 mRNA by binding to its 3′ UTR regions. • ZNF217 acts as an oncogene by activating the downstream PI3K/Akt/mTOR signal pathway. [ABSTRACT FROM AUTHOR]

Fat mass and obesity-associated protein (FTO) regulating the N6-methyladenine (m6A, the most pervasive epigenetic modification) levels within the nucleus has been identified as a potential biomarker for cancer diagnosis and prognosis. However, current methods for FTO detection are complicated or/and not sensitive enough for practical application. Herein, we propose a colorimetric biosensor for detecting FTO based on a delicate design of m6A demethylation-activated DNAzyme. Specifically, an m6A-blocked DNAzyme is constructed as a switch of the biosensor that can be turned on by target FTO. The decreased thermal stability resulting from substrate cleavage leads to a DNAzyme recycling to produce multiple primers. Then the rolling circle amplification (RCA) reactions can be initiated to generate G-quadruplex-DNAzymes catalyzing 2,2-azino-bis-(3-ethylben-zthiazoline-6-sulfonic acid (ABTS) oxidation which can be readily observed by the naked eye. Quantitative detection can also be achieved with a limit of detection (LOD) down to 69.9 fM, exhibiting higher sensitivity than previous reports. Therefore, this biosensor opens a simple and sensitive way to achieve visual assay of FTO via triple signal amplification. In addition, our biosensor has been successfully applied to FTO detection in clinical samples, which shows great potential in clinical molecular diagnostics. [Display omitted] [ABSTRACT FROM AUTHOR]

Amyloid-β (Aβ) is thought to be a critical pathologic factor of retinal pigment epithelium (RPE) degeneration in age-related macular degeneration (AMD). Aβ induces inflammatory responses in RPE cells and recent studies demonstrate the N6-methyladenosine (m6A) regulatory role in RPE cell inflammation. m6A is a reversible epigenetic posttranslational modification, but its relationship with Aβ-induced RPE degeneration is yet to be thoroughly investigated. The present study explored the role and mechanism of m6A in Aβ-induced RPE degeneration model. This model was induced via intravitreally injecting oligomeric Aβ and the morphology of its retina was analyzed. One of m6A demethylases, the fat mass and obesity-associated (FTO) gene expression, was assessed. An m6A-messenger RNA (mRNA) epitranscriptomic microarray was employed for further bioinformatic analyses. It was confirmed that Aβ induced FTO upregulation within the RPE. Hypopigmentation alterations and structural disorganization were observed in Aβ-treated eyes, and inhibition of FTO exacerbated retinal degeneration and RPE impairment. Moreover, the m6A-mRNA epitranscriptomic microarray suggested that protein kinase A (PKA) was a target of FTO, and the PKA/cyclic AMP-responsive element binding (CREB) signaling pathway was involved in Aβ-induced RPE degeneration. m6A-RNA binding protein immunoprecipitation confirmed that FTO demethylated PKA within the RPE cells of Aβ-treated eyes. Altered expression of PKA and its downstream targets (CREB and brain-derived neurotrophic factor) was confirmed by quantitative reverse-transcription polymerase chain reaction and Western blot analyses. Hence, this study's findings shed light on FTO-mediated m6A modification in Aβ-induced RPE degeneration and indicate potential therapeutic targets for AMD., (© 2022 International Federation for Cell Biology.)

Schematic presentation of Sensitive Detection of Fat Mass and Obesity-Associated Protein (FTO). FTO can specifically demethylate the substrate probe, and the emerging double strands can be cleaved by DpnII which is a methylation-sensitive restriction endonuclease. The polymerase TdT in turn recognizes the free 3′OH and initiates polymerization to produce a mass of G-quadruplexes. Thioflavin T (ThT) can then be embedded into G-quadruplexes, producing a strong fluorescence. [Display omitted] • A highly sensitive biosensor for FTO detection is developed. • The biosensor can provide an efficient strategy without labeling and separation. • This biosensor has remarkable potential for other epigenetic modification enzymes in clinical application. Fat mass and obesity-associated protein (FTO) can catalyze the demethylation of N6-methyladenosine (m6A) motif in ssDNA or RNA, which plays a crucial role in maintaining the relative stability of m6A modification. Meanwhile, dysregulation of FTO expression can lead to cancers and cardiovascular diseases. So quantitative analysis of FTO is of great significance for the research related with its biological functions and application in disease diagnosis. Herein, we propose a highly sensitive biosensor for FTO detection based on terminal deoxynucleotidyl transferase (TdT)-mediated signal amplification. To be specific, FTO can specifically demethylate the substrate probe, and the emerging double strands can be cleaved by DpnII which is a methylation-sensitive restriction endonuclease. The polymerase TdT in turn recognizes the free 3′OH and initiates polymerization to produce a mass of G-quadruplexes. Thioflavin T (ThT) can then be embedded into G-quadruplexes, producing a strong fluorescence. Experimental results manifest that the fluorescence intensity is positively correlated with the abundance of FTO. Moreover, this biosensor also has good specificity and anti-interference, and the limit of detection (LOD) is down to 30.9 fM. Therefore, our biosensor can provide an efficient strategy for FTO analysis without labeling and separation, showing great potential in clinical application. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) is the most prevalent mRNA modification which plays crucial roles in various biological processes, but its role in cementogenesis remains largely unknown. Here, using time-series transcriptomic analysis, we reveal that mRNA m6A demethylase Fat mass and obesity-associated protein (FTO) is involved in cementogenesis. Knocking down FTO decreases cementoblast differentiation and mineralization in both OCCM-30 cellular model and murine ectopic bone formation model. Mechanistically, we find that FTO directly binds Runt-related transcription factor 2 (Runx2) mRNA, an important cementogenesis factor, thus protecting it from YTH domain-containing family protein 2 (YTHDF2) mediated degradation, when cementoblasts are differentiating. Knocking down YTHDF2 restores the expression of Runx2 in FTO-knockdown cells. Moreover, under inflammatory conditions, TNF-α inhibits cementoblast differentiation and mineralization partly through FTO/RUNX2 axis. Collectively, our study reveals an important regulatory role of FTO/RUNX2 axis in normal and pathological cementogenesis. • Time-series transcriptomic analysis reveals that m6A demethylase FTO is involved in cementoblast differentiation. • Knocking down FTO decreases cementoblast differentiation and mineralization. • FTO binds Runx2 mRNA to protect it from YTHDF2-mediated degradation when cementoblasts are differentiating. • TNF-α inhibits cementoblast differentiation and mineralization partially through FTO/RUNX2 axis. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) is a common disorder without effective therapy yet. Renal ischemia/reperfusion (I/R) injury is a common cause of AKI. MicroRNA miR-192-5p has been previously reported to be upregulated in AKI models. However, its functional role in renal I/R injury is not fully understood. This study aimed to investigate the effects and the underlying mechanism of miR-192-5p in renal I/R progression. Hypoxia/reoxygenation (H/R)-induced cell injury model in HK-2 cells and I/R-induced renal injury model in mice were established in this study. Cell counting kit-8 assay was performed to determine cell viability. Quantitative real-time PCR and western blot analysis were performed to detect gene expressions. Hematoxylin-eosin and periodic acid-Schiff staining were performed to observe the histopathological changes. Enzyme-linked immunosorbent assay was performed to detect the kidney markers' expression. In vivo and in vitro results showed that miR-192-5p was up-regulated in the I/R-induced mice model and H/R-induced cell model, and miR-192-5p overexpression exacerbated I/R-induced renal damage. Then, the downstream target of miR-192-5p was analyzed by combining the differentially expressed mRNAs and the predicted genes and confirmed using a dual-luciferase reporter assay. It was found that miR-192-5p was found to regulate fat mass and obesity-associated (FTO) protein expression by directly targeting the 3' untranslated region of FTO mRNA. Moreover, in vivo and in vitro studies unveiled that FTO overexpression alleviated renal I/R injury and promoted HK-2 cell viability via stimulating autophagy flux. In conclusion, miR-192-5p aggravated I/R-induced renal injury by blocking autophagy flux via down-regulating FTO.

Understanding the effects of environmental pollutants on RNA methylation and its relative enzymatic activity can provide new markers for the evaluation of the ecotoxicological effects of environmental pollutants. To achieve this aim, sensitive and selective detection technique for m6A and its relative enzyme is necessary. Herein, an antibody-free enzyme-assisted photoelectrochemical (PEC) biosensor for detection of m6A and fat mass and obesity-associated protein (FTO) was constructed based on enzymatic m6A structural change, where Se-MoSe 2 nanoflower was selected as the photoactive material, gold nanoparticles (AuNPs) was used as recognition reagent, dm6AMP (N6-dithiolsitolmethyladenosine monophosphate, product of enzymatic m6A structural change and RNA hydrolysis) was used as the target molecule, and ZnSe was employed as signal amplification unit with the matched energy bandgap with Se-MoSe 2 nanoflower. This biosensor can simultaneously identify m6AMP and FTO protein with high sensitivity and specificity. The effect of three kinds of antibiotics (Griseofulvin, sulfadiazine, lincomycin) on the content of m6A in the total RNA of the roots and leaves of rice seedlings were firstly investigated using PEC technique. Then, the effect of four heavy metals (AsO 4 3-, Cd2+, Hg2+, Pb2+) on FTO protein activity was investigated with the IC 50 value of 43.61, 55.58, 50.48 and 35.99 μg/L, respectively. [Display omitted] • Photoactivity of MoSe 2 was improved by Se and ZnSe. • The mechanism for the increased photoactivty was investigated. • An antibody-free PEC biosensor was constructed based on Se-MoSe 2 and ZnSe. • The detection limits were 6.3 pM for m6A RNA and 0.78 ng/L for FTO. • The effect of heavy metal ions on FTO oxidation activity was assessed [ABSTRACT FROM AUTHOR]

Background: Over the past three decades, with substantial changes in lifestyle, the tendency to gain weight has increased, which is resulting in significant consequences affecting an individual's well-being. The fat mass and obesity-associated (FTO) gene is involved in food intake and energy expenditure and plays a crucial role in regulating homeostasis and controlling energy expenditure by hindering signals that generate from the brain. Edible seaweeds have been shown to enhance satiety owing to their health benefits., Methods: Extensive screening of plant-derived anti-obesity compounds and seaweed compounds was conducted and validated for ADME properties and toxicity prediction. Further, the top ranked compounds were docked against the FTO protein to identify potential inhibitors and were subjected to molecular dynamic simulation studies to understand the binding stability of ligand protein complex. Finally, MM/PBSA studies were performed to calculate the binding free energy of the protein-ligand complexes., Results: Through the virtual screening of 1,210 compounds, 443 compounds showed good docking scores less than -7.00 kcal/mol. Drug likeness screenings of 443 compounds showed that only 369 compounds were in accordance with these properties. Further toxicity prediction resulted in 30 non-toxic compounds. Molecular docking studies revealed four top ranked marine compounds. Finally, RL074 (2-hydroxyluzofuranone B) and RL442 (10-acetoxyangasiol) from marine red alga Laurencia sp showed good stability from molecular dynamic simulation studies. MM/PBSA results revealed that BT012 (24ε-hydroperoxy-6β-hydroxy-24-ethylcholesta-4,-28(29)-dien-3-one), an oxygenated fucosterol from brown alga Turbinaria conoides , possessed higher binding energy. Hence, with all the data obtained it could be concluded that three seaweed compounds, BT012, RL074 and RL442, may act as a potential anti-obesity lead compound in targeting FTO., Competing Interests: The authors declare that they have no competing interests., (© 2022 Prabhakar and Davis G.)

Uveitis, a vision-threatening inflammatory disease worldwide, is closely related to resident microglia. Retinal microglia are the main immune effector cells with strong plasticity, but their role in uveitis remains unclear. N6-methyladenosine (m 6 A) modification has been proven to be involved in the immune response. Therefore, we in this work aimed to identify the potentially crucial m 6 A regulators of microglia in uveitis. Through the single-cell sequencing (scRNA-seq) analysis and experimental verification, we found a significant decrease in the expression of fat mass and obesity-associated protein (FTO) in retinal microglia of uveitis mice and human microglia clone 3 (HMC3) cells with inflammation. Additionally, FTO knockdown was found to aggravate the secretion of inflammatory factors and the mobility/chemotaxis of microglia. Mechanistically, the RNA-seq data and rescue experiments showed that glypican 4 (GPC4) was the target of FTO, which regulated microglial inflammation mediated by the TLR4/NF-κB pathway. Moreover, RNA stability assays indicated that GPC4 upregulation was mainly regulated by the downregulation of the m 6 A "reader" YTH domain family protein 3 (YTHDF3). Finally, the FTO inhibitor FB23-2 further exacerbated experimental autoimmune uveitis (EAU) inflammation by promoting the GPC4/TLR4/NF-κB signaling axis, and this could be attenuated by the TLR4 inhibitor TAK-242. Collectively, a decreased FTO could facilitate microglial inflammation in EAU, suggesting that the restoration or activation of FTO function may be a potential therapeutic strategy for uveitis., (© 2022 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.)

During osteoporosis, fat mass and obesity-associated protein (FTO) promotes the shift of bone marrow mesenchymal stem cells to adipocytes and represses osteoblast activity. However, the role and mechanisms of FTO on osteoclast formation and bone resorption remain unknown. In this study, we investigated the effect of FTO on RAW264.7 cells and bone marrow monocytes (BMMs)-derived osteoclasts in vitro and observed the influence of FTO on ovariectomized (OVX) mice model to mimic postmenopausal osteoporosis in vivo. Results found that FTO was up-regulated in BMMs from OVX mice. Double immunofluorescence assay showed co-localization of FTO with tartrate-resistant acid phosphatase (TRAP) in femurs of OVX mice. FTO overexpression enhanced TRAP-positive osteoclasts and F-actin ring formation in RAW264.7 cells upon RANKL stimulation. The expression of osteoclast differentiation-related genes, including nuclear factor of activated T cells c1 (NFATc1) and c-FOS, was upregulated in BMMs and RAW264.7 cells after FTO overexpression. FTO overexpression induced the phosphorylation and nuclear translocation of factor-kappa B (NF-κB) p65 in BMMs and RAW264.7 cells exposed to RANKL. ChIP and dual-luciferase assays revealed that FTO overexpression contributed to RANKL-induced binding of NF-κB to NFATc1 promoter. Rescue experiments suggested that FTO overexpression-mediated osteoclast differentiation was suppressed after intervention with a NF-κB inhibitor pyrrolidine dithiocarbamate. Further in vivo evidence revealed that FTO knockdown increased bone trabecula and bone mineral density, inhibited bone resorption and osteoclastogenesis in osteoporotic mice. Collectively, our research demonstrates that downregulated FTO inhibits bone resorption and osteoclastogenesis through NF-κB inactivation, which provides a novel reference for osteoporosis treatment. • FTO is upregulated in BMMs isolated from the femurs of osteoporotic mice. • Knockdown of FTO inhibits RANKL-induced osteoclast differentiation. • Knockdown of FTO inhibits NF-κB activation and osteoclast-specific gene expression. • FTO may be a potential therapeutic gene for osteoporosis. [ABSTRACT FROM AUTHOR]

The rapid spread of SARS-CoV-2 and the resulting pandemic has led to a spasmodic search for approaches able to limit the diffusion of the disease. The epigenetic machinery has aroused considerable interest in the last decades, and much evidence has demonstrated that this type of modification could regulate the early stages of viral infection. Recently it was reported that N6-methyladenosine (m6A) influences SARS-CoV-2 replication, although its role remains to be further investigated. The knockdown of enzymes involved in the m6A pathway could represent an optimal strategy to deepen the epigenetic mechanism. In the present study, we blocked the catalytic activity of the fat mass and obesity-associated protein (FTO) by using the selective inhibitor rhein. We observed a strong broad-spectrum reduction of infectivity caused by various coronaviruses, including SARS-CoV-2. This effect could be due to the modulation of m6A levels and could allow identification of this modification as a new therapeutic target to treat SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

N6-methyladenosine (m6A) modification, the most prevalent form of RNA methylation, modulates gene expression post-transcriptionally. Di-(2-ethylhexyl) phthalate (DEHP) is a common environmental endocrine disrupting chemical that induces testicular injury due to the inhibition of the demethylase fat mass and obesity-associated protein (FTO) and increases the m6A modification. How FTO-mediated m6A modification in testicular Leydig cell injury induced by DEHP remains unclear. Here, the TM3 Leydig cell line was treated with mono-(2-ethylhexyl) phthalate (MEHP), the main metabolite of DEHP in the body, as well as FB23-2, an inhibitor of FTO. Decreased levels of testosterone in the culture supernatant, significantly increased apoptosis, and a remarkable upregulation of global m6A modification were found in both TM3 cells treated with MEHP and FB23-2. Transcriptome sequencing showed that both treatments significantly induced apoptosis-associated gene expression. Methylated RNA immunoprecipitation sequencing showed that the Leydig cell injury induced by upregulated m6A modification could be associated with multiple physiological disorders, including histone acetylation, reactive oxygen species biosynthesis, MAPK signaling pathway, hormone secretion regulation, autophagy regulation, and male gonadal development. Overall, the inhibition of FTO-mediated up-regulation of m6A could be involved in MEHP-induced Leydig cell apoptosis. Image 1 • MEHP increases global RNA m6A modification in TM3 Leydig cells. • MEHP alters expression of FTO in TM3 Leydig cells. • Leydig cells treated with MEHP or FB23-2 demonstrated a similar damage phenotype. • Apoptosis is vital in MEHP toxicity mediated by increased m6A modification. • Multiple disorders may be involved in MEHP-induced Leydig cell apoptosis. Increased m6A modification of RNA methylation related to the inhibition of demethylase FTO is associated with MEHP-induced Leydig cell injury. [ABSTRACT FROM AUTHOR]

Aim: To reveal transcriptome-wide N6-methyladenosine (m 6 A) methylome of coronary artery disease (CAD). Materials & methods: The m 6 A levels of RNA from peripheral blood mononuclear cells measured by colorimetry were significantly decreased in CAD cases. Transcriptome-wide m 6 A methylome profiled by methylated RNA immunoprecipitation sequencing (MeRIP-seq) identified differentially methylated m 6 A sites within both mRNAs and lncRNAs between CAD and control group. Results: Bioinformatic analysis indicated that differentially methylated genes were involved in the pathogenesis of atherosclerosis. MeRIP-quantitative real-time PCR assay confirmed the reliability of MeRIP-seq data. Finally, the rat carotid artery balloon injury model was performed to confirm the role of m 6 A demethylase FTO in neointima formation. Conclusion: Our study provided a resource of differentially methylated m 6 A profile for uncovering m 6 A biological functions in the pathogenesis of CAD.

Non-small cell lung cancer (NSCLC) represents one of the primary causes of cancer-related mortality all over the world. Following our initial finding of the upregulated expression of E2F transcription factor-1 (E2F1) in the NSCLC-related microarray, this study aimed to explore the regulatory role of E2F1 and underlying mechanism in NSCLC development. NSCLC cell viability, migration, and invasion were evaluated utilizing Cell Counting Kit 8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), wound-healing, and Transwell assays. Loss- and gain-function assays were performed to determine the effects of the fat mass and obesity-associated protein (FTO)/E2F1/neural epidermal growth factor-like 2 (NELL2) axis on NSCLC cell behaviors in vitro and NSCLC tumor growth in vivo . E2F1 was highly expressed in both NSCLC tissues and cells. E2F1 augmented the viability, migration, and invasion of NSCLC cells, which was attributable to E2F1 transcriptionally activating NELL2. FTO upregulated the expression of E2F1 by inhibiting the m6A modification of E2F1. The FTO/E2F1/NELL2 axis modulated NSCLC cell viability, migration, and invasion in vitro as well as affected NSCLC tumor growth and metastasis in vivo . The FTO/E2F1/NELL2 axis may impart pro-tumorigenic effects on the cell behavior of NSCLC cells and thus accelerate NSCLC progression., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Long non‑coding RNAs (lncRNAs) are involved in the regulation of esophageal squamous cell carcinoma (ESCC) progression. However, the function and mechanism of lncRNA cancer susceptibility candidate 15 (CASC15) are poorly defined. In the present study, tumor and normal adjacent tissues were collected from 45 patients with ESCC. Expression levels of CASC15, fat mass and obesity‑associated (FTO) protein and single‑minded 2 (SIM2) were examined via reverse transcription‑quantitative PCR and western blot assays. Cell proliferation and apoptosis were evaluated via MTT, flow cytometry and caspase‑3 activity assays, respectively. Additionally, an ESCC mouse xenograft model was used to assess the function of CASC15 in vivo. The interaction between FTO and CASC15/SIM2 was analyzed via RNA immunoprecipitation and RNA pull‑down assays. The results revealed that CASC15 expression was elevated in ESCC tissues, and patients with ESCC exhibiting high CASC15 expression had a poor prognosis. CASC15‑knockdown inhibited ESCC cell proliferation and facilitated apoptosis. Additionally, CASC15‑knockdown decreased the growth of ESCC xenograft tumors. CASC15 decreased SIM2 stability via FTO‑mediated demethylation. Additionally, FTO loss markedly weakened CASC15‑mediated pro‑proliferative and anti‑apoptotic effects in ESCC cells. SIM2 downregulation weakened the effect of CASC15‑knockdown on cell proliferation and inhibited the increase of the apoptotic rate and caspase‑3 activity induced by CASC15 depletion in ESCC cells. In conclusion, CASC15 promoted ESCC tumorigenesis by decreasing SIM2 stability via FTO‑mediated demethylation.

The alkaloids containing a carbazole nucleus are an established class of natural products with wide range of biological activities. A combination of thermodynamic and enzymatic activity studies provides an insight into the recognition of Clausine E by the fat mass and obesity‐associated protein (FTO). The binding of Clausine E to FTO was driven by positive entropy and negative enthalpy changes. Results also indicated that the hydroxyl group was crucial for the binding of small molecules with FTO. The structural and thermodynamic information provides the basis for the design of more effective inhibitors for FTO demethylase activity. [ABSTRACT FROM AUTHOR]

In this study, the interaction between 3-phenyl-1H-indazole ( 1a ) and the fat mass and obesity-associated (FTO) protein was confirmed by isothermal titration calorimetry (ITC). The structure feature of 1a was different from our previously reported FTO inhibitors (radicicol, N-CDPCB and CHTB); the Cl and diol group in structure motif is critical for inhibitors to bind to FTO. In order to test whether there is specificity for the interaction between FTO and 1a , the interactions between 1a and four important proteins (human serum albumin (HSA), pepsin, catalase and trypsin) were investigated by ITC, spectroscopy and molecular docking methods. ITC results showed spontaneous exothermic reactions occurring between 1a and the proteins except trypsin under investigated conditions. The order of the binding affinity of 3-phenyl-1H-indazole is catalase > HSA > FTO > pepsin. Comparison between ITC and spectral results was made. This work will provide the basis for the design of novel inhibitors for FTO. Abbreviations CAT catalase DMSO dimethyl sulfoxide FTO fat mass and obesity-associated protein HSA human serum albumin Pep pepsin Try trypsin Communicated by Ramaswamy H. Sarma.