RESUMO
FOXO1, a transcription factor downstream of the insulin/insulin like growth factor axis, has been linked to protein degradation. Elevated expression of FOXO orthologs can also prevent the aggregation of cytosine adenine guanine (CAG)-repeat disease causing polyglutamine (polyQ) proteins but whether FOXO1 targets mutant proteins for degradation is unclear. Here, we show that increased expression of FOXO1 prevents toxic polyQ aggregation in human cells while reducing FOXO1 levels has the opposite effect and accelerates it. Although FOXO1 indeed stimulates autophagy, its effect on polyQ aggregation is independent of autophagy, ubiquitin-proteasome system (UPS) mediated protein degradation and is not due to a change in mutant polyQ protein turnover. Instead, FOXO1 specifically downregulates protein synthesis rates from expanded pathogenic CAG repeat transcripts. FOXO1 orchestrates a change in the composition of proteins that occupy mutant expanded CAG transcripts, including the recruitment of IGF2BP3. This mRNA binding protein enables a FOXO1 driven decrease in pathogenic expanded CAG transcript- and protein levels, thereby reducing the initiation of amyloidogenesis. Our data thus demonstrate that FOXO1 not only preserves protein homeostasis at multiple levels, but also reduces the accumulation of aberrant RNA species that may co-contribute to the toxicity in CAG-repeat diseases.
Assuntos
Proteína Forkhead Box O1/genética , Peptídeos/genética , Agregação Patológica de Proteínas/genética , Proteínas de Ligação a RNA/genética , Adenina/metabolismo , Proteínas Amiloidogênicas , Autofagia/genética , Citosina/metabolismo , Proteína Forkhead Box O1/biossíntese , Regulação da Expressão Gênica/genética , Guanina/metabolismo , Células HEK293 , Humanos , Proteínas Mutantes/genética , Peptídeos/toxicidade , Agregação Patológica de Proteínas/patologia , Biossíntese de Proteínas/genética , Proteólise , RNA Mensageiro/genética , Repetições de Trinucleotídeos/genéticaRESUMO
This study attempted to analyze the alterations in the mRNA expression levels of autophagy- and apoptosis-related genes in the forkhead box transcription factor O (FOXO) pathway in schizophrenia patients before and after olanzapine treatment. For a total of 32 acute schizophrenic inpatients, clinical data with PANSS were obtained before and after four weeks of olanzapine treatment (mean dose 14.24 ± 4.35 mg/d) along with data from 32 healthy volunteers. The mRNA expression levels of the FOXO pathway genes were measured by real-time qPCR after fasting venous blood was collected and analyzed. The mRNA expression levels of FOXO1, FOXO3A, FASLG, and BCL2L11 were observed to be significantly decreased in acute schizophrenia patients. After four weeks of olanzapine treatment, the expression levels of the first three genes were further reduced, but BCL2L11 expression levels were not significantly changed. The pairwise correlations between the mRNA expression level of FASLG and those of the other three genes were not observed in acute schizophrenia patients, while these relationships were observed in healthy controls. After olanzapine treatment, the FASLG mRNA expression level was restored and exhibited a pairwise correlation with the FOXO3A and BCL2L11 mRNA expression levels but not with the FOXO1 mRNA expression level, and FASLG mRNA expression was also correlated with the duration of the disease. The statuses and correlations of the mRNA expression levels of FOXO pathway-related genes were altered in schizophrenia patients and were affected by olanzapine treatment and the duration of the disease.
Assuntos
Antipsicóticos/uso terapêutico , Apoptose/fisiologia , Autofagia/fisiologia , Fatores de Transcrição Forkhead/biossíntese , Olanzapina/uso terapêutico , Esquizofrenia/tratamento farmacológico , Adolescente , Adulto , Antipsicóticos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2/biossíntese , Proteína 11 Semelhante a Bcl-2/genética , Proteína Ligante Fas/biossíntese , Proteína Ligante Fas/genética , Feminino , Proteína Forkhead Box O1/biossíntese , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O3/biossíntese , Proteína Forkhead Box O3/genética , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Olanzapina/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Esquizofrenia/sangue , Esquizofrenia/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Resultado do Tratamento , Adulto JovemRESUMO
MiRNAs affect various biological pathways associated with the development, progression, clinical outcome and treatment response improvement in cervical cancer. This study was performed to evaluate the effects of miRNA 96 on cervical cancer and to clarify the mechanism. Vivo and vitro experiments were conducted in our trial. MiR-96 is upregulated in cervical cancer cell lines and cervical cancer tissues and is correlated with clinical features in cervical cancer patients. Overexpression of miR-96 enhances proliferation of cervical cancer cells, while inhibiting miR-96 reduces the proliferation of cervical cancer cells. Inhibition of miR-96 significantly decreased the percentage of cells in the S phase and increased the percentage of cells in G1/G0 peak in both SiHa and CaSki cells compared with NC cells and decreased the expressions of p21, p27 and cyclin D1. FOXO1 3'-UTR was sub cloned into a luciferase reporter vector and the putative miR-96 binding site in the FOXO1 3'-UTR was mutated. Treated with miR-96 inhibitor consistently enhanced the luciferase activity of the FOXO1 3'-UTR luciferase reporter plasmids in both SiHa and CaSki cells, whereas mutations in the miR-96-binding site abolished the effect. Vivo experiment also support these results. Therefore, inhibition of miR-96 might suppress growth, proliferation of CC cells and promote apoptosis of CC cells both in vitro and in vivo.
Assuntos
Proteína Forkhead Box O1/biossíntese , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/metabolismo , Neoplasias do Colo do Útero/patologia , Animais , Apoptose/genética , Proliferação de Células/genética , Feminino , Proteína Forkhead Box O1/genética , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismoRESUMO
BACKGROUND: Hepatic ER stress is a risk factor of insulin resistance and type 2 diabetes. X-box binding protein 1 spliced (XBP1s), a transcription factor, plays a key role in ameliorating insulin resistance and maintaining glucose homeostasis. Unfortunately, the short half-life of the protein dampens its clinical application, and the specific site of lysine residue that could be ubiquitinated and involved in the degradation of XBP1s remains elusive. METHODS AND RESULTS: Here, we identified K60 and K77 on XBP1s as two pivotal ubiquitin sites required for its proteasome-dependent degradation. We also constructed a double mutant form of XBP1s (K60/77R) and found that it showed higher capacity in resisting against ubiquitin-mediated protein degradation, increasing nuclear translocation, enhancing transcriptional activity, suppressing ER stress and promoting Foxo1 degradation, compared to that of wild type XBP1s (WT). Consistently, overexpression of the K60/77R XBP1s mutant in DIO mice increased the ability to reduce ER stress and decrease Foxo1 levels, thus contributed to maintaining glucose homeostasis. CONCLUSION: Our results suggest that delaying the degradation of XBP1s by preventing ubiquitination might provide a strategic approach for reducing ER stress as an anti-diabetes therapy.
Assuntos
Ubiquitinação/genética , Proteína 1 de Ligação a X-Box/genética , Animais , Dieta Hiperlipídica , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Proteína Forkhead Box O1/biossíntese , Proteína Forkhead Box O1/genética , Glucose/metabolismo , Teste de Tolerância a Glucose , Células HEK293 , Homeostase , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Obesidade/genética , Complexo de Endopeptidases do Proteassoma , Translocação Genética , Ubiquitinação/fisiologiaRESUMO
Forkhead box, class O, 1A (FOXO1A) is an important factor related to some human malignancies. We tested the association between FOXO1A cytoplasmic expression and World Health Organization grading system in primary brain tumors (PBTs). First of all, Western blot analysis was also performed in normal brain tissue and U87MG, LN229, GBM8401, and U118MG glioma cell lines protein lysates. Then, in order to realize FOXO1A gene expression in gliomas, U87MG, LN229, GBM8401 mRNA were applied to performed quantitative reverse transcription polymerase chain reaction (RT-PCR). At last, the immunohistochemical (IHC) analysis of FOXO1A was performed in 8 non-neoplastic brain tissues and 126 PBTs. The immunostain scores were obtained as the degree of cytoplasmic FOXO1A intensity multiplied by the percentage of positively stained tumor area. On the basis of the results of these in vitro studies, marked increase FOXO1A protein and mRNA expressions in glioma cell lines than in normal human tissue. On the view point of IHC stains, the average immunostain score of FOXO1A in all PBTs was significantly higher than non-neoplastic brain tissues. In addition, the immunostain scores of FOXO1A in high grade were higher than low-grade gliomas. Furthermore, higher cytoplasmic expression of FOXO1A might indicate the shorter overall survival rate in gliomas. Furthermore, FOXO1A expression was associated with isocitrate dehydrogenase I /2, ATRX, and p53 mutation by IHC staining. Therefore, the application of FOXO1A IHC stain might have some benefits to improve diagnostic accuracy, predict tumor progression, and evaluate survival time in glioma patients.
Assuntos
Neoplasias Encefálicas , Proteína Forkhead Box O1/biossíntese , Regulação Neoplásica da Expressão Gênica , Glioma , Proteínas de Neoplasias/biossíntese , Adulto , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Glioma/metabolismo , Glioma/mortalidade , Glioma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Valor Preditivo dos Testes , Taxa de SobrevidaRESUMO
Neuronal apoptosis induced by oxidative stress is a major pathological process that occurs after cerebral ischemia-reperfusion. Calycosin-7-O-ß-D-glucoside (CG) is a representative component of isoflavones in Radix Astragali (RA). Previous studies have shown that CG has potential neuroprotective effects. However, whether CG alleviates neuronal apoptosis through antioxidant stress after ischemia-reperfusion remains unknown. To investigate the positive effects of CG on oxidative stress and apoptosis of neurons, we simulated the ischemia-reperfusion process in vitro using an immortalized hippocampal neuron cell line (HT22) and oxygen-glucose deprivation/reperfusion (OGD/R) model. CG significantly improved cell viability and reduced oxidative stress and neuronal apoptosis. In addition, CG treatment upregulated the expression of SIRT1, FOXO1, PGC-1α, and Bcl-2 and downregulated the expression of Bax. In summary, our findings indicate that CG alleviates OGD/R-induced damage via the SIRT1/FOXO1/PGC-1α signaling pathway. Thus, CG maybe a promising therapeutic candidate for brain injury associated with ischemic stroke.
Assuntos
Apoptose/fisiologia , Proteína Forkhead Box O1/biossíntese , Glucose/deficiência , Glucosídeos/farmacologia , Isoflavonas/farmacologia , Estresse Oxidativo/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossíntese , Sirtuína 1/biossíntese , Animais , Apoptose/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVE: The aim of this study was to investigate the effect of long non-coding ribonucleic acid (lncRNA) maternally expressed gene 3 (MEG3) on retinopathy in diabetic rats by regulating the expression of forkhead transcription factor 01 (Fox01). MATERIALS AND METHODS: All rats were randomly divided into three groups, including the control group (n=10), diabetes mellitus (DM) group (n=10) and lncRNA MEG3 transfection group (n=10). The expressions of Fox01 and interleukin-1ß (IL-1ß) in the three groups were detected using immunohistochemical staining, quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting. RESULTS: Microscopic examinations showed that the retinal structure was clear and complete, the inner limiting membrane surface was smooth, and the cells were arranged orderly with uniform structure in the control group. In the DM group, the retinal ganglia were slightly thickened, the histiocytes were sparse and arranged disorderly, and the edema of the outer plexiform layer (OPL) was significant. Meanwhile, there was abnormal microvascular dilatation without neovascularization. In lncRNA MEG3 transfection group, the edema of retinal OPL was significantly alleviated when compared with the DM group, showing statistically significant differences (p<0.05). The results of immunohistochemical staining showed that the expressions of Fox01 and IL-1ß in the inner plexiform layer and inner nuclear layer increased markedly in the DM group and lncRNA MEG3 transfection group when compared with those in the control group (p<0.05). However, they were both significantly declined in lncRNA MEG3 transfection group when compared with the DM group (p<0.05). Furthermore, Western blotting and qRT-PCR indicated that the protein and mRNA expressions of Fox01 and IL-1ß in the retina of DM group and lncRNA MEG3 transfection group were remarkably higher than the control group (p<0.05). However, they were remarkably declined in lncRNA MEG3 transfection group when compared with the DM group (p<0.05). CONCLUSIONS: LncRNA MEG3 plays an important role in retinopathy in diabetic rats. In addition, it can ameliorate retinopathy in diabetic rats by inhibiting the expressions of IL-1ß and Fox01.
Assuntos
Diabetes Mellitus Experimental/genética , Retinopatia Diabética/genética , Proteína Forkhead Box O1/biossíntese , RNA Longo não Codificante/genética , Animais , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/patologia , Regulação da Expressão Gênica , Interleucina-1beta/biossíntese , RNA Longo não Codificante/metabolismo , Ratos , TransfecçãoRESUMO
Studies have shown that promoting the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts could protect against osteoporosis. Increasing evidence demonstrates that long non-coding RNAs (lncRNAs) participate in BMSC osteogenic differentiation. This study aimed to investigate the role and underlying mechanism of growth arrest-specific transcript 5 (GAS5) in osteogenic differentiation. The mechanism was mainly focused on miR-135a-5p/FOXO1 pathway by gain- and loss-of function tests. GAS5 and FOXO1 expression was decreased, whereas miR-135a-5p expression was increased, in the BMSCs from osteoporotic mice. Levels of GAS5 and FOXO1 were increased and miR-135a-5p expression was decreased during osteogenic differentiation of BMSCs. Overexpression of GAS5 promoted, whereas knockdown of GAS5 suppressed, BMSC osteogenic differentiation. As for the mechanism, GAS5 functioned as a competing endogenous RNA for miR-135a-5p to regulate FOXO1 expression. In conclusion, GAS5 promoted osteogenesis of BMSCs by regulating the miR-135a-5p/FOXO1 axis. This finding suggests that targeting GAS5 may be a useful therapy for treating postmenopausal osteoporosis.
Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular , Proteína Forkhead Box O1/biossíntese , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/biossíntese , Osteogênese , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Animais , Células da Medula Óssea/citologia , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células-Tronco Mesenquimais/citologia , Camundongos , RNA Longo não Codificante/genéticaRESUMO
Recent studies indicate that FoxO1 has roles in female reproductive system, especially in maternal endometrium. Although various cellular aspects and molecular pathways have been identified, the exact molecular characteristics of embryo implantation are still not completely understood. In this study, we aimed to investigate uterine expression and regulation of FoxO1 during peri-implantation period in mice. Experimental mouse models including, normal pregnancy, pseudopregnancy, artificial decidualization, and delayed implantation and activation were performed. Our results showed that FoxO1 expression was spatiotemporal in mouse endometrial tissue throughout peri-implantation period and its expression was significantly upregulated in luminal and glandular epithelium at the time of implantation. Moreover, on day 5 morning (09:00 AM) of pregnancy, expression of FoxO1 was cytoplasmic in endometrial luminal epithelial cells where embryo homing takes place. With progressing time on day 5 evening (19:00 PM) of pregnancy FoxO1 expression was nuclear in luminal epithelium at implantation site. Pseudopregnancy and artificial decidualization models indicated that FoxO1 expression was regulated by pregnancy hormones. Delayed implantation and activation model indicated that FoxO1 expression at the time of implantation is dependent upon activation status of blastocyst due to E2 induction and uterine sensitivity to implantation. In conclusion, our findings highlight a perspective for FoxO1 expression and regulation in mouse uterus during peri-implantation period indicating that its expression is regulated by implanting embryo and pregnancy hormones.
Assuntos
Decídua/metabolismo , Implantação Tardia do Embrião/fisiologia , Proteína Forkhead Box O1/biossíntese , Regulação da Expressão Gênica/fisiologia , Gravidez/fisiologia , Pseudogravidez/metabolismo , Animais , Blastocisto/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Neurodegenerative disorders such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by impairment of autophagy. Cellular survival is dependent on efficient clearance of phosphorylated α-synuclein, which accumulates as fibrils in the neuronal cytoplasm as Lewy bodies (LBs). The forkhead box O 1 (FOXO1) is a member of the FOXO family that functions in various intracellular processes including regulation of autophagy. Transcriptional activation of FOXO1 has been reported to initiate autophagy by inhibiting the expression of Mechanistic Target of Rapamycin (mTOR), mediated by sestrin 3 (SESN3) and tuberous sclerosis complex 2 (TSC2). Although many autophagy-related proteins are known to be incorporated into LBs, no report has documented the involvement of these autophagy modulators (FOXO1, SESN3 and TSC2) in the pathogenesis of PD and DLB. In the present study, we performed immunostaining and Western blot analysis using the brains of normal controls and patients with PD and DLB in order to clarify the involvement of FOXO1, SESN3 and TSC2 in LBs. Our study demonstrated for the first time the presence of FOXO1, SESN3 and TSC2 in brainstem-type LBs. The expression levels of these proteins in the brain did not differ between the normal controls and patients with PD or DLB. We further utilized mice model to investigate the effect of α-synuclein overexpression on these proteins, and found that TSC2 was significantly increased in α-synuclein transgenic mice relative to wild type mice at 9 weeks of age, but not at 30 weeks of age. Together with expression data showing gradual increase of these molecules with age in wild type mice, these findings suggest that autophagy modulators are incorporated into LBs and that the expression of these proteins can be increased by various factors including aging.
Assuntos
Envelhecimento/metabolismo , Autofagia/fisiologia , Proteína Forkhead Box O1/biossíntese , Proteínas de Choque Térmico/biossíntese , Doença por Corpos de Lewy/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/biossíntese , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Animais , Tronco Encefálico/metabolismo , Tronco Encefálico/patologia , Feminino , Células HeLa , Humanos , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Doença por Corpos de Lewy/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-IdadeRESUMO
Sterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that plays a central role in regulating expression of genes of carbohydrate and fatty acid metabolism in the liver. SREBP-1 activity is essential for the control of insulin-induced anabolic processes during the fed state. In addition, SREBP-1 regulates expression of key molecules in the insulin signaling pathway, including insulin receptor substrate 2 (IRS2) and a subunit of the phosphatidylinositol 3-kinase (PI3K) complex, PIK3R3, suggesting that feedback mechanisms exist between SREBP-1 and this pathway. Nevertheless, the overall contribution of SREBP-1 activity to maintain insulin signal transduction is unknown. Furthermore, Akt is a known activator of mTORC1, a sensor of energy availability that plays a fundamental role in metabolism, cellular growth and survival. We have silenced SREBP-1 and explored the impact on insulin signaling and mTOR in mice under fed, fasted and refed conditions. No alterations in circulating levels of insulin were observed. The studies revealed that depletion of SREBP-1 had no impact on IRS1Y612, AktS473, and downstream effectors GSK3αS21 and FoxO1S256 during the fed state. Nevertheless, reduced levels of these molecules were observed under fasting conditions. These effects were not associated with changes in phosphorylation of mTOR. Overall, our data indicate that the contribution of SREBP-1 to maintain insulin signal transduction in liver is modest.
Assuntos
Insulina/fisiologia , Fígado/metabolismo , Transdução de Sinais/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 1/fisiologia , Animais , Glicemia/análise , Células Cultivadas , Metabolismo Energético/genética , Jejum/metabolismo , Proteína Forkhead Box O1/biossíntese , Proteína Forkhead Box O1/genética , Vetores Genéticos , Glucoquinase/metabolismo , Gluconeogênese/genética , Quinase 3 da Glicogênio Sintase/biossíntese , Quinase 3 da Glicogênio Sintase/genética , Hepatócitos/metabolismo , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Período Pós-Prandial/fisiologia , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Activation of the transcription factor hypoxia inducible factor1α (HIF-1α) is considered critical for the stimulation of osteogenic markers including runtrelated transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin, which are closely associated with forkhead boxclass O1 (Foxo1) levels in osteoblasts. The present study explored the associations between HIF1α and Foxo1 in the regulation of cell viability, proliferation and apoptosis of osteoblasts. Osteoblasts obtained from children's iliac cancellous bone were used in the present study, which were confirmed by immunofluorescence staining for the osteoblast marker osteocalcin. The results revealed that the levels of reactive oxygen species and apoptosis were markedly increased in cells with knockdown of HIF1α. By contrast, these were reduced in response to overexpressed HIF1α. In addition, HIF1α overexpression significantly stimulated cell viability, which was suppressed by silencing HIF1α. HIF1α overexpression also significantly increased the transcriptional and translational levels of Foxo1. Conversely, silencing HIF1α markedly suppressed the expression levels of Foxo1. Furthermore, silencing HIF1α reduced the expression of osteogenic markers, including Runx2, ALP and osteocalcin. Runx2 and ALP expression induced by HIF1α were markedly reversed by Foxo1 small interfering (si)RNA, whereas osteocalcin was not significantly affected by Foxo1 siRNA. Therefore, the cooperation of and interactions between HIF1α and Foxo1 may be involved in the regulation of osteoblast markers, and serve a pivotal role in the proliferation and apoptosis of osteoblast. The HIF1αinduced expression of Runx2 and ALP may be completely dependent on the expression levels of Foxo1, and in turn, osteocalcin may be partially dependent on Foxo1 expression.
Assuntos
Apoptose/fisiologia , Osso Esponjoso/metabolismo , Proliferação de Células/fisiologia , Proteína Forkhead Box O1/biossíntese , Regulação da Expressão Gênica/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ílio/metabolismo , Osteoblastos/metabolismo , Antígenos de Diferenciação/biossíntese , Osso Esponjoso/citologia , Pré-Escolar , Feminino , Humanos , Ílio/citologia , Masculino , Osteoblastos/citologiaRESUMO
Immunity following an acutely resolved infection or the long-term equipoise of chronic viral infections often depends on the maintenance of antigen-specific CD8+ T cells, yet the ongoing transcriptional requirements of these cells remain unclear. We show that active and continuous programming by FOXO1 is required for the functional maintenance of a memory population. Upon Foxo1 deletion following resolution of an infection, memory cells rapidly lost their characteristic gene expression, gradually declined in number, and were impaired in self-renewal. This was extended to chronic infections, as a loss of FOXO1 during a persistent viral infection led to a rapid decline of the TCF7 (a.k.a. TCF1)-expressing memory-like subset of CD8+ T cells. We further establish FOXO1 regulation as a characteristic of human memory CD8+ T cells. Overall, we show that the molecular and functional longevity of a memory T cell population is actively maintained by the transcription factor FOXO1.
Assuntos
Proteína Forkhead Box O1/imunologia , Memória Imunológica/imunologia , Linfócitos T/imunologia , Doença Aguda , Animais , Doença Crônica , Proteína Forkhead Box O1/biossíntese , Proteína Forkhead Box O1/sangue , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/metabolismoRESUMO
It has been shown that microRNAs (miRNAs) greatly affect the functions of vascular smooth muscle cells (VSMC), but the effects of mRNAs under diabetic conditions remain unclear.Using a model of diabetic db/db mice, we studied the functions of microRNA-135a (miR-135a) during VSMC dysfunction.Compared to control WT mice, miR-135a expression in VSMC was significantly increased while the level of forkhead box O1 (FOXO1) protein decreased significantly. After transfecting miR-135a mimics into VSMC, the expression of FOXO1 was decreased, while cyclooxygenase-2 (COX-2) and monocyte chemoattractant protein-1 (MCP-1) expression levels were increased, thus promoting the interaction between monocytes and WT VSMC. On the other hand, transfection of an miR-135a inhibitor reversed the activated interaction between monocytes and db/db VSMC. The pro-inflammatory responses could also be enhanced by using siRNAs to silence the FOXO1 gene in WT VSMC, suggesting a negative regulatory role of FOXO1. FOXO1 siRNAs and miR-135a mimics could both enhance the transcriptional activity of COX-2 promoter. Using chromatin immunoprecipitation, we found that in db/db VSMC, the occupancy in promoter regions of inflammatory genes by FOXO1 was reduced.miR-135a increased the inflammatory responses of VSMC involved in complications of vascular diseases by downregulating the expression of FOXO1.
Assuntos
Regulação para Baixo , Proteína Forkhead Box O1/genética , Regulação da Expressão Gênica , Inflamação/genética , MicroRNAs/genética , Miócitos de Músculo Liso/metabolismo , Doenças Vasculares/genética , Animais , Western Blotting , Células Cultivadas , DNA/genética , Modelos Animais de Doenças , Proteína Forkhead Box O1/biossíntese , Imuno-Histoquímica , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/biossíntese , Miócitos de Músculo Liso/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doenças Vasculares/metabolismo , Doenças Vasculares/patologiaRESUMO
Growth arrest special 5 (GAS5) is a long non-coding RNA reported to function as an inhibitor in various tumors including cervical cancer. However, the molecular mechanism of GAS5 involved in cervical cancer progression remains far from being elucidated. The expression of GAS5, forkhead box protein O1 and phosphatase and tensin homolog was examined by quantitative reverse transcription polymerase chain reaction qRT-PCR. cell growth, invasion, and apoptosis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, transwell invasion assay, and flow cytometry analysis, respectively. The interaction between GAS5 and miR-196a or miR-205 was confirmed by luciferase reporter assay, RNA immunoprecipitation assay, and qRT-PCR. Xenograft tumor experiments were performed to validate the biological role of GAS5 and its molecular mechanism in cervical cancer in vivo. GAS5 expression was decreased in cervical cancer tissues and cells. GAS5 overexpression suppressed cervical cancer cell proliferation, invasion, and apoptosis. GAS5 was able to directly bind to miR-196a and miR-205 to downregulate their expression. Moreover, GAS5 induced forkhead box protein O1 and phosphatase and tensin homolog expression by repressing miR-196a and miR-205, respectively. Exogenous expression of GAS5 hindered tumor growth in vivo by downregulating miR-196a and miR-205. Upregulation of GAS5 suppressed cell proliferation, invasion, and apoptosis of cervical cancer cells by downregulating miR-196a and miR-205, contributing to our understanding the pathogenesis of cervical cancer and development of long non-coding RNA-mediated clinical therapy against this disease.
Assuntos
Carcinogênese/genética , MicroRNAs/biossíntese , RNA Longo não Codificante/genética , Neoplasias do Colo do Útero/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Proteína Forkhead Box O1/biossíntese , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , MicroRNAs/genética , PTEN Fosfo-Hidrolase/biossíntese , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: The objective of the study was to investigate the role of microRNA-9 (miR-9) targeting forkhead box O1 (FOXO1) in the proliferation, migration, and invasion of breast cancer cells. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the expressions of miR-9 and FOXO1 mRNA in breast cancer tissues, normal breast tissues, breast cancer cell lines, and normal breast epithelial cells. After the up-regulation of miR-9 expression, qRT-PCR and Western blotting were used to determine the expression of FOXO1. The luciferase reporter gene assay was used to validate the target gene. The CCK-8 assay, scratch-wound healing assay, and Transwell invasion assay were used to investigate the changes in the proliferation, migration, and invasion of breast cancer cells, respectively. RESULTS: MicroRNA-9 expression was significantly up-regulated in breast cancer tissues and breast cancer cell lines when compared with normal breast tissues and normal breast epithelial cells (both P < 0.05). FOXO1 mRNA and protein expressions were substantially down-regulated in breast cancer tissues and breast cancer cell lines when compared with normal breast tissues and normal breast epithelial cells (both P < 0.05). There can be a negative correlation between miR-9 and FOXO1 mRNA in breast cancer. Luciferase reporter gene assay indicated that miR-9 can down-regulate FOXO1 expression at a post-transcriptional level through binding specifically to FOXO1 3'UTR. The results of CCK-8 assay, scratch-wound healing assay, and Transwell invasion assay revealed that the inhibition of miR-9 can suppress MCF7 cell proliferation, migration, and invasion. Additionally, the expression of miR-9 increased significantly whilst that of FOXO1 decreased substantially as the disease progressed (P < 0.05). CONCLUSIONS: Our study provides evidence that miR-9 can promote the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1.
Assuntos
Neoplasias da Mama/genética , Proteína Forkhead Box O1/biossíntese , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/biossíntese , Invasividade Neoplásica/genética , Adulto , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo , Feminino , Humanos , Células MCF-7 , Pessoa de Meia-IdadeRESUMO
Asbestos exposure causes malignant tumors such as lung cancer and malignant mesothelioma. Based on our hypothesis in which continuous exposure to asbestos of immune cells cause reduction of antitumor immunity, the decrease of natural killer cell killing activity with reduction of NKp46 activating receptor expression, inhibition of cytotoxic T cell clonal expansion, reduced CXCR3 chemokine receptor expression and production of interferon-γ production in CD4+ T cells were reported using cell line models, freshly isolated peripheral blood immune cells from health donors as well as asbestos exposed patients such as pleural plaque and mesothelioma. In addition to these findings, regulatory T cells (Treg) showed enhanced function through cell-cell contact and increased secretion of typical soluble factors, interleukin (IL)-10 and transforming growth factor (TGF)-ß, in a cell line model using the MT-2 human polyclonal T cells and its sublines exposed continuously to asbestos fibers. Since these sublines showed a remarkable reduction of FoxO1 transcription factor, which regulates various cell cycle regulators in asbestos-exposed sublines, the cell cycle progression in these sublines was examined and compared with that of the original MT-2 cells. Results showed that cyclin D1 expression was markedly enhanced, and various cyclin-dependent kinase-inhibitors were reduced with increased S phases in the sublines. Furthermore, the increase of cyclin D1 expression was regulated by FoxO1. The overall findings indicate that antitumor immunity in asbestos-exposed individuals may be reduced in Treg through changes in the function and volume of Treg.
Assuntos
Ciclina D1/imunologia , Proteína Forkhead Box O1/biossíntese , Neoplasias Pulmonares/imunologia , Mesotelioma/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/imunologia , Ciclina D1/biossíntese , Ciclina D1/sangue , Proteína Forkhead Box O1/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-10/biossíntese , Interleucina-10/sangue , Interleucina-10/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/patologia , Mesotelioma/sangue , Mesotelioma/induzido quimicamente , Mesotelioma/patologia , Mesotelioma Maligno , Receptor 1 Desencadeador da Citotoxicidade Natural/biossíntese , Receptor 1 Desencadeador da Citotoxicidade Natural/sangue , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Receptores CXCR3/biossíntese , Receptores CXCR3/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta/imunologiaRESUMO
BACKGROUND: In mammals, the IGF-1 pathway affects the phenotype of aging. Since the function of the immune system is modulated by IGF-1, it is plausible that immunosenescence might in part result from altered control by this pathway. We therefore examined whether the expression of IGF-1R, FOXO1, and FOXO3a in peripheral blood mononuclear cells (PBMC) changes with age and if this might be due to changes in the expression of select miRNAs. METHODS: The expression of IGF-1R, FOXO1, FOXO3a, as well as of miR-9, miR-96, miR-99a, miR-132, miR-145, and miR-182 was examined in PBMC of young (27.8 ± 3.7 years), elderly (65.6 ± 3.4 years), and long-lived (94.0 ± 3.7 years) Polish Caucasians using real-time PCR. mRNA/miRNA interactions were studied in HEK 293 cells using luciferase-expressing pmirGLO reporter vector. RESULTS: The median expression of IGF-1R decreased with age (p < 0.000001), as did the expression of FOXO1 (p < 0.000001), while the expression of FOXO3a remained stable. We also found an age-associated increase of the median expression of miR-96 (p = 0.002), miR-145 (p = 0.024) and miR-9 (p = 0.026), decrease of the expression of miR-99a (p = 0.037), and no changes regarding miR-132 and miR-182. Functional studies revealed that miR-96 and miR-182 interacted with human IGF-1R mRNA, and that miR-145 and miR-132 interacted with human FOXO1 mRNA. CONCLUSIONS: The age-associated higher expression of miR-96 and miR-145 might contribute to the lower expression of IGF-1R while the higher expression of miR-96, miR-145 and miR-9 might contribute to the lower expression of FOXO1 in peripheral blood mononuclear cells of aging humans. Sustained expression/function of FOXO3a but not of the other two genes might be important for the maintenance of the immune system function in these individuals.
Assuntos
Envelhecimento/genética , Proteína Forkhead Box O1/genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Receptores de Somatomedina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , DNA/genética , Feminino , Proteína Forkhead Box O1/biossíntese , Células HEK293 , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Masculino , MicroRNAs/biossíntese , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Receptor IGF Tipo 1 , Receptores de Somatomedina/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
The hepatitis C virus (HCV) core protein is critical in the development of hepatocellular carcinoma (HCC). Investigations on HCC have previously focused on microRNAs, a class of small noncoding RNAs, which are crucial in cancer development and progression. The present study aimed to investigate whether microRNA (miR)196a is aberrantly regulated by the HCV core protein, and whether miR196a is involved in the regulation of the aberrant proliferation of HCVHCC cells. In the study, miRNA expression was detected by quantitative polymerase chain reaction analysis. An AdHCV core adenovirus was constructed and cell proliferation was measured using a Cell Counting Kit-8 assay and a cell cycle assay following infection. The results of the present study demonstrated that the HCV core protein increased the expression of miR196a, and that overexpression of miR196a in the HepG2 and Huh7 HCC cell lines promoted cell proliferation by inducing the G1S transition. Furthermore, the present study demonstrated that forkhead box O1 (FOXO1) was directly regulated by miR196a, and was essential in mediating the biological effects of miR196a in HCC. The overexpression of FOXO1 markedly reversed the effect of miR196a in HCC cell proliferation. Taken together, the data obtained in the present study provided compelling evidence that elevated expression levels of miR196a by the HCV core protein can function as an oncomicroRNA during HCVinduced cell proliferation by downregulating the expression of FOXO1, indicating a potential novel therapeutic target for HCV-related HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Proteína Forkhead Box O1/biossíntese , Regulação Neoplásica da Expressão Gênica , Hepacivirus/metabolismo , Neoplasias Hepáticas/metabolismo , MicroRNAs/biossíntese , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/biossíntese , Regulação para Cima , Proteínas do Core Viral/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Proteína Forkhead Box O1/genética , Células Hep G2 , Hepacivirus/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , MicroRNAs/genética , Proteínas de Neoplasias/genética , RNA Neoplásico/genética , Proteínas do Core Viral/genéticaRESUMO
It has previously been demonstrated that curcumin possesses an antitumor activity, which is associated with its ability to induce G2/M cell cycle arrest and apoptosis. However the detailed underlying mechanisms remain unclear. The present study aimed to investigate the efficacy and underlying mechanism of curcumininduced cell cycle arrest and apoptosis in U87 human glioblastoma cells. By immunofluorescence staining, subcellular fractionation and western blotting, the present study demonstrated that curcumin was able to induce G2/M cell cycle arrest and apoptosis by increasing the expression levels of cyclin G2, cleaved caspase3 and Fas ligand (FasL), and decreasing the expression of cyclindependent kinase 1 (CDK1). In addition, increased expression of forkhead box protein O1 (FoxO1) and decreased expression of phosphorylated (p)FoxO1 were detected in the curcumintreated U87 cells. Curcumin was also able to induce the translocation of FoxO1 from the cytoplasm to the nucleus. Furthermore, following knockdown of FoxO1 expression in curcumintreated U87 cells using FoxO1 small interfering RNA, the expression levels of cyclin G2, cleaved caspase3 and FasL were inhibited; however, the expression levels of CDK1 were not markedly altered. Notably, following knockdown of CDK1 expression under normal conditions, the total expression of FoxO1 was not affected; however, pFoxO1 expression was decreased and FoxO1 nuclear expression was increased. Furthermore, curcumininduced G2/M cell cycle arrest and apoptosis could be attenuated by FoxO1 knockdown. These results indicated that curcumin may induce G2/M cell cycle arrest and apoptosis in U87 cells by increasing FoxO1 expression. The present study identified a novel mechanism underlying the antitumor effects of curcumin, and may provide a theoretical basis for the application of curcumin in glioma treatment.