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Objective To investigate the effect of chronic restraint stress on the expression of N6-methyladenosine (m6A)and related enzymes in the hippocampus of mice. Methods Twenty C57BL/6J male mice were randomly divided into control group and chronic restraint stress (CRS) group, the model group was given for 3 weeks chronic restraint stress to establish a mouse anxiety model. Open field test and elevated plus maze test were used to detect anxiety-like behavior; Immunohistochemistry and m6A RNA methylation assay were used to detect the expression changes of mouse hippocampal m6A; Western blotting and Real-time PCR were used to analyze hippocampal m6A related enzymes expression. Results 1.The behavioral results showed that, compared with the control group, the CRS group showed significantly reduced time spent in the center of the open field(P<0.01), the CRS group showed significantly reduced exploration time in the open arm of elevated plus maze (P<0.0001); 2. Immunohistochemical results showed that, compared with the control group, the hippocampal m6A content in the CRS group reduced significantly (P < 0.001); The results of the m6A RNA methylation assay showed that, compared with the control group, the CRS group showed significantly reduced amount of hippocampal m6A(P<0.05); 3. Real-time PCR results showed that the expression of hippocampal demethylase anaplastic lymphoma kinase B(AlkB) homolog 5(ALKBH5) (P<0.001) and fat mass and obestity associated protein(FTO) (P< 0.05) in the CRS group significantly up-regulated, the expression of methylase Wilms' tumour 1-associating protein (WTAP) (P<0.05) was significantly down-regulated compared with the control group; The expression of m6A methylation binding protein YTH domaincontaining family protein 3 (YTHDF3) (P < 0.05) and YTH domaincontaining protein 2 (YTHDC2) (P < 0.01) was significantly up-regulated. Western blotting result showed that, compared with the control group, the mouse hippocampal demethylase ALKBH5 (P < 0.05) and FTO (P < 0.05) expression in the CRS group significantly up-regulated, the expression of WTAP (P<0.01) was significantly down-regulated; m6A methylation binding protein YTHDF3 (P<0.01) and YTHDC2 (P<0.05) were significantly up-regulated. Conclusion In the anxiety model induced by chronic restraint stress, the expression of m6A in the hippocampus of mice is down-regulated. The mechanism may be related to the up-regulation of the m6A demethylase ALKBH5 and FTO or the down-regulation of the methylase WTAP.
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Objective:To explore the clinical significance of N6-methyladenine (m6A) in systemic lupus erythematosus (SLE) by comparing the changes in plasma levels of m6A modification related proteins [methyltransferase 3 (METTL3), methyltransferase 14 (METTL14), Wilms tumor 1 associated protein (WTAP), AlkB homologous protein 5 (ALKBH5), and fat mass and obesity-associated protein (FTO)] and m6A between patients with systemic lupus erythematosus (SLE) and healthy controls.Methods:A total of 64 SLE patients admitted to the Seventh Affiliated Hospital of Sun Yat-Sen University from May 2020 to June 2022 and 24 healthy volunteers during the same period were selected to compare and analyze the plasma levels of METTL3, METTL14, WTAP, ALKBH5, FTO and m6A between the two groups. The correlation between METTL3, WTAP, FTO levels and clinical indicators was analyzed.Results:The plasma METTL3 level of SLE patients was significantly higher than that of control group ( P<0.05), and the plasma WTAP and FTO levels were significantly lower than those of control group (all P<0.05). In SLE patients, plasma METTL3 level was negatively correlated with hemoglobin level ( r=-0.344, P<0.05), plasma FTO level was positively correlated with plasma IgM level ( r=0.337, P<0.05), and plasma IgA level was negatively correlated with SLE patients ( r=-0.286, P<0.05). The incidence of renal involvement and positive rate of plasma anti-histone antibody were higher in SLE patients with high METTL3 level (all P<0.05). The positive rates of plasma anti-dsDNA antibody, anti-SM antibody and AuaA antibody were higher in SLE patients with low FTO level (all P<0.05). Conclusions:The plasma METTL3 level in SLE patients are significantly increased, while the plasma WTAP and FTO levels are significantly reduced, which are related to various clinical indicators and may be related to the onset of SLE.
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@#N6-methyladenine (m6A) modification, the most abundant and dynamic chemical modification on messenger RNA, plays an essential role in physiological and pathological progress.Recent studies have found that tumor progression can be affected by altering the m6A modification level of target genes. Therefore, small molecule targeted m6A demethylase can be used as a new anti-tumor strategy.This review focuses on the regulatory mechanism of m6A demethylases, including fat mass and obesity-associated protein (FTO) and AlkB homlog 5 (ALKBH5), as well as their biological functions in tumors, and summarizes the research progress of their small molecule inhibitors.
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【Objective】 To explore the relationship of fat mass and obesity-associated protein (FTO) with the m6A modification and expression level of DKK2 in the process of myocardial fibrosis. 【Methods】 Cardiac fibroblasts (CFs) were grouped as follows: Control group, AngⅡ-treated group, AngⅡ+EV group (transfected with empty vector and negative control siRNA and then treated with AngⅡ), AngⅡ+FTO-O group (transfected with FTO overexpression vector and then treated with AngⅡ), and AngⅡ+FTO-O+DKK2 siRNA group (treated with AngⅡ after co-transfection of FTO overexpression vector and DKK2 siRNA). Mice were divided into the following groups: Control group (sham operation group), AMI group (constructing acute myocardial infarction model), AMI+EV group (AMI mice were intraperitoneally injected with nanoparticles containing empty vector), and AMI+FTO-O group (AMI mice were intraperitoneally injected with nanoparticles containing FTO overexpression vector). Then, the expressions of FTO and DKK2 were detected by fluorescence quantitative PCR and Western blotting, the m6A modification level of DKK2 was detected by RNA binding protein immunoprecipitation, the cell viability was detected by CCK-8, the cardiac function of AMI mice was evaluated, and the cardiac pathological changes of mice were detected by HE and Masson staining. 【Results】 AngⅡ inhibited the expression of FTO, thereby enhancing the m6A modification level of DKK2 and downregulating the expression of DKK2 (P<0.05). AngⅡ promoted cell viability and enhanced the expressions of α-SMA, collagen Ⅰ and collagen Ⅲ (P<0.05). FTO overexpression significantly blocked the above-mentioned regulatory effects of AngⅡ (P<0.05), but DKK2 siRNA could antagonize the effect of FTO overexpression on AngⅡ. The expressions of FTO and DKK2 were downregulated in AMI mice, and the m6A modification level of DKK2 was increased (P<0.05). When FTO was overexpressed, the expressions of FTO and DKK2 in AMI mice were significantly restored, the m6A modification level of DKK2 and myocardial fibrosis were significantly reduced (P<0.05), and the cardiac pathological changes were significantly improved. 【Conclusion】 FTO can promote the expression of DKK2 by reducing the m6A modification level of DKK2, thereby inhibiting the progression of myocardial fibrosis. This indicates that FTO/DKK2 pathway is a key pathway in regulating myocardial fibrosis.
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Background Chemical modification of RNA is a recent hotspot in the field of epigenetics, but the specific mechanism of chemical modification of RNA in aluminum neurotoxicity has not been fully reported. Objective To investigate the alterations of fat mass and obesity-associated protein (FTO), that demethylates N6-methyladenosine (m6A), and brain-derived neurotrophic factor (BDNF) in different brain regions of rats and rat adrenal pheochromocytoma differentiated cells (PC12 cells) following aluminum exposure. Methods Animal experiment: Twenty-four healthy male SD rats were randomly divided into a control group (normal saline) and 10, 20, and 40 μmol·kg−1 exposure groups according to body weight, with 6 rats in each group. Maltol aluminum [Al(mal)3] was injected intraperitoneally every other day for 3 months. Cell experiment: PC12 cells were divided into a control group and 100, 200, and 400 μmol·L−1 exposure groups exposed to Al(mal)3 for 24 h. After exposure, the learning and memory ability of rats was measured by water maze experiment, and the protein expression levels of FTO and BDNF in rat cortex (n=6) and hippocampus (n=6) samples as well as in PC12 cells (n=5) were determined by Western blotting. Results The results of water maze test showed that the escape latency of the 40 μmol·kg−1Al(mal)3 group was higher than those of the control group, the 10 μmol·kg−1Al(mal)3 group, and the 20 μmol·kg−1Al(mal)3 group on day 3, 4, and 5 of training (P<0.05). The retention time of the target quadrant of the 40 μmol·kg−1Al(mal)3 group was also reduced compared with that of the control group (P<0.05), indicating that aluminum exposure damaged the learning and memory ability of the rats. The Western blotting results showed that in the cortex, compared with the control group, the protein expression levels of FTO and BDNF in the aluminum treated groups were decreased (P<0.05). In the hippocampus, compared with the control group, the protein expression levels of FTO and BDNF in the 20 μmol·kg−1 and the 40 μmol·kg−1Al(mal)3 groups were decreased (P<0.05). In PC12 cells, compared with the control group, the protein expression levels of FTO and BDNF in the aluminum treated groups were decreased (P<0.05). Conclusion Aluminum-induced learning and memory impairment is related to a simultaneous reduction of FTO and BDNF protein expressions, suggesting that m6A methylation may be involved.
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Objective:To explore the role of the fat mass and obesity-associated protein (FTO) in human renal tubular epithelial cells (HK-2) suffering ischemia-reperfusion injury (IRI).Methods:The in vitro IRI mo-del was established in HK-2 cells by induction with antimycin A, A23187 and 2-deoxy-D-glucose.The cells were divided into control group and ischemia-reperfusion group (I/R group). The mRNA and protein expressions of FTO, B-cell lymphoma / leukemia 2(Bcl-2)-associated X(Bax), Bcl-2 and cleaved cysteinyl aspartate specific proteinase(cleaved Caspase-3) in HK-2 cells before and after IRI were detected by real-time fluorescent quantitative PCR(qPCR) and Western blot, respectively.Cell apoptosis was measured using flow cytometry.The level ofe N 6-methy-ladenosine (m 6A) RNA was detected by colorimetry. Results:(1) The mRNA expressions of FTO (0.15±0.05 vs.1.00±0.23) and Bcl-2 (0.14±0.07 vs.1.02±0.25) in I/R group were significantly lower than those in control group; While those of Bax (3.10±0.35 vs.1.00±0.13) and cleaved Caspase-3 (4.21±0.56 vs.1.00±0.09) were significantly higher ( t=6.28, 5.84, -9.83, and -9.84, respectively, all P<0.01). (2) The protein expressions of FTO (0.69±0.14 vs.1.37±0.02) and Bcl-2 (0.50±0.12 vs.1.25±0.21) were significantly lower in I/R group than those of control group; While those of Bax (1.04±0.08 vs.0.57±0.06) and cleaved Caspase-3 (0.99±0.05 vs.0.36±0.07) were significantly higher ( t=8.10, 5.49, -8.22, and -12.09, respectively, all P<0.05). (3) Compared with the control group, the apoptosis rate of HK-2 cells in I/R group was significantly higher [(61.70±1.01)% vs.(0.16±0.10)%, t=63.80, P<0.01]. (4) Compared with the control group, the percentage of m 6A modification level in total RNA in I/R group was significantly higher [(3.13±0.21)% vs.(1.10±0.26)%, t=-10.61, P<0.01]. Conclusions:FTO-mediated RNA m 6A modification may affect renal IRI by regulating the apoptosis of HK-2 cells.
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ABSTRACT Introduction: Recent studies have shown that the association of FTO rs9939609 gene polymorphism with obesity depends on the level of the individual's physical activity. However, there are some studies that evaluated physical fitness, health, and motor performance in relation to the rs9939609 FTO gene polymorphism. Objective: To evaluate how the rs9939609 FTO gene polymorphism affects the results of physical fitness tests related to health and athletic performance in schoolchildren after 4 months of intervention of physical exercise. Method: The rs9939609 FTO gene polymorphism was genotyped in a total of 36 schoolchildren from southern Brazil, aged 8 to 16 years. Body mass index (BMI), health-related physical fitness (cardiorespiratory fitness, abdominal strength/endurance, and flexibility) and motor performance (upper and lower limb strength, agility, and speed) were evaluated. The intervention included exercise strategies based on Physical Education, healthy eating, and oral and postural care. Results: In the experimental group, after the intervention, significant differences were noted in individuals with the TT genotype. These individuals showed improvements in abdominal strength (p=0.025), lower limb strength (p=0.037) and agility (p=0.021). For individuals with the AA/AT genotype, improvements in flexibility (p=0.026), abdominal strength (p=0.002), upper limb strength (p=0.008) and lower limb strength (p=0.001) were observed. However, these differences were not statistically significant when comparing the TT and AT/AA genotypes. Conclusions: The experimental group showed improvements in abdominal strength, lower limb strength, and speed. Yet, individuals with different genotypes (AA/AT and TT) for polymorphism rs9939609 exhibited similar values for indicators of physical fitness, health, and motor performance. Level of Evidence II; Lesser quality RCT.
RESUMO Introdução: Estudos recentes demonstraram que a associação do polimorfismo do gene FTO rs9939609 e a obesidade dependem do nível de atividade física de um indivíduo. No entanto, existem alguns estudos que avaliaram a aptidão física, a saúde e o desempenho motor com relação ao polimorfismo rs9939609 do gene FTO. Objetivo: Avaliar como o polimorfismo rs9939609 do gene FTO afeta os resultados dos testes de aptidão física relacionados com a saúde e o desempenho atlético em escolares após 4 meses de intervenção com exercícios físicos. Método: O polimorfismo rs9939609 do gene FTO foi genotipado em um total de 36 escolares do sul do Brasil, com idades entre 8 e 16 anos. O índice de massa corporal (IMC), a aptidão física relacionada com a saúde (aptidão cardiorrespiratória, força abdominal/resistência e flexibilidade) e desempenho motor (força de membros superiores e inferiores, agilidade e velocidade) foram avaliados. A intervenção teve como base estratégias de exercícios da Educação Física e alimentação saudável, além de cuidados bucais e posturais. Resultados: No grupo experimental, após a intervenção, observaram-se diferenças significativas em indivíduos com o genótipo TT. Esses indivíduos apresentaram melhorias na força abdominal (p = 0,025), força dos membros inferiores (p = 0,037) e agilidade (p = 0,021). Para os indivíduos com o genótipo AA/AT, foram observadas melhorias na flexibilidade (p = 0,026), força abdominal (p = 0,002), força dos membros superiores (p = 0,008) e força dos membros inferiores (p = 0,001). No entanto, essas diferenças não foram estatisticamente significantes ao se comparar os genótipos TT e AT/AA. Conclusões: O grupo experimental apresentou melhorias na força abdominal, força dos membros inferiores e velocidade. Contudo, indivíduos com diferentes genótipos (AA/AT e TT) para o polimorfismo rs9939609 exibiram valores semelhantes para indicadores de aptidão física, saúde e desempenho motor. Nível de Evidência II, ECRC de menor qualidade.
RESUMEN Introducción: Estudios recientes han demostrado que la asociación del polimorfismo del gen FTO rs9939609 y la obesidad dependen del nivel de actividad física de um individuo. Sin embargo, existen algunos estudios que evaluaron la aptitud física, la salud y el rendimiento motor con relación al polimorfismo rs9939609 del gen FTO. Objetivo: Evaluar cómo el polimorfismo rs9939609 del gen FTO afecta los resultados de las pruebas de aptitud física relacionadas con la salud y el desempeño atlético en escolares después de 4 meses de intervención con ejercicios físicos. Método: El polimorfismo rs9939609 del gen FTO fue genotipado en un total de 36 escolares del sur de Brasil, con edades entre 8 y 16 años. El índice de masa corporal (IMC), la aptitud física relacionada con la salud (aptitud cardiorrespiratoria, fuerza abdominal/resistencia y flexibilidad) y rendimiento motor (fuerza de las extremidades superiores e inferiores, agilidad y velocidad) fueron evaluados. La intervención tuvo como base estrategias de ejercicios de Educación Física y alimentación saludable, además de cuidados bucales y posturales. Resultados: En el grupo experimental, después de la intervención, se observaron diferencias significativas en individuos con el genotipo TT. Estos individuos presentaron mejoras en la fuerza abdominal (p = 0,025), fuerza de las extremidades inferiores (p = 0,037) y agilidad (p = 0,021). Para las personas con el genotipo AA/AT, se observaron mejoras en la flexibilidad (p = 0,026), fuerza abdominal (p = 0,002), fuerza de las extremidades superiores (p = 0,008) y fuerza de las extremidades inferiores (p = 0,001). Sin embargo, estas diferencias no fueron estadísticamente significativas al comparar los genotipos TT y AT/AA. Conclusiones: El grupo experimental presentó mejoras en la fuerza abdominal, fuerza de las extremidades inferiores y velocidad. No obstante, individuos con diferentes genotipos (AA/AT y TT) para el polimorfismo rs9939609 mostraron valores similares para indicadores de aptitud física, salud y rendimiento motor. Nivel de Evidencia II; ECRC de menor calidad.