FoxO1 mRNA Research Articles

Time Course of Proteolysis Biomarker Responses to Resistance, High-Intensity Interval, and Concurrent Exercise Bouts.

Godwin, JS, Telles, GD, Vechin, FC, Conceição, MS, Ugrinowitsch, C, Roberts, MD, and Libardi, CA. Time course of proteolysis biomarker responses to resistance, high-intensity interval, and concurrent exercise bouts. J Strength Cond Res 37(12): 2326-2332, 2023-Concurrent exercise (CE) combines resistance exercise (RE) and high-intensity interval exercise (HIIE) in the same training routine, eliciting hypertrophy, strength, and cardiovascular benefits over time. Some studies suggest that CE training may hamper muscle hypertrophy and strength adaptations compared with RE training alone. However, the underlying mechanisms related to protein breakdown are not well understood. The purpose of this study was to examine how a bout of RE, HIIE, or CE affected ubiquitin-proteasome and calpain activity and the expression of a few associated genes, markers of skeletal muscle proteolysis. Nine untrained male subjects completed 1 bout of RE (4 sets of 8-12 reps), HIIE (12 × 1 minute sprints at V̇ o2 peak minimum velocity), and CE (RE followed by HIIE), in a crossover design, separated by 1-week washout periods. Muscle biopsies were obtained from the vastus lateralis before (Pre), immediately post, 4 hours (4 hours), and 8 hours (8 hours) after exercise. FBXO32 mRNA expression increased immediately after exercise (main time effect; p < 0.05), and RE and CE presented significant overall values compared with HIIE ( p < 0.05). There was a marginal time effect for calpain-2 mRNA expression ( p < 0.05), with no differences between time points ( p > 0.05). No significant changes occurred in TRIM63/MuRF-1 and FOXO3 mRNA expression, or 20S proteasome or calpain activities ( p > 0.05). In conclusion, our findings suggest that 1 bout of CE does not promote greater changes in markers of skeletal muscle proteolysis compared with 1 bout of RE or HIIE.

Implications on growth performance, glucose metabolism, PI3K/AKT pathway, intestinal flora induced by dietary taurine in a high-carbohydrate diet for grass carp (Ctenopharyngodon idella).

An 8-week experiment was performed to investigate the influence on growth performance, plasma biochemistry, glucose metabolism and the insulin pathway of supplementation of dietary taurine to a high-carbohydrate diet for grass carp. In this study, fish were fed diets at one of two carbohydrate levels, 31·49 % (positive control) or 38·61 % (T00). The high-carbohydrate basal diet (T00), without taurine, was supplemented with 0·05 % (T05), 0·10 % (T10), 0·15 % (T15) or 0·20 % (T20) taurine, resulting in six isonitrogenous (30·37 %) and isolipidic (2·37 %) experimental diets. The experimental results showed that optimal taurine level improved significantly weight gain, specific growth rate (SGR), feed utilisation, reduced plasma total cholesterol levels, TAG and promoted insulin-like growth factor level. Glucokinase, pyruvate kinase and phosphoenolpyruvate carboxykinase activities showed a quadratic function model with increasing dietary taurine level, while hexokinase, fatty acid synthetase activities exhibited a positive linear trend. Optimal taurine supplementation in high-carbohydrate diet upregulated insulin receptor (Ir), insulin receptor substrate (Irs1), phosphatidylinositol 3-kinase (pi3k), protein kinase B (akt1), glycogen synthase kinase 3 β (gs3kβ) mRNA level and downregulated insulin-like growth factor (igf-1), insulin-like growth factor 1 receptor (igf-1R) and Fork head transcription factor 1 (foxo1) mRNA level. The above results suggested that optimal taurine level could improve growth performance, hepatic capacity for glycolipid metabolism and insulin sensitivity, thus enhancing the utilisation of carbohydrates in grass carp. Based on SGR, dietary optimal tributyrin taurine supplementation in grass carp was estimated to be 0·08 %.

The clock transcription factor BMAL1 is a key regulator of extracellular matrix homeostasis and cell fate in the intervertebral disc

The circadian clock in mammals temporally coordinates physiological and behavioural processes to anticipate daily rhythmic changes in their environment. Chronic disruption to circadian rhythms (e.g., through ageing or shift work) is thought to contribute to a multitude of diseases, including degeneration of the musculoskeletal system. The intervertebral disc (IVD) in the spine contains circadian clocks which control ∼6% of the transcriptome in a rhythmic manner, including key genes involved in extracellular matrix (ECM) homeostasis. However, it remains largely unknown to what extent the local IVD molecular clock is required to drive rhythmic gene transcription and IVD physiology. In this work, we identified profound age-related changes of ECM microarchitecture and an endochondral ossification-like phenotype in the annulus fibrosus (AF) region of the IVD in the Col2a1-Bmal1 knockout mice. Circadian time series RNA-Seq of the whole IVD in Bmal1 knockout revealed loss of circadian patterns in gene expression, with an unexpected emergence of 12 h ultradian rhythms, including FOXO transcription factors. Further RNA sequencing of the AF tissue identified region-specific changes in gene expression, evidencing a loss of AF phenotype markers and a dysregulation of ECM and FOXO pathways in Bmal1 knockout mice. Consistent with an up-regulation of FOXO1 mRNA and protein levels in Bmal1 knockout IVDs, inhibition of FOXO1 in AF cells suppressed their osteogenic differentiation. Collectively, these data highlight the importance of the local molecular clock mechanism in the maintenance of the cell fate and ECM homeostasis of the IVD. Further studies may identify potential new molecular targets for alleviating IVD degeneration.

MRNA level of antioxidant genes and activity of NADPH-generating enzymes in rotenone-induced parkinsonism in rats

Aim. To analyze the mRNA level of genes encoding antioxidant enzymes and the transcription factors Nrf2 and Foxo1 regulating their expression and the activity of glucose-6-phosphate dehydrogenase (G6PDH) and NADPdependent isocitrate dehydrogenase (NADP-IDH) and assess the correlation between these parameters, oxidative status, and motor coordination parameters in rats with rotenone-induced parkinsonism.Materials and methods. The study was performed on male Wistar rats aged 4–6 months and weighing 200–250 g. Parkinsonism was modeled by subcutaneous administration of rotenone for 10 days at a dose of 2.5 mg / kg. To confirm the development of the pathology, motor coordination tests and histological staining of the cerebral cortex and striatum with hematoxylin and eosin were used. The oxidative status was analyzed based on the levels of conjugated dienes, carbonyl amino acid residues in proteins, and α-tocopherol. The enzyme activity was studied spectrophotometrically by the formation of NADPH. Real-time PCR was used to analyze the level of gene mRNA.Results. During the study, an increase in serum and brain concentrations of conjugated dienes, carbonyl amino acid residues, and α-tocopherol was observed in the experimental group of rats compared to the controls. It could be associated with the redistribution of this compound between tissues during pathology development. The animals with experimental parkinsonism, in addition, were characterized by a decrease in the mRNA level of the Sod1, Gpx1, Gsr, Gsta2, Nfe2l2, and Foxo1 genes, as well as the activity of G6PDH and NADP-IDH. In the rats with experimental parkinsonism, a negative correlation of NADPH-IDH activity in the brain with serum α-tocopherol level and a positive correlation with Gpx1 and Foxo1 mRNA levels in the striatum were found. The level of oxidatively modified proteins in the brain of the animals with PD was negatively correlated with the concentration of Gsta2 mRNA in the striatum, while the specific activity of G6PDH in the serum was characterized by the positive relationship with grip strength.Conclusion. The data obtained indicate that the inhibition of transcription of the genes encoding antioxidant enzymes and regulatory factors Nrf2 and Foxo1 contributed significantly to the development of oxidative stress in PD. A decrease in the activity of G6PDH and NADP-IDH led to a decrease in the availability of NADPH, which is a limiting factor in the functioning of the glutathione antioxidant system. Obviously, the inhibition of G6PDH and NADP-IDH was also an important pathogenic factor in the progression of the pathology. Along with a decrease in the content of antioxidant gene mRNA in the brain tissues, the level of α-tocopherol increased in the rats with parkinsonism, which could be the result of an imbalance in the functioning of antioxidant system.

Treatment of porcine ovarian follicles with tert-butyl hydroperoxide as an ovarian senescence model in vitro.

Ovarian aging is the main reason of female reproductive problems. Excessive oxidative stress can induce ovarian senescence and follicular atresia, thereby reducing the reproductive performance. Follicles were divided into five groups for in vitro culture based on the duration of stimulation with tert-butyl hydroperoxide (t-BHP)-control group and groups 1 h, 2 h, 6 h, and 12 h. The results revealed that the ratio of progesterone (P4) to estradiol (E2) was increased after 24 and 36 h of follicle culture, shifting follicles toward atresia (P < 0.05). Stimulated by 200 μM t-BHP, follicles showed progressive aging phenotype. Senescence-associated β-galactosidase staining (SA-β-Gal) showed a significant increase in the number of positive cells (P < 0.05). Reactive oxygen species were also significantly upregulated (P < 0.05). t-BHP treatment for 6 h induced significant increases in Caspase 3, P53, and Foxo1 mRNA and protein levels (P < 0.05) and significant decreases in SOD mRNA and protein levels (P < 0.05). Transcriptome sequencing analysis of the follicles showed that the aged and treatment groups were clustered together in hierarchical clustering. Correlation analysis indicated significant changes at the transcriptome level in the treatment groups versus the control group. The common differentially expressed genes in the treatment groups were enriched in three growth-factor signaling pathways associated with cell proliferation and apoptosis (P53, mTOR, and MAPK). In conclusion, induction of follicular senescence by treatment with 200 μM t-BHP for 6 h is an effective in vitro model to simulate ovarian senescence in sows.

The role of m6A demethylase FTO in chemotherapy resistance mediating acute myeloid leukemia relapse

Acute myeloid leukemia (AML) is the most common hematopoietic malignancies, and chemotherapy resistance is one of the main causes of relapse. Because of lower survival rate for patients with relapse, it is pivotal to identify etiological factors responsible for chemo-resistance. In this work, direct MeRIP-seq analysis of sequential samples at stage of complete remission (CR) and relapse identifies that dysregulated N6-methyladenosine (m6A) methylation is involved in this progression, and hypomethylated RNAs are related to cell differentiation. m6A demethylase FTO is overexpressed in relapse samples, which enhances the drug resistance of AML cells in vivo and in vitro. In addition, FTO knockdown cells exhibit stronger capacity of differentiation towards granules and myeloid lineages after cytosine arabinoside (Ara-C) treatment. Mechanistically, FOXO3 is identified as a downstream target of FTO, the hypomethylation of FOXO3 mRNA affects its RNA degradation and further reduces its own expression, which ultimately result in attenuated cell differentiation. Collectively, these results demonstrate that FTO-m6A-FOXO3 is the main regulatory axis to affect the chemotherapy resistance of AML cells and FTO is a potential therapeutic target of chemotherapy resistance in AML.

Eryngium billardieri extract affects cardiac gene expression of master regulators of cardiomyaopathy in rats with high fatdiet-induced insulin resistance

For years, numerous studies have focused on identifying approaches to increase insulin sensitivity by modifying the signaling factors. In the present study, we examined the effects of Eryngium billardieri extract, as an anti-diabetic herbal medication, on the heart mRNA level of Akt serine/threonine kinase (Akt), mechanistic target of rapamycin kinase (mTOR), peroxisome proliferator-activated receptor gamma (PPARγ), and Forkhead box o1 (Foxo1) in rats with high-fat diet (HFD)-induced insulin resistance (IR). We also assessed the anti-diabetic effects of E.billardieri extract in rats with insulin resistance. Twenty-seven male Wistar rats were divided into two groups. Nine rats were fed a normal diet (control group), and 18 rats were fed an HFD for 13 weeks (HFD group). To confirm the induction of insulin resistance, the oral glucose tolerance test (OGTT) was performed and homeostatic model assessment for insulin resistance (HOMA-IR) was calculated. Then rats with IR were randomly divided into the following groups: the HFD group, which continued an HFD, and the group treated with E.billardieri extract, which received the extract at a concentration of 50mg/kg for 30 days. On the 30th day, the animals were sacrificed and serum samples were collected for biochemistry analyses. Furthermore, the expression of Akt, mTOR, PPARγ, and Foxo1 was measured in heart tissue using the real-time polymerase chain reaction (PCR) method. Real-time PCR analyses revealed that an HFD can significantly decrease the expression level of Akt, mTOR, and PPARγ in the heart tissue. However, an HFD significantly increased the expression level of Foxo1 in the HFD group compared to the control group (P<0.05). In addition, our data showed that the administration of E. billardieri extract significantly enhanced the mRNA levels of Akt, PPARγ, and mTOR in the heart tissue compared to the HFD group (P<0.05), while it significantly decreased the Foxo1 mRNA levels (P<0.01). Given that Akt, mTOR, PPARγ, and Foxo1 are critical factors in insulin resistance, the present study suggests that E.billardieri could probably be used as an alternative treatment for IR as a major feature of metabolic syndrome.

Circulating cardiac MicroRNAs safeguard against dilated cardiomyopathy.

Cardiac-resident or -enriched microRNAs (miRNAs) could be released into the bloodstream becoming circulating cardiac miRNAs, which are increasingly recognized as non-invasive and accessible biomarkers of multiple heart diseases. However, dilated cardiomyopathy (DCM)-associated circulating miRNAs (DACMs) and their roles in DCM pathogenesis remain largely unexplored. Two human cohorts, consisting of healthy individuals and DCM patients, were enrolled for serum miRNA sequencing (10vs. 10) and quantitative polymerase chain reaction validation (46vs. 54), respectively. Rigorous screening strategy was enacted to define DACMs and their potentials for diagnosis. DCM mouse model, different sources of cardiomyocytes, adeno-associated virus 9 (AAV9), gene knockout, RNAscope miRNA in situ hybridization, mRFP-GFP-LC3B reporter, echocardiography and transmission electron microscopy were adopted for mechanistic explorations. Serum miRNA sequencing revealed a unique expression pattern for DCM circulating miRNAs. DACMs miR-26a-5p, miR-30c-5p, miR-126-5p and miR-126-3p were found to be depleted in DCM circulation as well as heart tissues. Their expressions in circulation and heart tissues were proven to be correlated significantly, and a combination of these miRNAs was suggested potential values for DCM diagnosis. FOXO3, a predicted common target, was experimentally demonstrated to be co-repressed within cardiomyocytes by these DACMs except miR-26a-5p. Delivery of a combination of miR-30c-5p, miR-126-5p and miR-126-3p into the murine myocardium via AAV9 carrying an expression cassette driven by cTnT promoter, or cardiac-specific knockout of FOXO3 (Myh6-CreERT2 , FOXO3 flox+/+ ) dramatically attenuated cardiac apoptosis and autophagy involved in DCM progression. Moreover, competitively disrupting the interplay between DACMs and FOXO3 mRNA by specifically introducing their interacting regions into murine myocardium crippled the cardioprotection of DACMs against DCM. Circulating cardiac miRNA-FOXO3 axis plays a pivotal role in safeguarding against myocardial apoptosis and excessive autophagy in DCM development, which may provide serological cues for DCM non-invasive diagnosis and shed light on DCM pathogenesis and therapeutic targets.

Silencing SIRT1 reduces 5-fluorouracil resistance of cholangiocarcinoma cells by inhibiting the FOXO1/Rab7 autophagy pathway

To investigate the mechanism by which SIRT1 silencing reduces 5-fluorouracil (5-FU) resistance of cholangiocarcinoma cells and the role of FOXO1/Rab7 autophagy pathway in mediating this effect. Human cholangiocarcinoma HCCC-9810 cells were treated with 50, 100, 150, and 200 μg/mL 5-FU to construct a 5-FU-resistant cell model, whose expressions of SIRT1, FOXO1 and Rab7 were detected with immunofluorescence assay, Western blotting and RTqPCR, and the expression levels of autophagy related proteins (Beclin1, LC3, and p62) were detected with Western blotting. The 5-FU resistant cells were transfected with a SIRT1 siRNA, and the changes in 5-Fu resistance and migration ability of the cells were evaluated using CCK-8 assay and wound healing assay; The changes in FOXO1 and Rab7 mRNA levels and protein expressions of SIRT1, FOXO1, Rab7, Beclin1, LC3 and P62 were detected with RT-qPCR and Western blotting. Treatments with 5-FU at 50, 100, 150, and 200 μg/mL all inhibited the proliferation of HCCC-9810 cells. Immunofluorescence assay revealed significantly enhanced SIRT1 expression in 5-FU-resistant HCC-9810 cells, and Western blotting also showed significantly up-regulated protein expressions of SIRT1, Rab7, P62, FOXO1 and Beclin 1 (P < 0.001) and an increased LC3II/LC3I ratio in the cells (P < 0.001). The mRNA levels of SIRT1, Rab7 and FOXO1 were also up-regulated in 5-Fu-resistant cells (P < 0.05). SIRT1 silencing significantly attenuated 5-FU resistance and migration ability of HCCC-9810 cells, and obviously decreased the protein expressions of SIRT1, Rab7, P62, FOXO1 and Beclin1 and the LC3II/LC3I ratio as well (P < 0.001). FOXO1 and Rab7 mRNA levels were significantly decreased in 5-FU-resistant HCC-9810 cells after SIRT1 silencing (P < 0.05). Silencing SIRT1 attenuates 5-FU resistance in HCC-9810 cells by inhibiting the activation of the FOXO1/Rab7 autophagy pathway.

MiR-27b attenuates mitochondrial oxidative stress and inflammation in endothelial cells

MiR-27b is highly expressed in endothelial cells (EC) but its function in this context is poorly characterized. This study aims to investigate the effect of miR-27b on inflammatory pathways, cell cycle, apoptosis, and mitochondrial oxidative imbalances in immortalized human aortic endothelial cells (teloHAEC), human umbilical vein endothelial cells (HUVEC), and human coronary artery endothelial cells (HCAEC) exposed to TNF-α. Treatment with TNF-α downregulates the expression of miR-27b in all EC lines, promotes the activation of inflammatory pathways, induces mitochondrial alteration and reactive oxygen species accumulation, fostering the induction of intrinsic apoptosis. Moreover, miR-27b mimic counteracts the TNF-α-related cytotoxicity and inflammation, as well as cell cycle arrest and caspase-3-dependent apoptosis, restoring mitochondria redox state, function, and membrane polarization. Mechanistically, hsa-miR-27b-3p targets the 3′untranslated regions of FOXO1 mRNA to downregulate its expression, blunting the activation of the Akt/FOXO1 pathway. Here, we show that miR-27b is involved in the regulation of a broad range of functionally intertwined phenomena in EC, suggesting its key role in mitigating mithochondrial oxidative stress and inflammation, most likely through targeting of FOXO1. Overall, results reveal for the first time that miR-27b could represent a possible target for future therapies aimed at improving endothelial health.

Zingerone reduces sodium arsenite-induced nephrotoxicity by regulating oxidative stress, inflammation, apoptosis and histopathological changes

Arsenic is widely available in the environment and arsenic toxicity is a public health problem of serious concern worldwide. Zingerone is a promising phytochemical with various pharmacological effects. In this study, the potential protective effect of zingerone against sodium arsenite (NaAsO2, SA) induced nephrotoxicity was investigated. Thirty-five male Sprague-Dawley rats were divided into five different groups as control, zingerone, SA, SA + zingerone 25, SA + zingerone 50. SA was administered alone at a dose of 10 mg/kg for 14 days or given 30 min before zingerone (25 mg/kg or 50 mg/kg) treatment. At the end of the experiment, the kidney tissues was examined biochemically, molecularly and microscopically. SA toxicity was associated with increased malondialdehyde level, whereas glutathione, superoxide dismutase, catalase, and glutathione peroxidase were decreased. Administration of SA caused inflammation in the kidney tissue by upregulation of NF-κB and IL-1β, TNF-α, IL-6, iNOS, COX-2, MAPK14, MAPK15, JNK. SA administration caused apoptosis in the kidney by upregulating caspase-3 and Bax levels and downregulating Bcl-2, and autophagy by activating beclin-1. Also, SA administration showed a suppressive effect on AKT2 and FOXO1 mRNA transcript levels. All these factors impair kidney function and increase creatinine and urea levels, resulting in pathological changes and a decrease in nephrin. Treatment with zingerone at doses of 25 and 50 mg/kg significantly reduced oxidative stress, inflammation, apoptosis and autophagy in kidney tissue. In addition, it was confirmed by histological evaluation as well as serum urea and creatinine levels that kidney damage due to SA toxicity can be modulated by zingerone administration.

Up-regulation of microRNA-183 reduces FOXO1 expression in gastric cancer patients with Helicobacter pylori infection.

The aim of the study is to detect the expression of FOXO1 mRNA and protein in samples from gastric cancer patients with Helicobacter pylori (H. pylori) infection, and to investigate the relationship between FOXO1 expression and miR-183 expression. Twenty-six gastric cancer patients with H. pylori infection and 26 gastric cancer patients without H. pylori infection were included into experimental group and control group, respectively. Tumor tissues and peripheral blood were collected from all subjects. QRT-PCR was used to determine the expression of miRNA and mRNA. Western blotting was carried out to measure protein expression. Dual luciferase reporter assay was used to identify direct interaction between miRNA and 3'-UTR of mRNA. Cell proliferation was examined by CCK-8 assay. FOXO1 mRNA and protein expression was down-regulated in gastric cancer patients, being possibly related to H. pylori infection. The expression of miR-183 in tumor tissues and serum from gastric cancer patients with H. pylori infection was elevated, and probably regulated the expression of FOXO1 by direct targeting. Stimulation by H. pylori up-regulated the expression of miR-183 in gastric cancer AGS cells, and reduced the levels of FOXO1 mRNA and protein. Inhibition of miR-183 elevated the expression of FOXO1 and suppressed the proliferation of AGS cells. The present study demonstrates that the expression of FOXO1 in tumor tissues and blood from gastric cancer patients with H. pylori infection is significantly down-regulated, and may be related to the up-regulation of miR-183. H. pylori may regulate FOXO1 expression through miR-183 to affect the pathological process of gastric cancer.

Effects of fasting on FOXO3 expression as an anti-aging biomarker in the liver

BackgroundAging is a multifactorial degenerative process that can be modulated by fasting through activation of the Fork-head transcription factor of the O class 3 (FOXO3), which plays an important role in increasing lifespans. However, the effects of different fasting durations on the expression of FOXO3 in the liver has not yet been reported. ObjectiveThis study analyzed the effects of different fasting durations on the FOXO3 expression and its pathway by measuring sirtuin1 (SIRT1), insulin-like growth factor-1 (IGF-1), and superoxide dismutase (SOD) activity in the liver. MethodsNew Zealand white rabbits were used to mimic the effects of fasting on humans. The rabbits were divided into the control, intermittent fasting (IF), and prolonged fasting (PF) groups. Both fasting groups were interspersed with the non-fasting phase for 8 h. This treatment was conducted for 6 days. On Day 7, all the rabbits were sacrificed, and their livers were taken to measure the FOXO3 and SIRT1 mRNA expressions, the IGF-1 protein level, and the SOD activity level. ANOVA, multiple comparison, and Pearson's correlation were performed for statistical analysis. ResultsThe FOXO3 and SIRT1 mRNA expressions were significantly higher in the IF group than in the control group. The FOXO3 expression was also 2.5 times higher in the IF group than in the PF group. There was a positive correlation between the FOXO3 and SIRT1 mRNA expressions. The IGF-1 protein level was significantly lower in the IF and PF groups than in the control group. The SOD-specific activity level was significantly higher in the IF group than in the control and PF groups. ConclusionsIntermittent fasting significantly increased the FOXO3 and SIRT1 mRNA expressions and the SOD activity level in the livers of the rabbits and significantly decreased the circulating and hepatic IGF-1. Therefore, intermittent fasting may give a protective intervention effect towards aging.

FOXO3 gene hypermethylation and its marked downregulation in breast cancer cases: A study on female patients.

FOXO3, a member of the FOX transcription factor family, is frequently described as being deregulated in cancer. Additionally, notable role of FOXO3 can be easily recognized in the process of ageing and survival. Even though various studies have been done to acknowledge the tumour-suppressive or oncogenic role of FOXO3 in cancer, still there exist a lack of understanding in terms of cancer prognosis and treatment. Therefore, to provide better insight, our study aims to evaluate the role and function of FOXO3 in breast cancer in Indian female patients. We examined the FOXO3 expression levels in breast cancer samples by analyzing mRNA and protein expression along with its clinicopathological parameters. A total of 127 cases of breast cancer with equal normal cases (n=127) were assessed with methylation (MS-PCR), Immunohistochemistry (IHC), mRNA expression using Real-time PCR was analysed and 66.14% cases at mRNA level were found to be downregulated, while 81.10% of cases had little or very little protein expression. Our data state, the promoter hypermethylation of the FOXO3 gene and the downregulated protein expression are significantly correlated (p=0.0004). Additionally, we found a significant correlation between the level of FOXO3 mRNA with ER (p=0.04) and status of lymph node (p=0.01) along with this. Data suggests the prognostic significance and the tumour-suppressive role of FOXO3 in breast cancer cases studied in India. However, there is a need for the extended research targeting FOXO3 to measure its clinical potential and develop well-defined therapeutic strategies.

THE FOXO3 LONGEVITY GENOTYPE PROTECTS AGAINST MECHANISMS OF CELLULAR AGING IN OKINAWANS

Abstract The genetic association of FOXO3 genotypes with human longevity is well established, although the mechanism is not fully understood. We now report on the relationship of the FOXO3 longevity variant rs2802292 with telomere length, telomerase activity, FOXO3 expression and inflammatory cytokine levels in men and women. In agreement with earlier work, the FOXO3 longevity variant conferred protection against telomere shortening of peripheral blood mononuclear cells from adults aged 55 and older. This was accompanied by higher levels of telomerase activity in carriers of the longevity-associated FOXO3 G-allele (P=0.015). FOXO3 mRNA expression increased slightly with age in both young (P=0.02) and old (P=0.08) G-allele carriers. Older female G-allele carriers displayed a modest decline in levels of pro-inflammatory cytokine IL-6 with age (P=0.07). In contrast, older male G-allele carriers displayed an age-dependent increase in levels of anti-inflammatory cytokine IL-10 with age (P=0.04). Thus, FOXO3 may act through several different pro-longevity mechanisms, which may differ by age and sex.

Melatonin Mitigates Cisplatin-Induced Ovarian Dysfunction via Altering Steroidogenesis, Inflammation, Apoptosis, Oxidative Stress, and PTEN/PI3K/Akt/mTOR/AMPK Signaling Pathway in Female Rats.

Ovarian damage and fertility impairment are major side effects of chemotherapy in pre-menopausal cancer patients. Cisplatin is a widely used chemotherapeutic drug. The present study was designed to assess the ameliorative effects of melatonin as an adjuvant for fertility preservation. Thirty-two adult female Wistar rats were divided randomly into four equal groups: Control, Melatonin, Cisplatin (CP) treated, and CP + Melatonin treated. The cisplatin-treated group showed decreased body and ovarian weights, decreased serum E2 and AMH, increased serum LH and FSH, reduced ovarian levels of SOD, CAT, GSH, and TAC, and increased ovarian MDA. The histopathological examination of the cisplatin-treated group showed deleterious changes within ovarian tissue in the form of damaged follicles and corpus luteum, hemorrhage, and inflammatory infiltrates with faint PAS reaction in zona pellucida, increased ovarian collagen deposition, and marked expression of caspase-3 immune reaction in granulosa and theca cells, stroma, and oocytes. Alongside, there was a significant downregulation in the mRNA expression of steroidogenic enzymes, IL10, AMPK, PI3K, AKT, mTOR, and PTEN, while TGF-β1, IL1β, IL6, TNF-α, NF-Kβ, P53, p38-MAPK, JNK, and FOXO3 mRNA expressions were upregulated in cisplatin-treated rats' ovarian tissue. Coadministration of cisplatin-treated rats with melatonin reversed these changes significantly. In conclusion, melatonin's antioxidant, anti-inflammatory, and anti-apoptotic activities could modulate ovarian disturbances induced by cisplatin and preserve fertility.

Circular RNA circFoxo3 Promotes Granulosa Cell Apoptosis Under Oxidative Stress Through Regulation of FOXO3 Protein.

Oxidative stress leads to ovarian functional decline by inducing granulosa cell (GC) apoptosis. Circular RNA circFoxo3 acts as a critical factor in regulating cell cycle and apoptosis, and cellular senescence in tumor cells. However, function of circFoxo3 is little understood in oxidative stress-induced injury of follicular GCs. In this study, we aimed to illustrate the regulation pattern of circFoxo3 in GCs under oxidative stress. CircFoxo3 was confirmed to be expressed in both human and mouse GCs by amplification with divergent primers and sequencing. In vitro and in vivo ovarian oxidative stress model, the expression of circFoxo3, FOXO3 protein, and its downstream targets were examined by quantitative real-time PCR and Western blotting, respectively. Knockdown of circFoxo3 was performed to evaluate the effects of circFoxo3-mediated GC apoptosis in vitro. RNA pull-down was used to discover the protein that interacted with circFoxo3 so as to illustrate the mechanism of circFoxo3 in GCs. Our results demonstrated that circFoxo3 was significantly upregulated in hydrogen peroxide (H2O2)-treated GCs and a 3-nitropropionic acid (3-NP)-induced mouse model of ovarian oxidative stress. Protein level of transcriptional factor FOXO3 was also remarkably increased in both in vitro and in vivo oxidative stress model, but FOXO3 mRNA expression revealed no significant difference. Knockdown of endogenous circFoxo3 downregulated FOXO3 protein level and blocked H2O2-induced cell apoptosis. CircFoxo3 could pull down high levels of MDM2 protein that induced FOXO3 ubiquitination and degradation. Furthermore, knockdown of MDM2 and circFoxo3 showed remarkably higher level of apoptosis when compared with the knockdown of circFoxo3 alone. Our study suggested that circFoxo3 regulated FOXO3 protein level in GCs by reducing interactions between FOXO3 and MDM2. In conclusion, circFoxo3 was positively associated with FOXO3 protein and jointly played crucial roles in mediating GC apoptosis induced by oxidative stress.

Hyperglycemia affects neuronal differentiation and Nestin, FOXO1, and LMO3 mRNA expression of human Wharton's jelly mesenchymal stem cells of children from diabetic mothers

Pregestational Diabetes Mellitus (PDM) during pregnancy constitutes an unfavorable embryonic and fetal development environment, with a high incidence of congenital malformations (CM). Neural tube defects are the second most common type of CM in children of diabetic mothers (CDM), who also have an elevated risk of developing neurodevelopmental disorders. The mechanisms that lead to these neuronal disorders in CDM are not yet fully understood. The present study aimed to know the effect of hyperglycemia on proliferation, neuronal differentiation percentage, and expression of neuronal differentiation mRNA markers in human umbilical cord Wharton's jelly mesenchymal stem cells (hUCWJMSC) of children from normoglycemic pregnancies (NGP) and PDM. We isolated and characterized hUCWJMSC by flow cytometry, immunofluorescence, RT-PCR and were induced to differentiate into adipocytes, osteocytes, and neurons. Proliferation assays were performed to determine the doubling time, and Nestin, TUBB3, FOXO1, KCNK2, LMO3, and MAP2 mRNA gene expression was assessed by semiquantitative RT-PCR. Hyperglycemia significantly decreased proliferation and neuronal differentiation percentage in NGP and PDM cells treated with 40 mM d-glucose. Nestin mRNA expression decreased under control glycemic conditions, while FOXO1, KCNK2, LMO3, and MAP2 mRNA expression increased during neuronal differentiation in both NGP and PDM cells. On the other hand, under hyperglycemic conditions, Nestin was significantly decreased in cells from NGP but not in cells from PDM, while mRNA expression of FOXO1 and LMO3 was significantly increased in cells from NGP, but not in cells from PDM. We found evidence that maternal PDM, with hyperglycemia in culture, affects the biological properties of fetal cells. All these results could be part of fetal programming.

Dietary DHA Enhanced the Textural Firmness of Common Carp (Cyprinus carpio L.) Fed Plant-Derived Diets through Restraining FoxO1 Pathways.

Omega-3 fatty acids have a positive effect on the muscle textural firmness of fish, while the intrinsic mechanism is poorly understood. To investigate the potential mechanism of textural modification caused by dietary docosahexaenoic acid ( DHA) in common carp (Cyprinus carpio L.), three plant-derived diets with varying DHA levels (0%, 0.5%, 1%, D1-D3) were prepared to feed juveniles (initial weight 15.27 ± 0.77 g) for 8 weeks, and the muscular texture, fibers density, and transcriptome were analyzed. The results showed that the growth performance, muscular DHA content, fibers density, and texture of the fish fed diets D2 and D3 were significantly ameliorated compared with the fish fed diet D1. The muscular transcriptome profiles indicated that the up-regulated genes of fish fed dietary DHA mainly in response to muscle proliferation, as well as the FoxO pathway, were significantly enriched in the D2 and D3 groups. Consistent with this, the Quantitative Real-Time PCR (qRT-PCR ) assays indicated that the expression of myogenic regulatory factors (myog, myod, mrf4, mrf5) was up-regulated in the high-DHA groups. Additionally, the expression of foxo1 (inhibitor of myofiber development) mRNA was down-regulated, while its negative regulatory pathway (MAPK and PI3K) was activated in the D2 and D3 groups. The results suggested that the DHA supplementation is beneficial to modifying the muscular textural firmness of common carp fed plant-derived diets, which could be attributed to the inhibition of FoxO1 pathways.

The N6-methyladenosine writer WTAP contributes to the induction of immune tolerance post kidney transplantation by targeting regulatory T cells

AbstractN6-methyladenosine (m6A) modification is involved in diverse immunoregulation, while the relationship between m6A modification and immune tolerance post kidney transplantation remains unclear. Expression of Wilms tumor 1-associating protein (WTAP), an m6A writer, was firstly detected in tolerant kidney transplant recipients (TOL). Then the role of WTAP on regulatory T (Treg) cell differentiation and function in CD4+ T cells from kidney transplant recipients with immune rejection (IR) was investigated. The potential target of WTAP and effect of WTAP on immune tolerance in vivo were subsequently verified. WTAP was upregulated in CD4+ T cells of TOL and positively correlated with Treg cell proportion. In vitro, WTAP overexpression promoted Treg cell differentiation and enhanced Treg cell-mediated suppression toward naïve T cells. Forkhead box other 1 (Foxo1) was identified as a target of WTAP. WTAP enhanced m6A modification of Foxo1 mRNA in coding sequence (CDS) region, leading to up-regulation of Foxo1. Overexpression of m6A demethylase removed the effect of WTAP overexpression, while Foxo1 overexpression reversed these effects. WTAP overexpression alleviated allograft rejection in model mice, as evidenced by reduced inflammatory response and increased Treg population. Our study suggests that WTAP plays a positive role in induction of immune tolerance post kidney transplant by promoting Treg cell differentiation and function.