Fat mass and obesity-associated protein (FTO)-induced upregulation of flotillin-2 (FLOT2) contributes to cancer aggressiveness in diffuse large B-cell lymphoma (DLBCL) via activating the PI3K/Akt/mTOR signal pathway.

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.

N6-methyladenosine demethylase fat mass and obesity-associated protein suppresses hyperglycemia-induced endothelial cell injury by inhibiting reactive oxygen species formation via autophagy promotion.

INTRODUCTION: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Targeting FTO induces colorectal cancer ferroptotic cell death by decreasing SLC7A11/GPX4 expression.

Ferroptosis is a newly identified iron-dependent form of death that is becoming increasingly recognized as a promising avenue for cancer therapy. N6-methyladenosine (m6A) is the most abundant reversible methylation modification in mRNA contributing to tumorigenesis. However, the crucial role of m6A modification in regulating ferroptosis during colorectal cancer (CRC) tumorigenesis remains elusive. Herein, we find that m6A modification is increased during ferroptotic cell death and correlates with the decreased m6A demethylase fat mass and obesity-associated protein (FTO) expression. Functionally, we demonstrate that suppressing FTO significantly induces CRC ferroptotic cell death, as well as enhancing CRC cell sensitivity to ferroptosis inducer (Erastin and RSL3) treatment. Mechanistically, high FTO expression increased solute carrier family 7 member 11 (SLC7A11) or glutathione peroxidase 4 (GPX4) expressions in an m6A-YTHDF2 dependent manner, thereby counteracting ferroptotic cell death stress. In addition, we identify Mupirocin as a novel inhibitor of FTO, and Mupirocin induces CRC ferroptosis and inhibits tumor growth. Clinically, the levels of FTO, SLC7A11, and GPX4, are highly correlated expression in CRC tissues. Our findings reveal that FTO protects CRC from ferroptotic cell death in promoting CRC tumorigenesis through triggering SLC7A11/GPX4 expression.

FTO Positively Regulates Odontoblastic Differentiation via SMOC2 in Human Stem Cells from the Apical Papilla under Inflammatory Microenvironment.

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.

Long non-coding RNA GATA6-AS1 is mediated by N6-methyladenosine methylation and inhibits the proliferation and metastasis of gastric cancer.

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.

Exploring new frontiers: cell surface vimentin as an emerging marker for circulating tumor cells and a promising therapeutic target in advanced gastric Cancer.

BACKGROUND: Circulating tumor cells (CTCs) hold immense promise in guiding treatment strategies for advanced gastric cancer (GC). However, their clinical impact has been limited due to challenges in identifying epithelial-mesenchymal transition (EMT)-CTCs using conventional methods. METHODS: To bridge this knowledge gap, we established a detection platform for CTCs based on the distinctive biomarker cell surface vimentin (CSV). A prospective study involving 127 GC patients was conducted, comparing CTCs enumeration using both EpCAM and CSV. This approach enabled the detection of both regular and EMT-CTCs, providing a comprehensive analysis. Spiking assays and WES were employed to verify the reliability of this marker and technique. To explore the potential inducer of CSV+CTCs formation, a combination of Tandem Mass Tag (TMT) quantitative proteomics, m6A RNA immunoprecipitation-qPCR (MeRIP-qPCR), single-base elongation- and ligation-based qPCR amplification method (SELECT) and RNA sequencing (RNA-seq) were utilized to screen and confirm the potential target gene. Both in vitro and in vivo experiments were performed to explore the molecular mechanism of CSV expression regulation and its role in GC metastasis. RESULTS: Our findings revealed the potential of CSV in predicting therapeutic responses and long-term prognosis for advanced GC patients. Additionally, compared to the conventional EpCAM-based CTCs detection method, the CSV-specific positive selection CTCs assay was significantly better for evaluating the therapeutic response and prognosis in advanced GC patients and successfully predicted disease progression 14.25 months earlier than radiology evaluation. Apart from its excellent role as a detection marker, CSV emerges as a promising therapeutic target for attenuating GC metastasis. It was found that fat mass and obesity associated protein (FTO) could act as a potential catalyst for CSV+CTCs formation, and its impact on the insulin-like growth factor-I receptor (IGF-IR) mRNA decay through m6A modification. The activation of IGF-I/IGF-IR signaling enhanced the translocation of vimentin from the cytoplasm to the cell surface through phosphorylation of vimentin at serine 39 (S39). In a GC mouse model, the simultaneous inhibition of CSV and blockade of the IGF-IR pathway yielded promising outcomes. CONCLUSION: In summary, leveraging CSV as a universal CTCs marker represents a significant breakthrough in advancing personalized medicine for patients with advanced GC. This research not only paves the way for tailored therapeutic strategies but also underscores the pivotal role of CSV in enhancing GC management, opening new frontiers for precision medicine.

Fat mass and obesity-associated protein (FTO) mediated m6A modification of circFAM192A promoted gastric cancer proliferation by suppressing SLC7A5 decay.

Gastric cancer (GC) is a common malignant tumor worldwide, especially in East Asia, with high incidence and mortality rate. Epigenetic modifications have been reported to participate in the progression of gastric cancer, among which m6A is the most abundant and important chemical modification in RNAs. Fat mass and obesity-associated protein (FTO) is the first identified RNA demethylase but little is known about its role in gastric cancer. In our study, data from TCGA and clinical samples showed that FTO was highly expressed in gastric cancer tissues. Kaplan-Meier plotter suggested that patients with the high level of FTO had a poor prognosis. In vitro and in vivo experiments confirmed the role of FTO in promoting gastric cancer cell proliferation. Mechanistically, we found that FTO bound to circFAM192A at the specific site and removed the m6A modification in circFAM192A, protecting it from degradation. CircFAM192A subsequently interacted with the leucine transporter solute carrier family 7 member 5 (SLC7A5) and enhancing its stability. As a result, an increased amount of SLC7A5 was on the membrane, which facilitated leucine uptake and activated the mTOR signaling pathway. Therefore, our study demonstrated that FTO promoted gastric cancer proliferation through the circFAM192A/SLC7A5 axis in the m6A-dependent manner. Our study shed new light on the role of FTO in gastric cancer progression.

The m6A demethylase fat mass and obesity-associated protein mitigates pyroptosis and inflammation in doxorubicin-induced heart failure via the toll-like receptor 4/NF-κB pathway.

Background: Doxorubicin (Dox) can induce cardiotoxicity, thereby restricting the utility of this potent drug. Herein, the study ascertained the mechanism of the N6-methyladenosine (m6A) demethylase fat mass and obesity-associated protein (FTO) in pyroptosis and inflammation during Dox-induced heart failure (HF). Methods: Serum samples were collected from HF patients for detection of the expression of FTO and toll-like receptor 4 (TLR4). Dox-treated H9C2 cardiomyocytes were chosen for in vitro HF modeling, followed by measurement of FTO and TLR4 expression. Cardiomyocytes were detected for viability, apoptosis, spatial distribution of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), and the levels of lactic dehydrogenase, inflammatory factors, oxidative stress markers, and pyroptosis-related proteins. The m6A levels of mRNA were examined. RNA immunoprecipitation (RIP) and mRNA stability measurement were used to determine mRNA and protein expression, and RNA m6A dot blot and methylated-RIP assay were performed to detect m6A methylation levels. The expression of p-NF-κB p65 and p-IκB-α was measured by western blotting. Results: In the serum of HF patients, FTO was elevated while TLR4 was decreased. Dox treatment reduced FTO expression and increased m6A methylation levels and TLR4 expression in H9C2 cells. Overexpression of FTO and knockdown of TLR4 reduced apoptosis, cytotoxicity, inflammation, pyroptosis, oxidative stress, NLRP3 co-localization, and fluorescence intensity in Dox-induced H9C2 cells. Mechanistically, FTO resulted in reduced binding activity of YTHDF1 to TLR4 mRNA via m6A demethylation of TLR4, thus declining TLR4, p-NF-κB p65, and p-IκB-α expression. TLR4 knockdown counteracted the effects of FTO knockdown on Dox-induced H9C2 cells. Conclusions: FTO alleviated Dox-induced HF by blocking the TLR4/NF-κB pathway.

FTO attenuates TNF-α-induced damage of proximal tubular epithelial cells in acute pancreatitis-induced acute kidney injury via targeting AQP3 in an N6-methyladenosine-dependent manner.

BACKGROUND: Acute kidney injury (AKI) is a frequent complication of severe acute pancreatitis (SAP). Previous investigations have revealed the involvement of FTO alpha-ketoglutarate-dependent dioxygenase (FTO) and aquaporin 3 (AQP3) in AKI. Therefore, the aim of this study is to explore the association of FTO and AQP3 on proximal tubular epithelial cell damage in SAP-induced AKI. METHODS: An in-vitro AKI model was established in human proximal tubular epithelial cells (PTECs) HK-2 via tumor necrosis factor-α (TNF-α) induction (20 ng/mL), after which FTO and AQP3 expression was manipulated and quantified by quantitative real-time PCR and Western blotting. The viability and apoptosis of PTECs under various conditions, and reactive oxygen species (ROS), superoxide dismutase (SOD), and malonaldehyde (MDA) levels within these cells were measured using commercial assay kits and flow cytometry. Methylated RNA immunoprecipitation and mRNA stability assays were performed to elucidate the mechanism of FTO-mediated N6-methyladenosine (m6A) modification. Western blotting was performed to quantify ß-catenin protein levels in the PTECs. RESULTS: FTO overexpression attenuated the TNF-α-induced decrease in viability and SOD levels, elevated apoptosis, increased levels of ROS and MDA, and diminished TNF-α-induced AQP3 expression and reduced ß-catenin expression, but its silencing led to contradictory results. FTO negatively modulates AQP3 levels in RTECs in an m6A-depednent manner and compromises AQP3 stability. In addition, all FTO overexpression-induced effects in TNF-α-induced PTECs were neutralized following AQP3 upregulation. CONCLUSION: FTO alleviates TNF-α-induced damage to PTECs in vitro by targeting AQP3 in an m6A-dependent manner.

MicroRNA-192-5p downregulates Fat Mass and Obesity-associated Protein to aggravate renal ischemia/reperfusion injury.

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.

FTO facilitates cancer metastasis by modifying the m6A level of FAP to induce integrin/FAK signaling in non-small cell lung cancer.

BACKGROUND: Emerging evidence suggests the critical roles of N6-methyladenosine (m6A) RNA modification in tumorigenesis and tumor progression. However, the role of m6A in non-small cell lung cancer (NSCLC) is still unclear. This study aimed to explore the role of the m6A demethylase fat mass and obesity-associated protein (FTO) in the tumor metastasis of NSCLC. METHODS: A human m6A epitranscriptomic microarray analysis was used to identify downstream targets of FTO. Quantitative real-time PCR (qRT‒PCR) and western blotting were employed to evaluate the expression levels of FTO and FAP in NSCLC cell lines and tissues. Gain-of-function and loss-of-function assays were conducted in vivo and in vitro to assess the effects of FTO and FAP on NSCLC metastasis. M6A-RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), luciferase reporter assays, and RNA stability assays were used to explore the mechanism of FTO action. Co-immunoprecipitation (co-IP) assays were used to determine the mechanism of FAP in NSCLC metastasis. RESULTS: FTO was upregulated and predicted poor prognosis in patients with NSCLC. FTO promoted cell migration and invasion in NSCLC, and the FAK inhibitor defactinib (VS6063) suppressed NSCLC metastasis induced by overexpression of FTO. Mechanistically, FTO facilitated NSCLC metastasis by modifying the m6A level of FAP in a YTHDF2-dependent manner. Moreover, FTO-mediated metastasis formation depended on the interactions between FAP and integrin family members, which further activated the FAK signaling. CONCLUSION: Our current findings provided valuable insights into the role of FTO-mediated m6A demethylation modification in NSCLC metastasis. FTO was identified as a contributor to NSCLC metastasis through the activation of the FAP/integrin/FAK signaling, which may be a potential therapeutic target for NSCLC. Video Abstract.

Emerging evidence suggests the crucial roles of N⁶-methyladenosine (m⁶A) RNA modification in tumorigenesis and progression. Nonetheless, the role of m⁶A in NSCLC remains unclear. The purpose of this study was to investigate the role of m⁶A demethylase fat mass and obesity-associated protein (FTO) in the tumor metastasis of non-small cell lung cancer (NSCLC). Results illustrated that FTO was upregulated and predicted poor prognosis in NSCLC patients. FTO promoted cell migration and invasion in NSCLC, and the FAK inhibitor defactinib (VS6063) suppressed NSCLC metastasis induced by overexpression of FTO. Mechanistically, FTO facilitated NSCLC metastasis by modifying the m⁶A level of FAP in a YTHDF2-dependent manner. Moreover, FTO-mediated metastasis formation depended on the interactions between FAP and integrin family members, which further activated the FAK signaling. Our current findings provided valuable insights into the role of FTO-mediated m⁶A demethylation modification in NSCLC metastasis. FTO was identified as a contributor to NSCLC metastasis through the activation of the FAP/integrin/FAK signaling, which may be a potential therapeutic target for NSCLC.

Glycogen synthase kinase-3ß mediates toll-like receptors 4/nuclear factor kappa-B-activated cerebral ischemia-reperfusion injury through regulation of fat mass and obesity-associated protein.

BACKGROUND: Glycogen synthase kinase-3ß (GSK3ß), 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 GSK3ß lentivirus and overexpression of FTO lentivirus (in vitro), TLR4 inhibitor (TAK242), and LiCl to regulate GSK3ß, FTO, TLR4 expression, and GSK3ß activity, respectively. RESULTS: After GSK3ß 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 GSK3ß 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 GSK3ß in vivo, the reduction of FTO in mice suffering from MCAO was reversed. CONCLUSIONS: Our research shows that GSK3ß/FTO/TLR4 pathway contributes to cerebral I/R injury.

Increased m6A-RNA methylation and demethylase FTO suppression is associated with silica-induced pulmonary inflammation and fibrosis.

Silicosis is a severe worldwide occupational hazard, characterized with lung tissue inflammation and irreversible fibrosis caused by crystalline silicon dioxide. As the most common and abundant internal modification of messenger RNAs or noncoding RNAs, N6-methyladenosine (m6A) methylation is dysregulated in the chromic period of silicosis. However, whether m6A modification is involved in the early phase of silica-induced pulmonary inflammation and fibrosis and its specific effector cells remains unknown. In this study, we established a pulmonary inflammation and fibrosis mouse model by silica particles on day 7 and day 28. Then, we examined the global m6A modification level by m6A dot blot and m6A RNA methylation quantification kits. The key m6A regulatory factors were analyzed by RTqPCR, Western blot, and immunohistochemistry (IHC) in normal and silicosis mice. The results showed that the global m6A modification level was upregulated in silicosis lung tissues with the demethylase FTO suppression after silica exposure for 7 days and 28 days. METTL3, METTL14, ALKBH5, and other m6A readers had no obvious differences between the control and silicosis groups. Then, single-cell sequencing analysis revealed that thirteen kinds of cells were recognized in silicosis lung tissues, and the mRNA expression of FTO was downregulated in epithelial cells, endothelial cells, fibroblasts, and monocytes. These results were further confirmed in mouse lung epithelial cells (MLE-12) exposed to silica and in the peripheral blood mononuclear cells of silicosis patients. In conclusion, the high level of global m6A modification in the early stage of silicosis is induced by the downregulation of the demethylase FTO, which may provide a novel target for the diagnosis and treatment of silicosis.

PIAS1 upregulation confers protection against Cerulein-induced acute pancreatitis via FTO downregulation by enhancing sumoylation of Foxa2.

OBJECTIVE: This research discussed the specific mechanism by which PIAS1 affects acute pancreatitis (AP). METHODS: 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. RESULTS: 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. CONCLUSION: PIAS1 upregulation diminished FTO transcription by increasing Foxa2 SUMO modification, thereby ameliorating Cerulein-induced AP.

FTO-mediated m6A modification alleviates autoimmune uveitis by regulating microglia phenotypes via the GPC4/TLR4/NF-κB signaling axis.

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.

FTO alleviates cerebral ischemia/reperfusion-induced neuroinflammation by decreasing cGAS mRNA stability in an m6A-dependent manner.

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.

RNA sequencing reveals the mechanism of FTO in inhibiting inflammation and excessive proliferation of lipopolysaccharide-induced human glomerular mesangial cells.

Chronic glomerulonephritis (CGN) is a leading cause of end-stage renal disease in China; thus, there is an urgent need for effective therapeutic targets and strategies for CGN treatment. However, studies on CGN pathogenesis are limited. In this study, we found that the fat mass and obesity-associated protein (FTO) was significantly decreased in the lipopolysaccharide (LPS)-induced human glomerular mesangial cells (HGMCs) (P < 0.01) and kidney tissues of CGN patients (P < 0.05). Moreover, double-labeling immunofluorescence and flow cytometry assays demonstrated that the overexpression of FTO could inhibit inflammation and excessive proliferation of HGMCs. Furthermore, RNA-sequencing (RNA-seq) and real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that FTO overexpression induced differential expression of 269 genes (absolute fold change ≥ 2 and P-value < 0.05), including 143 upregulated and 126 downregulated genes. Further functional analysis of these differentially expressed genes by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses suggested that FTO possibly mediates its inhibitory function by regulating the mammalian target of rapamycin (mTOR) signaling pathway and substance metabolism. Lastly, analysis of the PPI network and further identification of the top 10 hub genes (RPS15, RPS18, RPL18A, GNB2L1, RPL19, EEF1A1, RPS25, FAU, UBA52, and RPS6) indicated that FTO mediates its function by affecting the ribosomal proteins. Therefore, in this study, we elucidated the important role of FTO in the regulation of inflammation and excessive proliferation of HGMCs, suggesting FTO administration as a suitable therapeutic intervention for CGN.

Metformin alleviates the cognitive impairment induced by benzo[a]pyrene via glucolipid metabolism regulated by FTO/FoxO6 pathway in mice.

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.

FTO-stabilized miR-139-5p targets ZNF217 to suppress prostate cancer cell malignancies by inactivating the PI3K/Akt/mTOR signal pathway.

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.

The m6A demethylase FTO promotes keloid formation by up-regulating COL1A1.

Background: Keloid is a dermal fibrotic disease characterized by excessive proliferation of dermal fibroblasts and deposition of excessive collagen. N6-methyladenosine (m6A) plays a significant role in numerous physiological and pathological regulatory processes in the human body. Fat mass and obesity-associated protein (FTO) is one of the most essential m6A demethylases. However, whether FTO has a regulatory role in keloid development remains to be determined. Methods: In this study, we investigated the effects of the m6A demethylase FTO on keloid formation by performing hematoxylin and eosin (H&E) staining, m6A dot blotting, transwell migration experiment, and methylated RNA immunoprecipitation quantitative polymerase chain reaction (MeRIP-qPCR) tests, as well as real-time PCR (RT-PCR) and Western blot assays. Results: The H&E staining indicated abnormal arrangement and proliferation of fibroblasts in the keloid tissue. The m6A dot blotting and qPCR revealed lower levels of m6A modification and increased expression of the m6A demethylases FTO in keloid tissue. Furthermore, overexpression of FTO promoted fibroblast migration as well as the expression of collagen type I alpha 1 chain (COL1A1) and α-smooth muscle actin (α-SMA). Mechanistic experiments demonstrated that FTO enhances keloid formation by modulating COL1A1 m6A modification and messenger RNA (mRNA) stability. In addition, this study also revealed the role of FTO in the therapeutic effect of glucocorticoids on keloids. Conclusions: Our study demonstrates that FTO upregulates COL1A1 expression via regulating COL1A1 m6A modification and maintaining mRNA stability, hence promoting keloid development and providing a potential new therapeutic target for the treatment of keloids.