Fat Mass And Obesity-associated Protein Expression Research Articles

SUMOylation modification of FTO facilitates oxidative damage response of arsenic by IGF2BP3 in an m6A-dependent manner

N6-methyladenosine (m6A) is the most common form of internal post-transcriptional methylation observed in eukaryotic mRNAs. The abnormally increased level of m6A within the cells can be catalyzed by specific demethylase fat mass and obesity-associated protein (FTO) and stay in a dynamic and reversible state. However, whether and how FTO regulates oxidative damage via m6A modification remain largely unclear. Herein, by using both in vitro and in vivo models of oxidative damage induced by arsenic, we demonstrated for the first time that exposure to arsenic caused a significant increase in SUMOylation of FTO protein, and FTO SUMOylation at lysine (K)− 216 site promoted the down-regulation of FTO expression in arsenic target organ lung, and therefore, remarkably elevating the oxidative damage via an m6A-dependent pathway by its specific m6A reader insulin-like growth factor-2 mRNA-binding protein-3 (IGF2BP3). Consequently, these findings not only reveal a novel mechanism underlying FTO-mediated oxidative damage from the perspective of m6A, but also imply that regulation of FTO SUMOylation may serve as potential approach for treatment of oxidative damage.

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.

Target-induced activation of dual DNAzyme recycling amplifications for sensitive electrochemical fat mass and obesity-associated protein assay

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 MboI 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.

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.

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. To investigate the effects of GATA6-AS1 on the proliferation, invasion and migration of gastric cancer cells and its mechanism of action. 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. 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. 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.

N6-methyladenosine demethyltransferase FTO mediated m6A modification of estrogen receptor alpha in non-small cell lung cancer tumorigenesis.

Fat mass and obesity-associated protein (FTO), which is closely linked with obesity and dietary intake, plays an important role in diet-related metabolic diseases. However, the underlying mechanism of the N6-methyladenosine (m6A) demethyltransferase FTO in tumor development and progression remains largely unexplored. Here, we demonstrated that FTO expression was largely lower in non-small cell lung cancer (NSCLC) samples than in adjacent healthy tissues, and its expression negatively correlated with poor prognosis. Gain- and loss-of-function assays revealed that FTO inhibited NSCLC tumor cell growth and metastasis in vitro and in vivo. Mechanistically, estrogen receptor alpha (ESR1) is a target of FTO, and increased FTO expression significantly impaired the m6A levels of ESR1 mRNA. There were two clear m6A modification sites (5247A and 5409A) in the 3' untranslated region (3'UTR) of ESR1, and FTO could decrease their methylation. Moreover, the m6A readers YTHDF1 and IGF2BP3 recognized and bound the m6A sites in ESR1 mRNA, thereby enhancing its stability and facilitating tumor growth. We also showed that ESR1 has good diagnostic value for NSCLC. In conclusion, we uncovered an important mechanism of epitranscriptomic regulation by the FTO-YTHDF1-IGF2BP3-ESR1 axis and identified the potential of m6A-dependent therapeutic strategies for NSCLC.

Fat mass and obesity-associated protein (FTO) affects midpalatal suture bone remodeling during rapid maxillary expansion.

The fat mass and obesity-associated protein (FTO) is an RNA demethylase that contributes to several physiological processes. Nonetheless, the impact of FTO on bone remodeling in the midpalatal suture while undergoing rapid maxillary expansion (RME) remains unclear. First, to explore the expression of FTO in the midpalatal suture during RME, six rats were randomly divided into two groups: Expansion group and Sham group (springs without being activated). Then, suture mesenchymal stem cells (SuSCs) were isolated as in vitro model. The expression of FTO was knocked down by small interfering RNA to study the effect of FTO on the osteogenic differentiation of SuSCs. Finally, to evaluate the function of FTO in the process of bone remodeling in the midpalatal suture, ten rats were randomly divided into two groups: FB23-2 group (10 μM, a small molecule inhibitor of FTO) and DMSO group (control). Increased arch width and higher expression of OCN and FTO in the midpalatal area were observed in expansion group (P < .05). In the in vitro model, the mRNA expression levels of Runx2, Bmp2, Col1a1, Spp1, and Tnfrsf11b were decreased (P < .05) upon knocking down FTO. Additionally, the protein levels of RUNX2 and OPN were also decreased (P < 0.05). Adding FB23-2, a small-molecule inhibitor targeting FTO, to the medium of SuSCs caused a decrease in the mRNA expression levels of Runx2, Bmp2, Col1a1, Spp1, and Tnfrsf11b (P < 0.05). There was a statistically significant difference in evaluating the expression of OCN and OPN on the palatal suture between the FB23-2 and DMSO groups (P < .05). The molecular mechanisms by which FTO regulates SuSCs osteogenesis remain to be elucidated. The FTO conditional knock out mouse model can be established to further elucidate the role of FTO during RME. FTO contributes to the osteogenic differentiation of SuSCs and plays a promoting role in midpalatal suture bone remodeling during the RME.

Construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues

Fat mass and obesity-associated protein (FTO) is a crucial eraser of RNA N6- methyladenosine (m6A) modification, and abnormal FTO expression level is implicated in pathogenesis of numerous cancers. Herein, we demonstrate the construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues. When FTO is present, it specifically erases the methyl group in m6A, inducing the cleavage of demethylated DNA by endonuclease DpnII and the generation of a single-stranded DNA (ssDNA) with a 3′-hydroxyl group. Subsequently, terminal deoxynucleotidyl transferase (TdT) promotes the incorporation of dTTPs into the ssDNA to obtain a long polythymidine (T) DNA sequence. The resultant long poly (T) DNA sequence can act as a template to trigger hyperbranched strand displacement amplification (HSDA), yielding numerous DNA fragments that may be stained by SYBR Gold to produce an enhanced fluorescence signal. This biosensor processes ultrahigh sensitivity with a detection limit of 1.65 × 10−10 mg/mL (2.6 fM), and it can detect the FTO activity in a single MCF-7 cell. Moreover, this biosensor can screen the FTO inhibitors, evaluate enzyme kinetic parameters, and discriminate the FTO expression levels in the tissues of breast cancer patients and healthy persons.

FTO ameliorates doxorubicin-induced cardiotoxicity by inhibiting ferroptosis via P53–P21/Nrf2 activation in a HuR-dependent m6A manner

Doxorubicin (DOX)-induced cardiotoxicity seriously limits its clinical applicability, and no therapeutic interventions are available. Ferroptosis, an iron-dependent regulated cell death characterised by lipid peroxidation, plays a pivotal role in DOX-induced cardiotoxicity. N6-methyladenosine (m6A) methylation is the most frequent type of RNA modification and involved in DOX-induced ferroptosis, however, its underlying mechanism remains unclear. P21 was recently found to inhibit ferroptosis by interacting with Nrf2 and is regulated in a P53-dependent or independent manner, such as through m6A modification. In the present study, we investigated the mechanism underlying m6A modification in DOX-induced ferroptosis by focusing on P21. Our results show that fat mass and obesity–associated protein (FTO) down-regulation was associated with DOX-induced cardiotoxicity. FTO over-expression significantly improved cardiac function and cell viability in DOX-treated mouse hearts and H9C2 cells. FTO over-expression significantly inhibited DOX-induced ferroptosis, and the Fer-1 inhibition of ferroptosis significantly reduced DOX-induced cardiotoxicity. P21 was significantly upregulated by FTO and activated Nrf2, playing a crucial role in the anti-ferroptotic effect. FTO upregulated P21/Nrf2 in a P53-dependent manner by mediating the demethylation of P53 or in a P53-independent manner by mediating P21/Nrf2 directly. Human antigen R (HuR) is crucial for FTO-mediated regulation of ferroptosis and P53–P21/Nrf2. Notably, we also found that P21 inhibition in turn inhibited HuR and P53 expression, while HuR inhibition further inhibited FTO expression. RNA immunoprecipitation assay showed that HuR binds to the transcripts of FTO and itself. Collectively, FTO inhibited DOX-induced ferroptosis via P21/Nrf2 activation by mediating the m6A demethylation of P53 or P21/Nrf2 in a HuR-dependent manner and constituted a positive feedback loop with HuR and P53–P21. Our findings provide novel insight into key functional mechanisms associated with DOX-induced cardiotoxicity and elucidate a possible therapeutic approach.

Lactylation-driven FTO targets CDK2 to aggravate microvascular anomalies in diabetic retinopathy

Diabetic retinopathy (DR) is a leading cause of irreversible vision loss in working-age populations. Fat mass and obesity-associated protein (FTO) is an N6-methyladenosine (m6A) demethylase that demethylates RNAs involved in energy homeostasis, though its influence on DR is not well studied. Herein, we detected elevated FTO expression in vitreous fibrovascular membranes of patients with proliferative DR. FTO promoted cell cycle progression and tip cell formation of endothelial cells (ECs) to facilitate angiogenesis in vitro, in mice, and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetic microvascular leakage, and mediated EC–microglia interactions to induce retinal inflammation and neurodegeneration in vivo and in vitro. Mechanistically, FTO affected EC features via modulating CDK2 mRNA stability in an m6A-YTHDF2-dependent manner. FTO up-regulation under diabetic conditions was driven by lactate-mediated histone lactylation. FB23-2, an inhibitor to FTO’s m6A demethylase activity, suppressed angiogenic phenotypes in vitro. To allow for systemic administration, we developed a nanoplatform encapsulating FB23-2 and confirmed its targeting and therapeutic efficiency in mice. Collectively, our study demonstrates that FTO is important for EC function and retinal homeostasis in DR, and warrants further investigation as a therapeutic target for DR patients.

Fat mass and obesity-associated (FTO) gene is essential for insulin secretion and β-cell function: In vitro studies using INS-1 cells and human pancreatic islets

AimsIn this study, we investigated the role of the FTO gene in pancreatic β-cell biology and its association with type 2 diabetes (T2D). To address this issue, human pancreatic islets and rat INS-1 (832/13) cells were used to perform gene silencing, overexpression, and functional analysis of FTO expression; levels of FTO were also measured in serum samples obtained from diabetic and obese individuals. ResultsThe findings revealed that FTO expression was reduced in islets from hyperglycemic/diabetic donors compared to normal donors. This reduction correlated with decreased INS and GLUT1 expression and increased PDX1, GCK, and SNAP25 expression. Silencing of Fto in INS-1 cells impaired insulin release and mitochondrial ATP production and increased apoptosis in pro-apoptotic cytokine-treated cells. However, glucose uptake and reactive oxygen species production rates remained unaffected. Downregulation of key β-cell genes was observed following Fto-silencing, while Glut2 and Gck were unaffected. RNA-seq analysis identified several dysregulated genes involved in metal ion binding, calcium ion binding, and protein serine/threonine kinase activity. Furthermore, our findings showed that Pdx1 or Mafa-silencing did not influence FTO protein expression. Overexpression of FTO in human islets promoted insulin secretion and upregulated INS, PDX1, MAFA, and GLUT1 expression. Serum FTO levels did not significantly differ between individuals with diabetes or obesity and their healthy counterparts. ConclusionThese findings suggest that FTO plays a crucial role in β-cell survival, metabolism, and function and point to a potential therapeutic utility of FTO in T2D patients.

The FTO-CMPK2 Pathway in Fibroblast-like Synoviocytes Modulates Rheumatoid Arthritis Synovial Inflammation and Cartilage Homeostasis via mtDNA Regulation.

In rheumatoid arthritis (RA), a debilitating autoimmune disorder marked by chronic synovial inflammation and progressive cartilage degradation, fibroblast-like synoviocytes (FLS) are key pathogenic players. Current treatments targeting these cells are limited. Our study focused on the Fat Mass and Obesity-associated protein (FTO), known for its roles in cell proliferation and inflammatory response modulation, and its involvement in RA. We specifically examined the inflammatory regulatory roles of FTO and CMPK2, a mitochondrial DNA synthesis protein, in FLS. Utilizing a combination of in vitro and in vivo methods, including FTO inhibition and gene knockdown, we aimed to understand FTO's influence on RA progression and chondrocyte functionality. Our findings showed that increased FTO expression in RA synovial cells enhanced their proliferation and migration and decreased senescence and apoptosis. Inhibiting FTO significantly slowed the disease progression in our models. Our research also highlighted that the FTO-CMPK2 pathway plays a crucial role in regulating synovial inflammation through the mtDNA-mediated cGAS/STING pathway, affecting chondrocyte homeostasis. This study indicates that targeting the FTO-CMPK2 axis could be a promising new therapeutic strategy for managing RA.

RNA m6A dynamic modification mediated by nucleus-localized FTO is involved in follicular reserve.

In eukaryotic organisms, the most common internal modification of messenger RNA (mRNA) is N6-methyladenosine (m6A). This modification can be dynamically and reversibly controlled by specific enzymes known as m6A writers and erasers. The fat-mass and obesity-associated protein (FTO) catalyzes RNA demethylation and plays a critical role in various physiological and pathological processes. Our research identified dynamic alterations in both m6A and FTO during the assembly of primordial follicles, with an inverse relationship observed for m6A levels and nuclear-localized FTO expression. Application of Fto small interfering RNA (siRNA) altered the expression of genes related to cell proliferation, hormone regulation, and cell chemotaxis, and affected RNA alternative splicing. Overexpression of the full-length Fto gene led to changes in m6A levels, alternative splicing of Cdk5, cell proliferation, cell cycle progression, and proportion of primordial follicles. Conversely, overexpression of Fto lacking a nuclear localization signal (NLS) did not significantly alter m6A levels or primordial follicle assembly. These findings suggest that FTO, localized in the nucleus but not in the cytoplasm, regulates RNA m6A demethylation and plays a role in cell proliferation, cell cycle progression, and primordial follicle assembly. These results highlight the potential of m6A and its eraser FTO as possible biomarkers and therapeutic targets.

FTO up-regulation induced by MYC suppresses tumour progression in Epstein-Barr virus-associated gastric cancer.

Epstein-Barr virus-associated gastric cancer (EBVaGC) is regarded as a distinct molecular subtype of GC, accounting for approximately 9% of all GC cases. Clinically, EBVaGC patients are found to have a significantly lower frequency of lymph node metastasis and better prognosis than uninfected individuals. RNA N6-methyladenosine (m6A) modification has an indispensable role in modulating tumour progression in various cancer types. However, its impact on EBVaGC remains unclear. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and m6A dot blot were conducted to compare the m6A modification levels between EBVaGC and EBV-negative GC (EBVnGC) cells. Western blot, real-time quantitative PCR (RT-qPCR) and immunohistochemistry were applied to explore the underlying mechanism of the reduced m6A modification in EBVaGC. The biological function of fat mass and obesity-associated protein (FTO) was determined in vivo and in vitro. The target genes of FTO were screened by MeRIP-seq, RT-qPCR and Western blot. The m6A binding proteins of target genes were verified by RNA pulldown and RNA immunoprecipitation assays. Chromatin immunoprecipitation and Luciferase report assays were performed to investigate the mechanism how EBV up-regulated FTO expression. M6A demethylase FTO was notably increased in EBVaGC, leading to a reduction in m6A modification, and higher FTO expression was associated with better clinical outcomes. Furthermore, FTO depressed EBVaGC cell metastasis and aggressiveness by reducing the expression of target gene AP-1 transcription factor subunit (FOS). Methylated FOS mRNA was specifically recognized by the m6A 'reader' insulin-like growth factor 2 mRNA binding protein 1/2 (IGF2BP1/2), which enhanced its transcripts stability. Moreover, MYC activated by EBV in EBVaGC elevated FTO expression by binding to a specific region of the FTO promoter. Mechanistically, our work uncovered a crucial suppressive role of FTO in EBVaGC metastasis and invasiveness via an m6A-FOS-IGF2BP1/2-dependent manner, suggesting a promising biomarker panel for GC metastatic prediction and therapy.

Prenatal Nicotine Exposure Raises Male Blood Pressure via FTO-Mediated NOX2/ROS Signaling.

Cigarette smoking/nicotine exposure in pregnancy shows an increased risk of hypertension in offspring, but the mechanisms are unclear. This study tested the hypothesis that m6A RNA hypomethylation epigenetically regulates vascular NOX (NADPH oxidase) and reactive oxygen species production, contributing to the fetal programming of a hypertensive phenotype in nicotine-exposed offspring. Pregnant rats were exposed to episodic chronic intermittent nicotine aerosol (CINA) or saline aerosol control from gestational day 4 to day 21, and experiments were performed in 6-month-old adult offspring. Antenatal CINA exposure augmented Ang II (angiotensin II)-stimulated blood pressure response in male, but not female offspring. Moreover, CINA increased vascular NOX2 expression and superoxide production exclusively in male offspring. Inhibition of NOX2 with gp91ds-tat, both ex vivo and in vivo, mitigated the CINA-induced elevation in superoxide production and blood pressure response. Notably, CINA enhanced the expression of vascular m6A demethylase FTO (fat mass and obesity-associated protein), while reducing the total vascular m6A abundance and specific m6A methylation of the NOX2 gene. Additionally, ex vivo inhibition of FTO with FB23-2 attenuated CINA-induced increases in vascular NOX2 expression. In vitro experiments using human umbilical vein endothelial cells demonstrated that nicotine dose-dependently upregulated FTO and NOX2 protein abundance, which were reversed by treatment with the FTO inhibitor FB23-2 or FTO knockdown using siRNAs. This study uncovers a new mechanism: m6A demethylase FTO-mediated epigenetic upregulation of vascular NOX2 signaling in CINA-induced hypertensive phenotype. This insight could lead to a therapeutic target for preventing and treating developmental hypertension programming.

Overexpression of FTO inhibits excessive proliferation and promotes the apoptosis of human glomerular mesangial cells by alleviating FOXO6 m6A modification via YTHDF3-dependent mechanisms.

Background: N6-methyladenosine (m6A) is a prevalent post-transcriptional modification presented in messenger RNA (mRNA) of eukaryotic organisms. Chronic glomerulonephritis (CGN) is characterised by excessive proliferation and insufficient apoptosis of human glomerular mesangial cells (HGMCs) but its underlying pathogenesis remains undefined. Moreover, the role of m6A in CGN is poorly understood. Methods: The total level of m6A modification was detected using the m6A quantification assay (Colorimetric). Cell proliferation was assessed by EdU cell proliferation assay, and cell apoptosis was detected by flow cytometry. RNA sequencing was performed to screen the downstream target of fat mass and obesity-associated protein (FTO). MeRIP-qPCR was conducted to detect the m6A level of forkhead box o6 (FOXO6) in HGMCs. RIP assay was utilized to indicate the targeting relationship between YTH domain family 3 (YTHDF3) and FOXO6. Actinomycin D assay was used to investigate the stability of FOXO6 in HGMCs. Results: The study found that the expression of FTO was significantly reduced in lipopolysaccharide (LPS)-induced HGMCs and renal biopsy samples of patients with CGN. Moreover, FTO overexpression and knockdown could regulate the proliferation and apoptosis of HGMCs. Furthermore, RNA sequencing and cellular experiments revealed FOXO6 as a downstream target of FTO in regulating the proliferation and apoptosis of HGMCs. Mechanistically, FTO overexpression decreases the level of FOXO6 m6A modification and reduces the stability of FOXO6 mRNA in a YTHDF3-dependent manner. Additionally, the decreased expression of FOXO6 inhibits the PI3K/AKT signaling pathway, thereby inhibiting the proliferation and promoting apoptosis of HGMCs. Conclusion: This study offers insights into the mechanism through which FTO regulates the proliferation and apoptosis of HGMCs by mediating m6A modification of FOXO6 mRNA. These findings also suggest FTO as a potential diagnostic marker and therapeutic target for CGN.

Disruption of CerS6-mediated sphingolipid metabolism by FTO deficiency aggravates ulcerative colitis

Background and aimsDeregulation of RNA N6-methyladenosine (m6A) modification in intestinal epithelial cells (IECs) influences intestinal immune cells and leads to intestinal inflammation. We studied the function of fat mass-and obesity-associated...

Suppression of TLR4 prevents diabetic bone loss by regulating FTO-mediated m6A modification.

Toll-like receptor-4 (TLR4) has been implicated in the development and progression of diabetic osteoporosis. However, the mechanisms underlying TLR4-regulated bone metabolism in diabetes are yet to be fully understood. Epigenetic modifications have been indicated as a possible mechanism leading to increased risk of osteoporosis and bone fracture. As N6-methyladenosine (m6A) is the most common epigenetic modification in eukaryotic mRNAs, we hypothesized that TLR4 regulates m6A modification in bone tissues of diabetic rats, thereby potentially explaining the pathogenesis of diabetic bone loss. m6A sequencing (m6A-seq) was performed in samples of the femur of TLR4-wild type (TLR4WT) and TLR4-knockout (TLR4KO) diabetic rats to identify genes with differential m6A modifications that may be associated with the bone loss phenotype. We found that in TLR4KO rats, the rapid weight loss of diabetic rats was prevented, and bone mineral density (BMD) was significantly increased. m6A-seq and Gene Ontology enrichment analysis revealed that m6A-modified genes in the femur of TLR4KO diabetic rats were associated with regulation of biological processes such as osteoclast differentiation. qRT-PCR analysis on the expression levels of the m6A-modified methyltransferases and demethylases demonstrated that only the m6A demethylase fat mass and obesity-associated protein（FTO）was decreased. Using an osteoclast cell model, we confirmed that TLR4-mediated osteoclast differentiation was induced by glycolipid toxicity via inhibition of FTO expression. Taken together, these results suggest that inhibition of TLR4 may prevent diabetic bone loss via regulation of FTO-mediated m6A modification.

M6A eraser FTO impairs gemcitabine resistance in pancreatic cancer through influencing NEDD4 mRNA stability by regulating the PTEN/PI3K/AKT pathway

BackgroundGemcitabine resistance has brought great challenges to the treatment of pancreatic cancer. The N6-methyladenosine (m6A) mutation has been shown to have a significant regulatory role in chemosensitivity; however, it is not apparent whether gemcitabine resistance can be regulated by fat mass and obesity-associated protein (FTO).MethodsCells with established gemcitabine resistance and tissues from pancreatic cancer patients were used to evaluate FTO expression. The biological mechanisms of the effects of FTO on gemcitabine resistant cells were investigated using CCK-8, colony formation assay, flow cytometry, and inhibitory concentration 50. Immunoprecipitation/mass spectrometry, MeRIP-seq, RNA sequencing and RIP assays, RNA stability, luciferase reporter, and RNA pull down assays were employed to examine the mechanism of FTO affecting gemcitabine resistant pancreatic cancer cells.ResultsThe results revealed that FTO was substantially expressed in cells and tissues that were resistant to gemcitabine. Functionally, the gemcitabine resistance of pancreatic cancer could be enhanced by FTO, while its depletion inhibited the growth of gemcitabine resistant tumor cells in vivo. Immunoprecipitation/mass spectrometry showed that the FTO protein can be bound to USP7 and deubiquitinated by USP7, leading to the upregulation of FTO. At the same time, FTO knockdown significantly decreased the expression level of NEDD4 in an m6A-dependent manner. RNA pull down and RNA immunoprecipitation verified YTHDF2 as the reader of NEDD4, which promoted the chemoresistance of gemcitabine resistant cells. FTO knockdown markedly increased the PTEN expression level in an NEDD4-dependent manner and influenced the chemosensitivity to gemcitabine through the PI3K/AKT pathway in pancreatic cancer cells.ConclusionIn conclusion, we found that gemcitabine resistance in pancreatic cancer can be influenced by FTO that demethylates NEDD4 RNA in a m6A-dependent manner, which then influences the PTEN expression level and thereby affects the PI3K/AKT pathway. We also identified that the FTO level can be upregulated by USP7.

Construction of a Quantum-Dot-Based FRET Nanosensor through Direct Encoding of Streptavidin-Binding RNA Aptamers for N6-Methyladenosine Demethylase Detection.

N6-Methyladenosine (m6A) demethylases can catalyze the removal of the methyl modification on m6A, and it is closely associated with the occurrence, proliferation, differentiation, and metastasis of malignancies. The m6A demethylases (e.g., fat mass and obesity-associated protein (FTO)) may act as a cancer biomarker and are crucial for anticancer drug screening and early clinical diagnosis. Herein, we demonstrate the construction of a quantum-dot-based Förster resonance energy-transfer (FRET) nanosensor through direct encoding of streptavidin-binding RNA aptamers (SA aptamers) for m6A demethylase detection. This nanosensor employs multiple Cy5-molecule-labeled SA aptamers as the building materials to construct the 605QD-RNA-Cy5 nanoassembly, and it exploits the hinder effect of m6A upon elongation and ligation reactions to distinguish m6A-containing RNA probes from demethylated RNA probes. When m6A demethylase is present, the m6A-containing RNA probes are demethylated to generate the demethylated RNA probes, initiating strand extension and ligation reactions to yield a complete transcription template for SA aptamers. Subsequently, a T7-assisted cascade transcription amplification reaction is activated to transcribe abundant SA aptamers with the incorporation of multiple Cy5 fluorophores. The Cy5-incorporated SA aptamers can self-assembly onto the streptavidin-coated 605QD surface to obtain the 605QD-SA aptamer-Cy5 nanoassemblies, resulting in the generation of distinct FRET signals. This nanosensor exhibits ultrahigh sensitivity and excellent specificity, and it can detect endogenous FTO at the single-cell level. Furthermore, this nanosensor can precisely measure enzyme kinetic parameters, screen m6A demethylase inhibitors, and differentiate the FTO expression between breast cancer patients and healthy individual tissues, offering a versatile platform for clinical diagnostic and drug discovery.