FTO Protein Research Articles

Honokiol inhibits human osteosarcoma MG63 cell migration by upregulating FTO and Smad6 to promote autophagy

BackgroundOsteosarcoma (OS) is a common primary malignant tumor of bone, most commonly seen in children and adolescents, which has a low survival rate and is a serious threat to patients' lives. Honokiol (HKL) is the main active components of Magnolia officinalis, which have significant anti-tumor properties. The aim of this study was to observe the autophagic and migratory effects of HKL on MG63 cells and to investigate whether the mechanism of action was related to FTO and Smad6. MethodsFirstly, we cultured MG63 cells in vitro and intervened with different concentrations of HKL to detect cell activity by CCK8, apoptosis by flow cytometry, cell migration ability by scratch assay, cell invasion ability by transwell assay and MMP2, P62, LC3 I/II, FTO and Smad6 protein expression by Western blot. ResultsHKL inhibited MG63 cells activity and that this effect was dose and time dependent. Although there was no significant effect on apoptosis and invasive ability, HKL could act through effects such as promoting cell autophagy and inhibiting migration. HKL increased the protein expression levels of FTO, Smad6, MMP2, LC3 I/II and P62, and this effect was reduced after silencing of Smad6. ConclusionsHKL induced autophagy and inhibited cell migration in MG63 cells by increasing the expression of FTP and Smad6. It can be seen that HKL may be a promising drug for the treatment of OS.

Fat mass and obesity associated gene and homeobox transcription factor iriquois-3 mRNA profiles in the metabolic tissues of zebrafish are modulated by feeding and food deprivation

Fat mass and obesity associated gene (FTO) has been strongly associated with obesity, and it is functionally linked to the homeobox transcription factor iriquois-3 (IRX3). In mammals, FTO and IRX3 are involved in the regulation of food intake and metabolism. This study aimed to determine whether FTO and IRX3are affected by feeding and food unavailability. FTO and IRX3 mRNA and protein were found widely distributed in all tissues examined, including the brain, muscle, gut, and liver. Postprandial increase in the abundance of FTO and IRX3 mRNAs was observed in metabolic tissues of both male and female zebrafish at 1 h post-feeding. Meanwhile, their expression in the brain and gut decreased at 3 h post-feeding, reaching preprandial levels. Additionally, FTO and IRX3 mRNA abundance in examined tissues increased after 7 days of food deprivation, but substantially decreased after refeeding for 24 h. In summary, we report that both FTO and IRX3 are meal-sensitive genes in zebrafish. The fasting-induced increase suggests a possible appetite regulatory role for FTO and IRX3 in zebrafish. These findings highlight the importance of FTO and IRX3 in appetite and metabolic regulation in zebrafish.

Comprehensive analysis of the m6A demethylase FTO in endothelial dysfunction by MeRIP sequencing

N6-methyladenosine (m6A) is the most general post-transcriptional modification of eukaryotic mRNAs and long-stranded non-coding RNAs. In this process, It has been shown that FTO associates with the m6A mRNA demethylase and plays a role in diabetic vascular endothelial dysfunction. In the present study, we detected FTO protein expression in HUVECs by Western blot and found that FTO was highly expressed in all disease groups relative to the control group. To explore the mechanism of FTO in T2DM vasculopathy, we performed an analysis by methylated RNA immunoprecipitation sequencing (MeRIP-seq) to elucidate the role of aberrant m6A modification and mRNA expression in endothelial dysfunction. The results showed 202 overlapping genes with varying m6A modifications and varied mRNA expression, and GO and KEGG enrichment analysis revealed that these genes were predominantly enriched in pathways associated with T2DM complications and endothelial dysfunction. By an integrated analysis of MeRIP-seq and RNA-seq results, the IGV plots showed elevated kurtosis of downstream candidate gene modifications, which may be downstream targets for FTO to exercise biological functions. HOXA9 and PLAU mRNA expression levels were significantly down after FTO inhibition. In the current work, we set up a typological profile of the m6A genes among HUVECs as well as uncovered a hidden relationship between RNA methylation modifications for T2DM vasculopathy-associated genes. Taken together, this study indicates that endothelial functional impairment is present in T2DM patients and may be related to aberrant expression of FTO.

Risk of Fat Mass- and Obesity-Associated Gene-Dependent Obesogenic Programming by Formula Feeding Compared to Breastfeeding.

It is the purpose of this review to compare differences in postnatal epigenetic programming at the level of DNA and RNA methylation and later obesity risk between infants receiving artificial formula feeding (FF) in contrast to natural breastfeeding (BF). FF bears the risk of aberrant epigenetic programming at the level of DNA methylation and enhances the expression of the RNA demethylase fat mass- and obesity-associated gene (FTO), pointing to further deviations in the RNA methylome. Based on a literature search through Web of Science, Google Scholar, and PubMed databases concerning the dietary and epigenetic factors influencing FTO gene and FTO protein expression and FTO activity, FTO's impact on postnatal adipogenic programming was investigated. Accumulated translational evidence underscores that total protein intake as well as tryptophan, kynurenine, branched-chain amino acids, milk exosomal miRNAs, NADP, and NADPH are crucial regulators modifying FTO gene expression and FTO activity. Increased FTO-mTORC1-S6K1 signaling may epigenetically suppress the WNT/β-catenin pathway, enhancing adipocyte precursor cell proliferation and adipogenesis. Formula-induced FTO-dependent alterations of the N6-methyladenosine (m6A) RNA methylome may represent novel unfavorable molecular events in the postnatal development of adipogenesis and obesity, necessitating further investigations. BF provides physiological epigenetic DNA and RNA regulation, a compelling reason to rely on BF.

Effect of Triclisia subcordata Oliv. (Menispermaceae) leaves on hormonal imbalance and genes expression in the ovaries of letrozole-induced polycystic rats via in vivo and computational approaches

The hormonal imbalance, insulin sensitivity, and ovarian function are among the biological processes linked to polycystic ovarian syndrome (PCOS). Hence, in PCOS ovaries, the impact of Triclisia subcordata Oliv. leaf (TSL) on hormone levels and the expression of the genes FTO, CYP11A, IRS, P53, and HSD17B2 was studied. Comparable to a normal control group, the rats treated with TSL methanol extract had the lowest levels of testosterone and the highest quantity of the follicle-stimulating hormone. In the ovaries of PCOS rats, TSL methanol extract strongly reduced HSD17B2 expression and significantly increased IRS expression. Furthermore, significant changes in FTO expression were detected, suggesting possible correlations with metabolic disorders, hormone dysregulation, and features of ovarian function. Additionally, P53 expression significantly decreased. GC-MS analysis revealed the presence of farnesol formate, 4-(4-ethylcyclohexyl) -1-pentyl, and cyclohexene as phytoconstituents. In T. subcordata leaves, cyclohexene, 4-(4-ethylcyclohexyl)-1-pentyl-, and farnesol formate showed a more unusual and specific binding to the p53 core, FTO protein, and cytochrome P450 CYP11A1 domains. According to the molecular dynamics study, one of the main mechanisms that have been proposed for the recovery of metabolic syndrome in affected individuals is the stabilization of proteins by the binding of these chemicals to the targeted structure. All these results suggest that Triclisia subcordata leaves may have complex impacts on PCOS-related molecular pathways, including restoring normalcy to insulin sensitivity, androgen metabolism, hormone balance, and ovarian function.

FTO is Associated with Patient Prognosis and Immune Infiltrates in Gastric Cancer and Regulates TGF-β Expression.

This study aimed to examine the associations of FTO expression with prognosis, tumor microenvironment (TME), immune cell infiltration, immune checkpoint genes, and relevant signaling pathways in GC. Furthermore, the relationship between FTO and TGF-β was studied in GC. The mRNA expression and clinical survival data of GC samples were obtained from The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD). TIMER2, TNM plot, and GEPIA database were used to analyze FTO expression. The associations of FTO with prognosis and clinicopathologic features were assessed using the Kaplan-Meier plotter and UALCAN database, respectively. The R software was employed to analyze its related signaling pathways and the associations with TME, immune cell infiltration, and immune checkpoint genes. GEPIA and ENCORI were used to examine the association of FTO with TGF-β expression. The SRAMP website was utilized to predict m6A modification of TGF-β. IHC, Western blot, and qPCR were used to analyze the expression levels of FTO and TGF-β in clinical gastric cancer tissue samples or gastric cancer cell lines. In addition, a m6A RNA methylation assay kit was used to determine m6A levels in gastric cancer cells. FTO mRNA and protein levels were significantly elevated in GC compared to normal gastric tissues. Kaplan-Meier survival analysis suggested that upregulated FTO was associated with a worse prognosis in GC. Upregulated FTO was markedly correlated with differentiation degree, lymph node metastasis, and clinical TNM stage. GO and KEGG pathway analyses revealed that FTO-associated molecules were enriched in neuroactive ligand-receptor interaction, calcium signaling, PI3k-Akt signaling, cAMP signaling pathways, and TGF-β signaling pathways, among others. The TME score was remarkably higher in the high-FTO group than in the low-FTO group. Furthermore, FTO expression had positive correlations with different types of immune cells and immune checkpoint genes. Moreover, FTO may regulate TGF-β in an m6A RNA modification manner in GC. FTO may become an independent predictive prognostic biomarker correlating with TME, immune cell infiltration, and immune checkpoint genes in gastric cancer and might influence GC progression by regulating TGF-β expression.

An α-ketoglutarate conformational switch controls iron accessibility, activation, and substrate selection of the human FTO protein

The fat mass and obesity-associated fatso (FTO) protein is a member of the Alkb family of dioxygenases and catalyzes oxidative demethylation of N6-methyladenosine (m6A), N1-methyladenosine (m1A), 3-methylthymine (m3T), and 3-methyluracil (m3U) in single-stranded nucleic acids. It is well established that the catalytic activity of FTO proceeds via two coupled reactions. The first reaction involves decarboxylation of alpha-ketoglutarate (αKG) and formation of an oxyferryl species. In the second reaction, the oxyferryl intermediate oxidizes the methylated nucleic acid to reestablish Fe(II) and the canonical base. However, it remains unclear how binding of the nucleic acid activates the αKG decarboxylation reaction and why FTO demethylates different methyl modifications at different rates. Here, we investigate the interaction of FTO with 5-mer DNA oligos incorporating the m6A, m1A, or m3T modifications using solution NMR, molecular dynamics (MD) simulations, and enzymatic assays. We show that binding of the nucleic acid to FTO activates a two-state conformational equilibrium in the αKG cosubstrate that modulates the O2 accessibility of the Fe(II) catalyst. Notably, the substrates that provide better stabilization to the αKG conformation in which Fe(II) is exposed to O2 are demethylated more efficiently by FTO. These results indicate that i) binding of the methylated nucleic acid is required to expose the catalytic metal to O2 and activate the αKG decarboxylation reaction, and ii) the measured turnover of the demethylation reaction (which is an ensemble average over the entire sample) depends on the ability of the methylated base to favor the Fe(II) state accessible to O2.

Targeting the oncogenic m6A demethylase FTO suppresses tumourigenesis and potentiates immune response in hepatocellular carcinoma

ObjectiveFat mass and obesity-associated protein (FTO), an eraser of N6-methyadenosine (m6A), plays oncogenic roles in various cancers. However, its role in hepatocellular carcinoma (HCC) is unclear. Furthermore, small extracellular vesicles...

LncRNA CCRR maintains Ca2+ homeostasis against myocardial infarction through the FTO-SERCA2a pathway.

Cardiac conduction regulatory RNA (CCRR) has been documented as an antiarrhythmic lncRNA in our earlier investigation. This study aimed to evaluate the effects of CCRR on SERCA2a and the associated Ca2+ homeostasis in myocardial infarction (MI). Overexpression of CCRR via AAV9-mediated delivery not only partially reversed ischemia-induced contractile dysfunction but also alleviated abnormal Ca2+ homeostasis and reduced the heightened methylation level of SERCA2a following MI. These effects were also observed in CCRR over-expressing transgenic mice. A conserved sequence domain of CCRR mimicked the protective function observed with the full length. Furthermore, silencing CCRR in healthy mice led to intracellular Ca2+ overloading of cardiomyocytes. CCRR increased SERCA2a protein stability by upregulating FTO expression. The direct interaction between CCRR and FTO protein was characterized by RNA-binding protein immunoprecipitation (RIP) analysis and RNA pulldown experiments. Activation of NFATc3 was identified as an upstream mechanism responsible for CCRR downregulation in MI. This study demonstrates that CCRR is a protective lncRNA that acts by maintaining the function of FTO, thereby reducing the m6A RNA methylation level of SERCA2a, ultimately preserving calcium homeostasis for myocardial contractile function in MI. Therefore, CCRR may be considered a promising therapeutic strategy with a beneficial role in cardiac pathology.

Abstract 3290: FTO mediates tumor growth and resistance to erlotinib in NSCLC

Abstract Lung cancer, among the most common cancer types, ranks first in cancer associated deaths. Nearly 85% of the lung cancer cases are non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR), an established marker for NSCLC, is frequently overexpressed on the cell surface. Amplifying mutations often render these receptors activated in a ligand independent manner, therefore, therapies such as erlotinib, an EGFR tyrosine kinase inhibitor (EGFR-TKI), target these activating mutations and inhibit tumor growth. Nevertheless, patients acquire resistance to EGFR TKI within 9-14 months of treatment. Recent studies show that fat mass and obesity- associated (FTO) gene, initially identified to increase the body mass index, plays a major role in cancer development and acquired therapy resistance in several cancer types. FTO demethylates m6A mRNA and modulates tumorigenesis, and drug resistance. In this study, we explored the functions of FTO in EGFR TKI resistance in NSCLC. To study the expression of FTO in erlotinib resistant cells, we used wildtype-EGFR NSCLC cell lines (H2170, H358) and mutant EGFR NSCLC cell lines (H1975, PC9) that were rendered resistant to erlotinib. Our RT-qPCR results demonstrated that FTO was upregulated by 1.7-3.0 fold in erlotinib resistant H2170, H358, PC9 and H1975 cells. FTO protein was upregulated by 1.3-1.6 fold as seen by western blotting, and by 1.7 to 2.6 fold in the nucleus of erlotinib resistant H1975, H2170 and PC9 cell lines by immunofluorescence. Furthermore, we investigated FTO inhibition with Dac51 (FTOi) on cell viability and cell migration properties. MTT cell viability assay showed a 9-17% decrease in cell viability when treated with a combination of Dac51 and erlotinib compared to Dac51 alone in erlotinib resistant H2170 and PC9 cells, respectively. The combinatory treatment showed an additive effect compared to the Dac51 and erlotinib treatments alone. Wound healing assay revealed an 18% open wound area after 48-hour treatment with both Dac51 and erlotinib compared to 0.1% after treatment with erlotinib alone in erlotinib resistant H2170 cells. To evaluate the effect of siRNA mediated FTO (siFTO) knockdown on cell viability and energy metabolism, MTT and luciferase assays were performed. MTT assay showed a 21-28% decrease in viability in erlotinib resistant H1975, H2170, PC9 cells, demonstrating increase in the erlotinib efficacy, as compared to the mock siRNA treatment. Luciferase assay showed an inverse relationship between the FTO and ATP, where the latter was upregulated by 1.3 to 3.4 folds in erlotinib resistant H1975, H2170 and PC9 cells, when treated with siFTO, suggesting its involvement in the energy requirements of the NSCLC cells. In conclusion, our study suggests FTO was upregulated in erlotinib resistant NSCLC cells on both the gene and protein levels. FTO could mediate resistance to EGFR TKI possibly through cell proliferation and migration and help with prognosis of NSCLC patients. Citation Format: Aayush Rastogi, Rong Qiu, Neelu Puri. FTO mediates tumor growth and resistance to erlotinib in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3290.

Mechanism of Fat Mass and Obesity-Related Gene-Mediated Heme Oxygenase-1 m6A Modification in the Recovery of Neurological Function in Mice with Spinal Cord Injury.

This study examined the mechanism of fat mass and obesity-related gene (FTO)-mediated heme oxygenase-1 (HO-1) m6A modification facilitating neurological recovery in spinal cord injury (SCI) mice. FTO/HO-1 was identified as a key regulator of SCI as well as a potential target for treatment of SCI. An SCI mouse was treated with pcDNA3.1-FTO/pcDNA3.1-NC/Dac51. An oxygen/glucose deprivation (OGD) cell model simulated SCI, with cells treated with pcDNA3.1-FTO/si-HO-1/Dac51. Motor function and neurobehavioral evaluation were assessed using the Basso, Beattie, and Bresnahan (BBB) scale and modified neurological severity score (mNSS). Spinal cord pathology and neuronal apoptosis were assessed. Further, FTO/HO-1 mRNA and protein levels, HO-1 mRNA stability, the interaction of YTHDF2 with HO-1 mRNA, neuronal viability/apoptosis, and HO-1 m6A modification were evaluated. Spinal cord injury mice exhibited reduced BBB, elevated mNSS scores, disorganized spinal cord cells, scattered nuclei, and severe nucleus pyknosis. pcDNA3.1-FTO elevated FTO mRNA, protein expression, and BBB score; reduced the mNSS score of SCI mice; decreased neuronal apoptosis; improvedthe cell arrangement; and improved nucleus pyknosis in spinal cord tissues. OGD decreased FTO expression. FTO upregulation ameliorated OGD-induced neuronal apoptosis. pcDNA3.1-FTO reduced HO-1 mRNA and protein and HO-1 m6A modification, while increasing HO-1 mRNA stability and FTO in OGD-treated cells. FTO upregulated HO-1 by modulating m6A modification. HO-1 downregulation attenuated the effect of FTO. pcDNA3.1-FTO/Dac51 increased the HO-1 m6A level in mouse spinal cord tissue homogenate, reduced BBB, boosted mNSS scores of SCI mice, aggravated nucleus pyknosis, and increased neuronal apoptosis in spinal cord tissues, confirming that FTO mediated HO-1 m6A modification facilitated neurological recovery in SCI mice. The fat mass and obesity-related gene modulates HO-1 mRNA stability by regulating m6A modification levels, thereby influencing HO-1 expression and promoting neurological recovery in SCI mice.

FTO protein regulates the TGF-β signalling pathway through RNA N6-methyladenosine modification to induce unexplained recurrent spontaneous abortion.

Recent studies have revealed the involvement of RNA m6A modification in embryonic development; however, the relationship between aberrant RNA m6A modification and unexplained recurrent spontaneous abortion (URSA) remains unclear. In this study, we analysed the level of RNA m6A modification in trophoblasts using dot blot, RNA m6A quantification, and MeRIP assays. By integrating data from the GEO database, RNA-Seq, and MeRIP-Seq, we examined the aberrant expression of m6A methyltransferases and their downstream molecules in chorionic villus (placental) tissues. RNA pull-down, RIP, and electrophoretic mobility shift assay were used to analyse the binding relationship between the YTHDC1 protein and MEG3. Additionally, RNA stability and BrU immunoprecipitation chase assays were utilised to elucidate the regulation of MEG3 stability by YTHDC1. ChIP and DNA pull-down RNA experiments were performed to elucidate the mechanism by which MEG3 targets EZH2 to the TGF-β1 promoter. The results showed that the expression of the m6A demethylase FTO protein was significantly increased in URSA trophoblasts, leading to inhibition of the MEG3 m6A modification and weakening of the stabilising effect of the m6A binding protein YTHDC1 on MEG3. Furthermore, MEG3 was found to bind simultaneously with the EZH2 protein and the TGF-β1 gene promoter, enabling the localisation of EZH2 protein to the TGF-β1 gene promoter and subsequent inhibition of TGF-β1 gene expression. In summary, our findings elucidate the mechanism by which FTO protein regulates the MEG3-TGF-β signalling pathway, thereby suppressing trophoblast invasion and proliferation in URSA trophoblast cells. These findings provide new insights for the treatment of URSA.

SAT632 The Functional Role And Regulatory Mechanism Of FTO M6A RNA Demethylase In Human Uterine Leiomyosarcoma

Abstract Disclosure: Q. Yang: None. A. Al-Hendy: None. Introduction: Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis, high rates of recurrence, and metastasis. However, the origin and molecular mechanism underlying driving its clinical and biological behavior remain to be determined. Recently, we and others have revealed the role of microRNAs, DNA methylation, and histone modifications contributing to the pathogenesis of uLMS. However, the connectivity between reversible RNA m6A methylation and uLMS remains unclear. In this study, we assessed the role and mechanism of the m6A demethylase fat mass- and obesity-associated protein (FTO) in the pathogenesis of uLMS. Materials and Methods: We performed immunohistochemistry (IHC) and immunoblot analysis to measure the m6A erasers FTO and ALKBH5 protein levels in LMS and adjacent myometrium tissues (n=8) and cell lines. We performed LMS cell proliferation and cell cycle phase distribution analysis. RNA-seq was performed to determine the molecular mechanism underlying m6A eraser inhibition in LMS cells (n=4 for each group). The R package limma+voom was used for this analysis. Hallmark pathway analysis was used to identify enriched pathways in response to FTO inhibition. Real-time PCR was performed to validate the differentially expressed genes. Ingenuity pathway analysis (IPA) was performed to determine the upstream regulators targeting cell cycle-related genes. We used a cutoff −1.5 > fold-change > 1.5 and an adjusted p-value of 0.05. In addition, data were analyzed by a two-tailed unpaired Student’s t-test between any two groups. Results: IHC analysis revealed that m6A demethylases FTO and ALKBH5 are aberrantly upregulated in uLMS tissues compared to adjacent myometrium (p<0.01, H-score). Furthermore, inhibition of FTO with its small, potent inhibitor (Dac51) decreased the uLMS proliferation dose-dependently via cell cycle arrest at G1. Notably, RNA-seq analysis revealed that inhibition of FTO with Dac51 exhibited a significant decrease in the expression of multiple CDK members and a significant increase in the expression of CDKN1A. Furthermore, IPA analysis revealed that transcription factors, including SP1 and E2F members are the upstream regulators targeting CDK members in response to Dac51 treatment. Moreover, Dac51 treatment altered the expression of several epigenetic regulators, including Sirt1 and 2, HDAC10 and SUV39H1, and rewiring of several critical pathways, including inflammation response, G2M checkpoint, and C-Myc signaling, among others, leading to suppression of uLMS phenotype. Conclusion: Our findings uncover the anti-uLMS function of a potent FTO inhibitor via reprogramming the oncogenic epitranscriptome. Therefore, targeted inhibition of FTO may provide an additional regulatory layer for a promising and novel strategy for treating patients with this aggressive uterine cancer. Presentation: Saturday, June 17, 2023

Obesity and Cancer

It is a key priority in public health to treat illnesses due to the rising frequency of obesity-related malignancies. Around 11.9% of male and 13.1% of female cancer cases worldwide have an obesity-related malignancy as their primary cause. In addition to colorectal, postmenopausal ovarian, breast, gallbladder, thyroid, and multiple myeloma cancers, endometrial, esophageal, hepatocellular carcinoma, gastric cancer, renal and pancreatic adenocarcinomas, and thyroid cancer, the relationship between obesity and cancer is clear in many anatomical sites. This article provides a thorough overview of the most recent epidemiological data on obesity and its connection to cancer in light of this urgent issue. The contradiction of weight increase and cancer risk and death. Furthermore, examining BMI differences in obesity-related malignancies offers a comprehensive grasp of the disease's complexities. These mechanisms include adipokine pathophysiology, cellular and micro environmental changes, ectopic fat accumulation, disruptions in dietary nutrients, circadian cycles, and potential factors like alteration of the intestinal flora and mechanical influences on weight. The conclusion's discussion of potential future approaches to prevention, detection, and treatment highlights the pressing need for more study and action on this important public health problem. The main objective is to increase knowledge of the complexity of obesity-related malignancies and increase awareness of it to create efficient methods for prevention and treatment. This review offers insight into how potential processes and risk factors combine on target tissues, ultimately causing them to take on a feature of cancer by addressing these important aspects. This review focuses on the Epidemiology of obesity influencing cancer, links between obesity and cancer, the influence of cancer by weight growth, somatometry, biological links, ectopic fat deposition, insulin sensitivity, adiponectin, obesity influencing widely occurring cancers, and FTO protein.

Cell Differentiation and Aging Lead To Up-Regulation of FTO, While the ALKBH5 Protein Level Was Stable During Aging but Up-Regulated During in vitro-Induced Cardiomyogenesis.

FTO and ALKBH5 proteins are essential erasers of N6-adenosine methylation in RNA. We studied how levels of FTO and ALKBH5 proteins changed during mouse embryonic development, aging, cardiomyogenesis, and neuroectodermal differentiation. We observed that aging in male and female mice was associated with FTO up-regulation in mouse hearts, brains, lungs, and kidneys, while the ALKBH5 level remained stable. FTO and ALKBH5 proteins were up-regulated during experimentally induced cardiomyogenesis, but the level of ALKBH5 protein was not changed when neuroectodermal differentiation was induced. HDAC1 depletion in mouse ES cells caused FTO down-regulation. In these cells, mRNA, carrying information from genes that regulate histone signature, RNA processing, and cell differentiation, was characterized by a reduced level of N6-adenosine methylation in specific gene loci, primarily regulating cell differentiation into neuroectoderm. Together, when we compared both RNA demethylating proteins, the FTO protein level undergoes the most significant changes during cell differentiation and aging. Thus, we conclude that during aging and neuronal differentiation, m6A RNA demethylation is likely regulated by the FTO protein but not via the function of ALKBH5.

Formulation of herbal tea using Cymbopogon citratus, Foeniculum vulgare and Murraya koenigii and its anti-obesity potential

ObjectivesThe term ‘herbal tea’ refers to a beverage made from the medicinal plants, herbs and spices. Herbal teas are non-caffeinated and consumed worldwide due to their therapeutic and healing properties. The present study aimed at the development of herbal tea formulation using Cymbopogon citratus leaves, Foeniculum vulgare seeds and Murraya koenigii leaves and its qualitative and quantitative evaluation. MethodsSensory evaluation of tea was carried out using 9point headonic Scale. Qualitative and quantitative phytochemical analysis, antioxidant activity using DPPH assay and antimicrobial activity using disc diffusion method was carried out. In addition to this molecular docking of different chemical compounds present in Cymbopogon citratus, Foeniculum vulgare and Murraya koenigii was performed against Alpha-ketoglutarate-dependent dioxygenase also called at mass and obesity-associated protein FTO. ResultsFormulation 1 possessed better sensory attributes than other formulations. Physicochemical analysis of extracts indicated the presence of 20–22% moisture content and 13–18% ash content in all tested extracts. Phytochemical analysis of extracts indicated the presence of various phytochemicals like alkaloids, tannins, phenols, flavonoids etc. in all tested extracts. Antioxidant potential of extracts increased with the increasing volume of extracts. All tested extracts possessed higher antimicrobial potential against S. aureus as compared to E. coli. It was found that herbal tea extract possessed highest antimicrobial potential against both tested strains as compared to individual plant extracts. FTIR analysis of extracts indicated the presence of various functional groups in the extracts. In silico analysis of compounds indicated that tested compounds possess good binding affinity with FTO protein and show a possible good replacement as synthetic drugs to prevent and treat obesity. ConclusionThe Cymbopogon citratus leaves, Foeniculum vulgare seeds and Murraya koenigii leaves could become a good source of constituents for formulation of herbal tea which was acceptable to consumers and possess good antimicrobial, antioxidant and anti-obesity potential.

FTO Promotes the Stemness of Gastric Cancer Cells.

The full name of the FTO gene is fat mass and obesity-associated gene. In recent years, it has also been found that FTO is involved in m6A demethylation and regulates the progression of multiple cancers, including gastric cancer. The cancer stem cell theory argues that cancer stem cells are key factors in cancer metastasis, and inhibiting the expression of stemness genes is a good method to inhibit metastasis of gastric cancer. Currently, the role of the FTO gene in regulating stemness of gastric cancer cells is still unclear. By analyzing public databases, it was discovered that FTO gene expression was increased in gastric cancer, and high expression of FTO was associated with poor prognosis of patients with gastric cancer. After gastric cancer stem cells were isolated, it was found that FTO protein expression was increased in gastric cancer stem cells; stemness of gastric cancer cells was reduced after the FTO gene knockdown; subcutaneous tumors of nude mice were smaller than those of the control group after FTO knockdown; and stemness of gastric cancer cells was enhanced after FTO was overexpressed by plasmid. By reviewing additional literature and experimental validation, we found that SOX2 may be the factor by which FTO promotes the stemness of gastric cancer cells. Therefore, it was concluded that FTO could promote the stemness of gastric cancer cells, and targeting FTO may be a potential therapeutic approach for patients with metastatic gastric cancer. CTR number: TOP-IACUC-2021-0123.

The role and mechanism of FTO in pulmonary vessels

ABSTRACT Pulmonary vascular remodeling (PVR) is the main factor of pulmonary hypertension (PH). The pathological characteristics of PVR are vascular smooth muscle hyperplasia, hypertrophy, and extensive damage. In vivo experiments, the expression of FTO in PH rat lung tissues of different rat models of hypoxia PH was observed by immunohistochemical method. mRNA microarray analysis was used to analyze the differential expressed genes in rat lung tissues. In vitro experiments, we developed models of overexpression and knockdown of FTO to study the effect of FTO protein expression on cell apoptotic, cell cycle, and the abundance of m6A. The expression of FTO was increased in PH rats. FTO knockdown can inhibit the proliferation of PASMCs, thereby regulating the cell cycle and reducing the expression of Cyclin D1 and the abundance of m6A, while overexpression of FTO leads to increased expression of Cyclin D1 and the abundance of m6A. FTO destroys the stability of Cyclin D1 by regulating the abundance of Cyclin D1 m6A, causing cell cycle arrest and inducing cell proliferation, thus inducing the occurrence and development of PVR in PH.

In silico Interaction of Rhamnus’ Flavonoids With Fat Mass And Obesity Associated Protein

The anti-obesity potential of various plant extracts and their associated bioactive compounds is well known. Molecular docking studies of FTO with flavonoids, using Orlistat (an anti-obesity drug) as a control, were performed to identify the effects of Rhamnus’ flavonoids with FTO (Obesity and obesity associated protein). Prior to molecular docking simulation, Rhamnus flavanoids were analysed using AutoDockTools (version 1.5.6). Docking simulations of the interaction of Rhamnus flavanoids with FTO were performed using AutoDock Vina version 1.1.2. Their binding affinities were obtained. BIOVIA Discovery Studio software was used to visualise the interaction between receptor and ligand. Our study approved the binding ability to FTO protein, and the affinity was as Aloe Emodin Dimer>Emodin>6-Methoxysorigenin. As a results, Rhamnus flavonoids have the remarkable ability to FTO protein, which means they are potent molecules as a potent FTO-inhibitor. Interestingly, Orlistat has lower affinity than Aloe-emodin dimer (-8.7 vs -10.8), which means aloe-emodin dimer more potent to bound the active site. In contrast, two other Rhamnnus flavonoids were shown lower binding affinity when compared to Orlistat.In conclution, Rhmanus phytomolecules able to bind to the catalytic site of FTO as well as “Orlistat” has been demonstrated by molecular docking. Thus, Rhamnus flavonoids especially “Aloe-emodin dimer” is a potent molecule to develop “anti-obesity drug”.

Bone Marrow Mesenchymal Stem Cells Reversed Ovarian Aging-related m6A RNA Methylation Modification Profile in Aged Granulosa Cells

BackgroundOvarian ageing causes endocrine disturbances and the degeneration of systemic tissue and organ functions to seriously affect women's physical and mental health, and effective treatment methods are urgently needed. Based on our previous studies using juvenile rhesus monkey bone marrow mesenchymal stem cells (BMMSCs) to treat ovarian ageing in rhesus monkey, we found that BMMSCs improved ovarian structure and function. This study continues to explore the mechanism by which BMMSCs reversed granulosa cell (GC) ageing.MethodsA GC ageing model and coculture system of BMMSCs were established, changes in the level of the N6-methyladenosine (m6A) methylation modification were detected, m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq) were performed, correlations between m6A peaks and mRNA expression were determined, and the expression of hub genes was identified using Q-PCR, immunofluorescence staining, and western blot.ResultsOur results showed that H2O2 successfully induced GC ageing and that BMMSCs reversed measures of GC ageing. BMMSCs increased the expression of the FTO protein and reduced the overall level of m6A. We identified 797 m6A peaks (348 hypomethylated and 449 hypermethylated peaks) and 817 differentially expressed genes (DEGs) (412 upregulated and 405 downregulated) after aged GCs were cocultured with BMMSCs, which significantly associated with ovarian function and epigenetic modification. The epigenetic repressive mark and important cell cycle regulator lysine demethylase 8 (KDM8) was downregulated at both the mRNA and protein levels, histone H3 was upregulated in aged GCs after BMMSC coculture, and KDM8 was upregulated after FTO was inhibited through FB23.ConclusionsOur study revealed an essential role for m6A in BMMSCs in reversing GC ageing, and FTO regulated KDM8 mediates histone H3 changes may as a novel regulatory mechanism in BMMSCs to reverse GC ageing.Graphical