FTO mRNA Research Articles

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.

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.

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-Mediated m6A Modification of FTH1 Inhibits Ferroptosis of Neurons in Neonatal Cerebral Hypoxic Ischemia.

FTO alpha-ketoglutarate dependent dioxygenase (FTO) is aberrantly expressed in brain disorders. However, the roles of FTO in neonatal hypoxic-ischemic brain injury (HIE) are still unclear. This study aims to investigate the potential of FTO in neonatal HIE. Oxygen-glucose deprivation (OGD) was used to establish HIE in vitro. mRNA levels were detected by real-time reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Protein expression was detected by Western blot. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), ferrous iron (Fe2+) and glutathione (GSH) was detected by specific kit. m6A sites were analyzed using SRAMP and further verify by methylated RNA immunoprecipitation (MeRIP) assay. Cell proliferation was determined by Cell Counting Kit-8 (CCK-8) assay. Cell death was determined by propidium iodide (PI) staining. FTO was downregulated in patients with neonatal HIE and OGD-treated neurons. Moreover, FTO mRNA expression was decreased in ferroptosis inducer, especially ferric ammonium citrate (FAC). However, overexpression of FTO inhibited the ferroptosis of neurons. Moreover, FTO-mediated N6-methyladenosine (m6A) modification of ferritin heavy chain 1 (FTH1) suppressed its mRNA expression and stability, inhibiting its protein expression. However, overexpression of FTH1 abrogated the effects of FTO and promoted the ferroptosis of neurons. In summary, FTO functions as a protective role in neonatal HIE via inhibiting FTH1 signaling. Thence, targeting may be a promising strategy for FTO neonatal HIE.

ALKBH5 promotes the development of lung adenocarcinoma by regulating the polarization of M2 macrophages through CDCA4

ObjectiveLung adenocarcinoma (LUAD) is the most common subtype of lung cancer, with high morbidity and mortality. N6-methyladenosine (m6A) is an important regulator of LUAD progression. Here, we investigated the potential biological functions of ALKBH5 (a m6A demethylated enzyme) and cell division cycle associated protein 4 (CDCA4) in the progression of LUAD. MethodsThe expressions of CDCA4, METTL3, ALKBH5, FTO, YTHDC2 and YTHDC1 mRNA and proteins in LUAD and adjacent tissues, as well as NCI-H1299 and NCI-H157 cells were detected by RT-qPCR and western blot. Meanwhile, the role of ALKBH5 and CDCA4 in macrophage polarization was explored through tumor formation in Lewis lung carcinoma (LLC) mice and the co-culture system of NCI-H1299 and NCI-H157/THP-1 cells. Cell characterization was further analyzed. The expression of Ki-67 in tumor tissue was tested by immunohistochemistry. The scale of M1 and M2 macrophages was determined by flow cytometry. ResultsCDCA4 was significantly overexpressed in NCI-H1299 and NCI-H157 cell lines compared with BEAS-2B cells. The fold enrichment of CDCA4 m6A level in the overexpression (oe)-METTL3 or short hairpin (sh)-ALKBH5 cells was enhanced. Overexpression of CDCA4 promoted the cell viability, proliferation and migration, and inhibited apoptosis, which was reversed by sh-ALKBH5 intervention. Overexpression of YTHDC2 (not YTHDC1) inhibited the effect of CDCA4 on sh-ALKBH5 cells. sh-CDCA4 inhibited tumor growth and weight of LLC cells in mice, and promoted M1/M2 ratio in LLC mice and NCI-H1299/THP-1 and NCI-H157/THP-1 co-culture systems. Oe-CDCA4 promoted the volume and weight of tumor and inhibited the M1/M2 ratio of tumor tissue in LLC mice, but was reversed by sh-ALKBH5 intervention. Conclusionm6A demethylase ALKBH5 promotes the development of LUAD through CDCA4 regulation of malignant characterization and M1/M2 macrophage polarization.

Down-regulation of the FTO gene in follicular fluid of infertile women with ovarian endometriosis

Objective To evaluate FTO concentrations in follicular fluid (FF) of women with ovarian endometriosis (OE) and controls women without OE undergoing in vitro fertilization-embryo transfer (IVF-ET). Methods FTO concentrations in FF were measured in 74 patients (37 in the control group and 37 in the OE group) by ELISA. We measured the expression of FTO in GCs of 40 patients (19 in the control group and 21 in the OE group) by RT-qPCR. The level of m6A in GCs was measured in 20 patients (10 in the control group and 10 in the OE group) by colorimetry. Results Compared with the control group, FTO concentrations in FF (6.92 ± 0.44 vs. 5.67 ± 0.40 ng/ml) (p <.05) and FTO mRNA level in GCs of OE group were decreased significantly (p <.05), and the level of m6A was increased (0.21 ± 0.01 vs. 0.17 ± 0.03 ng) (p >.05). Conclusions The FTO concentrations in FF of infertility women with OE are decreased, which may be related to the impaired oocyte quality in endometriosis patients.

Comprehensive analysis of N6-methyladenosine-related RNA methylation in the mouse hippocampus after acquired hearing loss

BackgroundThe mechanism underlying cognitive impairment after hearing loss (HL) remains unclear. N6-methyladenosine (m6A) is involved in many neurodegenerative diseases; however, its role in cognitive impairment after HL has not yet been investigated. Therefore, we aimed to analyze the m6A modification profile of the mouse hippocampus after HL exposure. A mouse model of neomycin-induced HL was established. An auditory brainstem-response test was utilized for detecting hearing threshold. The passive avoidance test was served as the mean for evaluating cognitive function. The m6A-regulated enzyme expression levels were analyzed by using reverse transcription quantitative real-time polymerase chain reaction and western blot analyses. RNA sequencing (RNA-Seq) and methylated RNA immunoprecipitation sequencing (MeRIP-Seq) were performed with the aim of investigating gene expression differences and m6A modification in the mouse hippocampus.ResultsNeomycin administration induced severe HL in mice. At four months of age, the mice in the HL group showed poorer cognitive performance than the mice in the control group. METTL14, WTAP, and YTHDF2 mRNA levels were downregulated in the hippocampi of HL mice, whereas ALKBH5 and FTO mRNA levels were significantly upregulated. At the protein level, METTL3 and FTO were significantly upregulated. Methylated RNA immunoprecipitation sequencing analysis revealed 387 and 361 m6A hypermethylation and hypomethylation peaks, respectively. Moreover, combined analysis of mRNA expression levels and m6A peaks revealed eight mRNAs with significantly changed expression levels and methylation.ConclusionsOur findings revealed the m6A transcriptome-wide profile in the hippocampus of HL mice, which may provide a basis for understanding the association between HL and cognitive impairment from the perspective of epigenetic modifications.

U-shaped association between serum IGF2BP3 and T2DM: A cross-sectional study in Chinese population.

To clarify the expression of N6-methyladenosine (m6 A) modulators involved in the pathogenesis of type 2 diabetes mellitus (T2DM). We further explored the association of serum insulin-like growth factor 2 mRNA-binding proteins 3 (IGF2BP3) levels and odds of T2DM in a high-risk population. The gene expression data set GSE25724 was obtained from the Gene Expression Omnibus, and a cluster heatmap was generated by using the R package ComplexHeatmap. Differential expression analysis for 13 m6 A RNA methylation regulators between nondiabetic controls and T2DM subjects was performed using an unpaired t test. A cross-sectional design, including 393 subjects (131 patients with newly diagnosed T2DM, 131 age- and sex-matched subjects with prediabetes, and 131 healthy controls), was carried out. The associations between serum IGF2BP3 concentrations and T2DM were modeled by restricted cubic spline and logistic regression models. Two upregulated (IGF2BP2 and IGF2BP3) and 5 downregulated (methyltransferase-like 3 [METTL3], alkylation repair homolog protein 1 [ALKBH1], YTH domain family 2 [YTHDF2], YTHDF3, and heterogeneous nuclear ribonucleoprotein [HNRNPC]) m6 A-related genes were found in islet samples of T2DM patients. A U-shaped association existed between serum IGF2BP3 levels and odds of T2DM according to cubic natural spline analysis models, after adjustment for body mass index, waist circumference, diastolic blood pressure, total cholesterol, and triglyeride. Multivariate logistic regression showed that progressively higher odds of T2DM were observed when serum IGF2BP3 levels were below 0.62 ng/mL (odds ratio 3.03 [95% confidence interval 1.23-7.47]) in model 4. Seven significantly altered m6 A RNA methylation genes were identified in T2DM. There was a U-shaped association between serum IGF2BP3 levels and odds of T2DM in the general Chinese adult population. This study provides important evidence for further examination of the role of m6 A RNA methylation, especially serum IGF2BP3 in T2DM risk assessment.

Overexpression of FTO alleviates osteoarthritis by regulating the processing of miR-515-5p and the TLR4/MyD88/NF-κB axis.

Osteoarthritis (OA) is regarded as the most prevalent chronic joint disease. Fat-mass and obesity-associated gene (FTO) is involved in OA alleviation. This study elucidated the role of FTO in OA and the associated mechanism. We established a cell injury model by stimulating human normal chondrocytes (C28/I2) with lipopolysaccharide (LPS), and measured cell viability, apoptosis, and inflammatory cytokines using CCK-8, flow cytometry, Western blot, and ELISA. TLR4, MyD88, p/t-p65, and p/t-IκBα levels, FTO, COX-2, and iNOS mRNA levels, and m6A methylation levels were measured by Western blot, RT-qPCR, and colorimetry. RNA immunoprecipitation and co-immunoprecipitation were conducted to confirm the interaction between FTO and DGCR8. pri-miR-515-5p process was regulated in an m6A-dependent manner. After predicting the presence of several binding sites between miR-515-5p and TLR4 on Targetscan, we further confirmed their relationship by dual-luciferase assay. OA rat models were established by monosodium iodoacetate injection. The pathological changes in knee joint were observed by HE staining. FTO was diminished in LPS-induced C28/I2 cells. With the increase of LPS concentration, cell viability was repressed, apoptosis rate was increased, and inflammatory markers were promoted, which were annulled by FTO overexpression. FTO interacted with DGCR8 and modulated the pri-miR-515-5p processing in an m6A-dependent manner. miR-515-5p silencing partially averted the inhibitory effect of FTO on LPS-induced cell injury. Given that TLR4 was a direct target of miR-515-5p, miR-515-5p inactivated the MyD88/NF-κB pathway by targeting TLR4. FTO overexpression improved cartilage structure in OA rats, reduced apoptosis, inhibited inflammation in synovial fluid, and repressed the TLR4/MyD88/NF-κB axis. FTO alleviated OA in an m6A-dependent manner via the miR-515-5p/TLR4/MyD88/NF-κB axis.

M6A RNA, FTO, ALKBH5 Expression in Type 2 Diabetic and Obesity Patients.

To investigate whether m6A content changes in type 2 diabetes mellitus (T2DM) and obese individuals and whether the relationship of m6A content with the mRNA expression levels of FTO and ALKBH5 genes. Cross-sectional study. Department of Internal Medicine, Firat University, Medical School, Elazig, Turkey, between January 2019 and January 2022. The study included 34 newly diagnosed patients with type 2 diabetes mellitus, 34 obese individuals, and 33 healthy individuals without any chronic and metabolic disease matched for age and gender. The global m6A RNA methylation, FTO, and ALKBH5 gene analyses of all the participants were performed. Total cholesterol, triglyceride, LDL, HDL, HbA1c, and insulin and glucose levels were measured. The median percentages of m6A RNA methylation in the control group, obese, and T2DM participants were 5.62%, 4.20%, and 5.21% respectively (p=0.004). The m6A RNA methylation percentage of the obese participants was significantly lower than controls (p=0.021). The FTO and ALKBH5 mRNA levels were significantly lower in obese and T2DM participants than in controls. There was a negative significant correlation between m6A RNA level and FTO i.e. (r=-0.291, p=0.003) and ALKBH5 (r=-0.321. p=0.001) levels. m6A RNA expression levels of obese individuals were lower than healthy controls. The FTO and ALKBH5 mRNA expressions were lower in both obese and T2DM participants compared to the healthy controls. There was no significant difference between obese and T2DM individuals in terms of m6A RNA expression, FTO and ALKBH5 mRNA expression. m6A RNA expression, FTO, and ALKBH5 levels have a potential role in obesity and diabetes mellitus. m6A RNA methylation, Epigenesis, Genetic, FTO, ALKBH5.

MiR-155 regulates m6A level and cell progression by targeting FTO in clear cell renal cell carcinoma

Although FTO, as an eraser of N6-methyladenosine (m6A), plays context-dependent tumor-suppressive and oncogenic roles in various cancer type, underlying molecular events of its aberrant expression in cancers is complex and still poorly understood. Here we show that miR-155 directly targets FTO to negatively regulate its expression and increased m6A level in ccRCC. Combining bioinformatics analysis and luciferase reporter assays, we identified that miR-155 directly bound to the 3′UTR of FTO mRNA and reduced FTO protein levels in ccRCC cells. Moreover, cell function assays, xenografts assays and m6A dot blot assays revealed that overexpression of miR-155 enhanced tumor cell proliferation and global mRNA m6A level, while decreasing apoptosis in a FTO-dependent manner. Collectively, our data demonstrates the functional importance of miR-155 in regulating FTO expression and global mRNA m6A level, and provides profound insights into ccRCC tumorigenesis.

Deregulation of N6-Methyladenosine RNA Modification and Its Erasers FTO/ALKBH5 among the Main Renal Cell Tumor Subtypes.

(1) Background: Methylation of N6-adenosine (m6A) is the most abundant messenger RNA (mRNA) modification in eukaryotes. We assessed the expression profiles of m6A regulatory proteins in renal cell carcinoma (RCC) and their clinical relevance, namely, as potential biomarkers. (2) Methods: In silico analysis of The Cancer Genome Atlas (TCGA) dataset was use for evaluating the expression of the m6A regulatory proteins among RCC subtypes and select the most promising candidates for further validation. ALKBH5 and FTO transcript and protein expression were evaluated in a series of primary RCC (n = 120) and 40 oncocytomas selected at IPO Porto. (3) Results: In silico analysis of TCGA dataset disclosed altered expression of the major m6A demethylases among RCC subtypes, particularly FTO and ALKBH5. Furthermore, decreased FTO mRNA levels associated with poor prognosis in ccRCC and pRCC. In IPO Porto’s cohort, FTO and ALKBH5 transcript levels discriminated ccRCC from oncocytomas. Furthermore, FTO and ALKBH5 immunoexpression differed among RCC subtypes, with higher expression levels found in ccRCC comparatively to the other RCC subtypes and oncocytomas. (4) Conclusion: We conclude that altered expression of m6A RNA demethylases is common in RCC and seems to be subtype specific. Specifically, FTO and ALKBH5 might constitute new candidate biomarkers for RCC patient management, aiding in differential diagnosis of renal masses and prognostication.

FTO Gene Polymorphisms Contribute to the Predisposition and Radiotherapy Efficiency of Nasopharyngeal Carcinoma.

BackgroundNasopharyngeal carcinoma (NPC) is mainly concentrated in East and Southeast Asia. This study aims to elucidate the potential associations of functional SNPs in the fat mass and obesity associated gene (FTO) with NPC risk and radiotherapy outcomes in a Chinese population.MethodsFunctional SNP rs1477196 G>A, rs9939609 T>A, rs7206790 C>G, and rs8047395 A>G were genotyped and evaluated for their associations with NPC risk and radiotherapy outcomes.ResultsBoth rs9939609 (allele A versus allele T: OR=1.59; 95% CI=1.17–2.17; P-value=0.003) and rs8047395 (allele G versus allele A: OR=0.76; 95% CI=0.64–0.9; P-value=0.002) were significantly associated with risk of NPC. GTEx showed risk allele A of rs9939609 and rs8047395 were significantly associated with higher FTO mRNA levels in skeletal muscle tissue, which also corroborated our findings. Meanwhile, both rs1477196 (allele A versus allele G: OR=1.64; 95% CI=1.09–2.49; P-value=0.019) and rs9939609 (allele A versus allele T: OR=0.61; 95% CI=0.43–0.87; P-value=0.006) were significantly associated with complete remission (CR) of NPC.ConclusionOur study identified that FTO polymorphisms contributed to the susceptibility and radiotherapy efficacy of NPC. These results shed light on the potential of establishing markers for predicting risk and personalized treatment of NPC.

Interplay between RNA Methylation Eraser FTO and Writer METTL3 in Renal Clear Cell Carcinoma Patient Survival.

m6A-methyltransferase METTL3 and demethylase FTO regulate gene expression by dynamically modifying RNA methylation. However, their clinical relevance in renal Clear Cell Carcinoma (CCRCC) has not been well elucidated. This study aims to investigate prognostic values of FTO and METTL3 mRNA and DNA methylation, their differential expression and associations with chemokines and inflammation-related genes in CCRCC. Kaplan-Meier survival curves and multivariate cox regression were performed for survival analyses, and random-effects meta-analysis was conducted for differential expression of FTO and METTL3 in CCRCC. A significantly negative correlation was observed between mRNA and DNA methylation for both FTO and METTL3. Survival analysis showed a superior survival in patients with either high FTO mRNA or low DNA methylation, or low METTL3 mRNA or high DNA methylation. The adjusted hazard ratios were 0.67 (95% CI: 0.49-0.91, p = 0.01) for high vs. low FTO mRNA, 2.17 (1.38-3.42, p = 0.0008) for high vs. low FTO DNA methylation, 1.97 (1.45-2.68, p < 0.0001) for high vs. low METTL3 mRNA, and 0.49 (0.31-0.79, p = 0.003) for high vs. low METTL3 DNA methylation, respectively. There was a significant interaction between FTO and METTL3 mRNA levels (p = 0.024). Upregulation of FTO and METTL3 with 1.64 folds (95% CI: 1.43-1.89) and 1.17 folds (1.02-1.35), respectively, was observed in CCRCC vs. normal kidney tissues. FTO and METTL3 mRNA showed opposite expressions of CD8+ T cell migration-related chemokines. Dysregulated FTO and METTL3 may be involved in the disease development and progression, affecting immune response in CCRCC. FTO and METTL3 expression and DNA methylation are potential prognostic markers of CCRCC.

FTO (Fat-Mass and Obesity-Associated Protein) Participates in Hemorrhage-Induced Thalamic Pain by Stabilizing Toll-Like Receptor 4 Expression in Thalamic Neurons.

Hemorrhage-caused gene changes in the thalamus likely contribute to thalamic pain genesis. RNA N6-methyladenosine modification is an additional layer of gene regulation. Whether FTO (fat-mass and obesity-associated protein), an N6-methyladenosine demethylase, participates in hemorrhage-induced thalamic pain is unknown. Expression of Fto mRNA and protein was assessed in mouse thalamus after hemorrhage caused by microinjection of Coll IV (type IV collagenase) into unilateral thalamus. Effect of intraperitoneal administration of meclofenamic acid (a FTO inhibitor) or microinjection of adeno-associated virus 5 (AAV5) expressing Cre into the thalamus of Ftofl/fl mice on the Coll IV microinjection–induced TLR4 (Toll-like receptor 4) upregulation and nociceptive hypersensitivity was examined. Effect of thalamic microinjection of AAV5 expressing Fto (AAV5-Fto) on basal thalamic TLR4 expression and nociceptive thresholds was also analyzed. Additionally, level of N6-methyladenosine in Tlr4 mRNA and its binding to FTO or YTHDF2 (YTH N6-methyladenosine RNA binding protein 2) were observed. FTO was detected in neuronal nuclei of thalamus. Level of FTO protein, but not mRNA, was time-dependently increased in the ipsilateral thalamus on days 1 to 14 after Coll IV microinjection. Intraperitoneal injection of meclofenamic acid or adeno-associated virus-5 expressing Cre microinjection into Ftofl/fl mouse thalamus attenuated the Coll IV microinjection–induced TLR4 upregulation and tissue damage in the ipsilateral thalamus and development and maintenance of nociceptive hypersensitivities on the contralateral side. Thalamic microinjection of AAV5-Fto increased TLR4 expression and elicited hypersensitivities to mechanical, heat and cold stimuli. Mechanistically, Coll IV microinjection produced an increase in FTO binding to Tlr4 mRNA, an FTO-dependent loss of N6-methyladenosine sites in Tlr4 mRNA and a reduction in the binding of YTHDF2 to Tlr4 mRNA in the ipsilateral thalamus. Our findings suggest that FTO participates in hemorrhage-induced thalamic pain by stabilizing TLR4 upregulation in thalamic neurons. FTO may be a potential target for the treatment of this disorder.

Expression of obesity-related genes in human spermatozoa affects the outcomes of reproductive treatments

To study the abundance of obesity-related gene (ORG) mRNA in human spermatozoa and its association with sperm quality parameters, embryonic development, and pregnancy rates after assisted reproduction treatment (ART). Cross-sectional study of spermatozoa ORG mRNA expression, and sperm and embryonic development parameters of infertile couples attending a single ART center. University, in collaboration with a medically assisted reproduction center. One hundred six couples seeking fertility treatment and receiving ART. Expression of spermatozoa ORG mRNA was assessed by quantitative reverse transcription-polymerase chain reaction. Sperm and embryonic development parameters were measured by board-certified embryologists. Serum β-human chorionic gonadotropin levels and fetal heartbeat detection on ultrasound were used to document biochemical and clinical pregnancy, respectively. Correlations between the abundance of ORG transcripts in spermatozoa and sperm quality, embryonic development, and achievement of pregnancy. The abundance of spermatozoa FTO mRNA was positively correlated with total sperm count (r = 0.5030), fertilization rate (r = 0.4854), embryo cleavage rate (r = 0.5705), and high-quality embryo rate (r = 0.6982). The abundance of spermatozoa MC4R transcript was negatively correlated with sperm viability (r = -0.3111) and positively correlated with biochemical pregnancy (r = 0.4420). The abundance of MC4R and GNPDA2 transcripts was higher in spermatozoa of men with asthenozoospermia and teratozoospermia than in those with normozoospermia. To our knowledge, this is the first report showing that the abundance of MC4R and FTO transcripts in spermatozoa is associated with sperm and embryo quality parameters, as well as pregnancy rates. Overall, these results further support the view that male factors beyond classic sperm quality parameters, namely the abundance of ORG transcripts, also affect the outcome of ART.

Hsa-let-7b-5p Inhibits Proliferation of Human Leukemia THP-1 Cells via FTO/m6A/MYC Signaling Pathway

To investigate the down-regulation effect of let-7b-5p on the expression of FTO in acute myeloid leukemia cell line THP-1 and inhibitory effect on THP-1 proliferation via m6A/MYC signaling pathway. The acute myeloid leukemia cell line THP-1 and the normal human peripheral blood mononuclear cells (PBMNC) were selected as subjects. The expression of let-7b-5p and FTO mRNA in those cells was detected by qPCR, further the expression of FTO protein in those cells was detected by Western blot. And, the luciferase reporter gene assay was used to verify the targeting effect of let-7b-5p on FTO. Finally, THP-1 cells were transfected respectively with let-7b-5p mimic, and PBMNC with let-7b-5p inhibitor, there after the C-MYC mRNA m6A enrichment level in transfected cells was analyzed by dot blot, the expression levels of let-7b-5p, FTO and c-MYC were assayed by RT-PCR, the expressions of FTO and c-MYC protein were verified by Western blot, and the proliferation level of cells after transfection was detected by MTT assay. Compared with PBMNC, the expression of let-7b-5p in THP-1 significantly decreased, while the expression of FTO was significantly increased (P<0.05). After transfection with let-7b-5p mimic combined with FTO 3'-UTR, the luciferase activity of transfected THP-1 cells significantly decreased, but the luciferase activity significantly increased after transfection with mutant 3'-UTR, which was significantly different from the negative control group(blank vector) (P<0.05). Let-7b-5p inhibitor down-regulated c-MYC mRNA m6A enrichment, and then up-regulated the expression of FTO in transfected PBMNC cells, the effects of which were significant (P<0.05). However, let-7b-5p mimic up-regulated c-MYC mRNA m6A enrichment level and down-regulated the expression of FTO in the transfected THP-1 cells, and the cell proliferation rate was significantly lower than that in the negative control group (blank vector) (P<0.05). Human acute myeloid leukemia cell line THP-1 low expresses the let-7b-5p, which regulates c-MYC expression through let-7b-5p-/FTO-/m6A axis and promotes the proliferation of leukemia cell line THP-1.

N6-Adenosine Methylation of Socs1 mRNA Is Required to Sustain the Negative Feedback Control of Macrophage Activation.

Bacterial infection triggers a cytokine storm that needs to be resolved to maintain the host's wellbeing. Here, we report that ablation of m6A methyltransferase subunit METTL14 in myeloid cells exacerbates macrophage responses to acute bacterial infection in mice, leading to high mortality due to sustained production of pro-inflammatory cytokines. METTL14 depletion blunts Socs1 m6A methylation and reduces YTHDF1 binding to the m6A sites, which diminishes SOCS1 induction leading to the overactivation of TLR4/NF-κB signaling. Forced expression of SOCS1 in macrophages depleted of METTL14 or YTHDF1 rescues the hyper-responsive phenotype of these macrophages invitro and invivo. We further show that LPS treatment induces Socs1 m6A methylation and sustains SOCS1 induction by promoting Fto mRNA degradation, and forced FTO expression in macrophages mimics the phenotype of METTL14-depleted macrophages. We conclude that m6A methylation-mediated SOCS1 induction is required to maintain the negative feedback control of macrophage activation in response to bacterial infection.

The association of dietary and plasma fatty acid composition with FTO gene expression in human visceral and subcutaneous adipose tissues.

The human obesity susceptibility gene, FTO, associates with body mass and obesity in humans through regulation of energy expenditure and intake. We aimed to determine how fatty acids in plasma and in diet associate with FTO gene expression in subcutaneous and visceral adipose tissues. In this study, 97 participants aged ≥ 18years were selected from patients admitted to the hospital for abdominal surgeries. Habitual dietary intake of participants was collected using a valid and reliable food frequency questionnaire (FFQ), from which the intake of fatty acids was quantified. Plasma fatty acids were assessed by gas-liquid chromatography. The mRNA expression of the FTO gene in visceral and subcutaneous adipose tissues obtained by biopsy was measured by Real-Time Quantitative Reverse Transcription PCR. Standardized β-coefficients were calculated by multivariable linear regression. After adjusting for age, homeostasis model insulin resistance index (HOMA-IR), and body mass index, total fatty acid intake was significantly associated with FTO gene expression in visceral (STZβ = 0.208, P = 0.037) and subcutaneous (STZβ = 0.236, P = 0.020) adipose tissues. Dietary intake of monounsaturated fatty acid (MUFA) and polyunsaturated fatty acids (PUFA) had positive significant associations with the expression of FTO in visceral (STZβ = 0.227, P = 0.023; STZβ = 0.346, P < 0.001, respectively) and subcutaneous (STZβ = 0.227, P = 0.026; STZβ = 0.274, P = 0.006, respectively) adipose tissues. There were no associations between plasma fatty acids and FTO mRNA expression in either subcutaneous or visceral adipose tissues. The weak association of dietary total fatty acids, MUFA, and PUFA with FTO gene expression in both adipose tissues may highlight the importance of dietary fatty acids composition along with total fat intake in relation to FTO gene expression.

High intensity exercise downregulates FTO mRNA expression during the early stages of recovery in young males and females

BackgroundPhysical exercise and activity status may modify the effect of the fat mass- and obesity-associated (FTO) genotype on body weight and obesity risk. To understand the interaction between FTO’s effect and physical activity, the present study investigated the effects of high and low intensity exercise on FTO mRNA and protein expression, and potential modifiers of exercise-induced changes in FTO in healthy-weighted individuals.MethodsTwenty-eight untrained males and females (25.4 ± 1.1 years; 73.1 ± 2.0 kg; 178.8 ± 1.4 cm; 39.0 ± 1.2 ml.kg.min− 1 VO2peak) were genotyped for the FTO rs9939609 (T > A) polymorphism and performed isocaloric (400 kcal) cycle ergometer exercise on two separate occasions at different intensities: 80% (High Intensity (HI)) and 40% (Low Intensity (LO)) VO2peak. Skeletal muscle biopsies (vastus lateralis) and blood samples were taken pre-exercise and following 10 and 90 mins passive recovery.ResultsFTO mRNA expression was significantly decreased after HI intensity exercise (p = 0.003). No differences in basal and post-exercise FTO protein expression were evident between FTO genotypes. Phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and Akt substrate of 160 kDa (AS160) were significantly increased following HI intensity exercise (p < 0.05). Multivariate models of metabolomic data (orthogonal two partial least squares discriminant analysis (O2PLS-DA)) were unable to detect any significant metabolic differences between genotypes with either exercise trial (p > 0.05). However, skeletal muscle glucose accumulation at 10 mins following HI (p = 0.021) and LO (p = 0.033) intensity exercise was greater in AA genotypes compared to TT genotypes.ConclusionOur novel data provides preliminary evidence regarding the effects of exercise on FTO expression in skeletal muscle. Specifically, high intensity exercise downregulates expression of FTO mRNA and suggests that in addition to nutritional regulation, FTO could also be regulated by exercise.Trial registrationACTRN12612001230842. Registered 21 November 2012 – Prospectively registered, https://www.anzctr.org.au/