RESUMEN
Repeated sevoflurane exposure in neonatal mice triggers neuroinflammation with detrimental effects on cognitive function. Yet, the mechanism of the sevoflurane-induced cytokine response is largely unknown. In this study, we reveal that 3-MA, an autophagy inhibitor, attenuated the sevoflurane-induced neuroinflammation and cognitive dysfunction, including the decreased freezing time and fewer platform crossings, in the neonate mice. 3-Methyladenine (3-MA) suppressed sevoflurane-induced expression of interleukin-6 and tumor necrosis factor-alpha in vitro. Moreover, sevoflurane activates IRF3, facilitating cytokine transcription in an AKT3-dependent manner. Mechanistically, sevoflurane-induced autophagic degradation of dehydrocholesterol-reductase-7 (DHCR7) resulted in accumulations of its substrate 7-dehydrocholesterol (7-DHC), mimicking the effect of sevoflurane on AKT3 activation and IRF3-driven cytokine expression. 3-MA significantly reversed sevoflurane-induced DHCR7 degradation, AKT phosphorylation, IRF3 activation, and the accumulation of 7-DHC in the hippocampal CA1 region. These findings pave the way for additional investigations aimed at developing novel strategies to mitigate postoperative cognitive impairment in pediatric patients.
RESUMEN
Focal malformations of cortical development (FMCDs) are brain disorders mainly caused by hyperactive mTOR signaling due to both inactivating and activating mutations of genes in the PI3K-AKT-mTOR pathway. Among them, mosaic and somatic activating mutations of the mTOR pathway activators are more frequently linked to severe form of FMCDs. A human stem cell-based FMCDs model to study these activating mutations is still lacking. Herein, we genetically engineer human embryonic stem cell lines carrying these activating mutations to generate cortical organoids. Mosaic and somatic expression of AKT3 activating mutations in cortical organoids mimicking the disease presentation with overproliferation and the formation of dysmorphic neurons. In parallel comparison of various AKT3 activating mutations reveals that stronger mutation is associated with more severe neuronal migratory and overgrowth defects. Together, we have established a feasible human stem cell-based model for FMCDs that could help to better understand pathogenic mechanism and develop novel therapeutic strategy.
Asunto(s)
Malformaciones del Desarrollo Cortical , Organoides , Proteínas Proto-Oncogénicas c-akt , Humanos , Organoides/metabolismo , Organoides/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/patología , Malformaciones del Desarrollo Cortical/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Transducción de Señal/genética , Corteza Cerebral/patología , Corteza Cerebral/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/genética , Mutación , Neuronas/metabolismo , Neuronas/patología , Línea CelularRESUMEN
BACKGROUND: As the most abundant modification in eukaryotic messenger RNAs (mRNAs), N6-methyladenosine (m6A) plays vital roles in many biological processes. METHODS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen for m6A targets in esophageal cancer cells and patients. The role of m6A RNA methylase in esophageal cancer was also analyzed using bioinformatics. In vitro and in vivo experiments were used to analyze gene expression and function. CCK-8, colony formation, cell apoptosis and immunofluorescence staining assays were performed to evaluate the proliferation, migration and invasion of esophageal cancer cells, respectively. Western blot analysis, RNA stability, RIP and luciferase reporter assays were performed to elucidate the underlying mechanism involved. RESULTS: We found that the m6A demethylase FTO was significantly upregulated in esophageal cancer cell lines and patient tissues. In vivo and in vitro assays demonstrated that FTO was involved in the proliferation and apoptosis of esophageal cancer cells. Moreover, we found that the m6A methyltransferase METTL14 negatively regulates FTO function in esophageal cancer progression. FTO alone is not related to the prognosis of esophageal cancer, and its function is antagonized by METTL14. By using transcriptome-wide m6A-seq and RNA-seq assays, we revealed that AKT3 is a downstream target of FTO and acts in concert to regulate the tumorigenesis and metastasis of esophageal cancer. Taken together, these findings provide insight into m6A-mediated tumorigenesis in esophageal cancer and could lead to the design of new therapeutic strategies.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Neoplasias Esofágicas , Metiltransferasas , Humanos , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Carcinogénesis , Transformación Celular Neoplásica , Desmetilación , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Metiltransferasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Germline gain of function variations in the AKT3 gene cause brain overgrowth syndrome with megalencephaly and diffuse bilateral cortical malformations. Here we report a child with megalencephaly, who is a carrier of a novel heterozygous missense variant in the AKT3 gene NM_005465.7:c.964G>T,p.Asp322Tyr. The phenotype of this patient is associated with pituitary deficiencies diagnosed at 2 years of age: growth hormone (GH) deficiency responsible for growth delay and central hypothyroidism. After 6 months of GH treatment, intracranial hypertension was noted, confirmed by the observation of papilledema and increased intracranial pressure, requiring the initiation of acetazolamide treatment and the discontinuation of GH treatment. This is the second reported patient described with megalencephaly and AKT3 gene variant associated with GH deficiency . Other endocrine disorders have also been reported in few cases with hypothyroidism and hypoglycemia. Pituitary deficiency may be a part of the of megalencephaly phenotype secondary to germline variant in the AKT3 gene. Special attention should be paid to growth in these patients and search for endocrine deficiency is necessary in case of growth retardation or hypoglycemia.
Asunto(s)
Mutación de Línea Germinal , Megalencefalia , Mutación Missense , Proteínas Proto-Oncogénicas c-akt , Humanos , Megalencefalia/genética , Megalencefalia/patología , Mutación Missense/genética , Proteínas Proto-Oncogénicas c-akt/genética , Mutación de Línea Germinal/genética , Masculino , Preescolar , Fenotipo , Hipotiroidismo/genética , Hipotiroidismo/patología , Hipotiroidismo/complicaciones , Femenino , Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/genéticaRESUMEN
Communication between adjacent endothelial cells is important for the homeostasis of blood vessels. We show that quiescent endothelial cells use Jagged1 to instruct neighboring endothelial cells to assume a quiescent phenotype and secure the endothelial barrier. This phenotype enforcement by neighboring cells is operated by R-Ras through activation of Akt3, which results in upregulation of a Notch ligand Jagged1 and consequential upregulation of Notch target genes, such as UNC5B, and VE-cadherin accumulation in the neighboring cells. These signaling events lead to the stable interaction between neighboring endothelial cells to continue to fortify juxtacrine signaling via Jagged1-Notch. This mode of intercellular signaling provides a positive feedback regulation of endothelial cell-cell interactions and cellular quiescence required for the stabilization of the endothelium.
Asunto(s)
Células Endoteliales , Proteínas de la Membrana , Proteínas Serrate-Jagged , Células Endoteliales/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al Calcio/genética , Péptidos y Proteínas de Señalización Intercelular , Receptores Notch/metabolismo , Proteína Jagged-1/genéticaRESUMEN
K-Ras is the most frequently mutated Ras variant in pancreatic, colon and non-small cell lung adenocarcinoma. Activating mutations in K-Ras result in increased amounts of active Ras-GTP and subsequently a hyperactivation of effector proteins and downstream signaling pathways. Here, we demonstrate that oncogenic K-Ras(V12) regulates tumor cell migration by activating the phosphatidylinositol 3-kinases (PI3-K)/Akt pathway and induces the expression of E-cadherin and neural cell adhesion molecule (NCAM) by upregulation of Akt3. In vitro interaction and co-precipitation assays identified PI3-Kα as a bona fide effector of active K-Ras4B but not of H-Ras or N-Ras, resulting in enhanced Akt phosphorylation. Moreover, K-Ras(V12)-induced PI3-K/Akt activation enhanced migration in all analyzed cell lines. Interestingly, Western blot analyses with Akt isoform-specific antibodies as well as qPCR studies revealed, that the amount and the activity of Akt3 was markedly increased whereas the amount of Akt1 and Akt2 was downregulated in EGFP-K-Ras(V12)-expressing cell clones. To investigate the functional role of each Akt isoform and a possible crosstalk of the isoforms in more detail, each isoform was stably depleted in PANC-1 pancreatic and H23 lung carcinoma cells. Akt3, the least expressed Akt isoform in most cell lines, is especially upregulated and active in Akt2-depleted cells. Since expression of EGFP-K-Ras(V12) reduced E-cadherin-mediated cell-cell adhesion by induction of polysialylated NCAM, Akt3 was analyzed as regulator of E-cadherin and NCAM. Western blot analyses revealed pronounced reduction of E-cadherin and NCAM in the Akt3-kd cells, whereas Akt1 and Akt2 depletion upregulated E-cadherin, especially in H23 lung carcinoma cells. In summary, we identified oncogenic K-Ras4B as a key regulator of PI3-Kα-Akt signaling and Akt3 as a crucial regulator of K-Ras4B-induced modulation of E-cadherin and NCAM expression and localization.
Asunto(s)
Adenocarcinoma , Neoplasias Pulmonares , Neoplasias Pancreáticas , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Moléculas de Adhesión de Célula Nerviosa , Cadherinas , Neoplasias Pulmonares/genética , Isoformas de Proteínas , Fosfatidilinositol 3-Quinasas/metabolismo , Pulmón/metabolismo , Neoplasias Pancreáticas/patologíaRESUMEN
BACKGROUND: Alveolar hypercoagulation and fibrinolytic inhibition are mainly responsible for massive alveolar fibrin deposition, which are closely related with refractory hypoxemia in acute respiratory distress syndrome (ARDS). Our previous study testified runt-related transcription factor (RUNX1) participated in the regulation of this pathophysiology in this syndrome, but the mechanism is unknown. We speculate that screening the downstream genes associated with RUNX1 will presumably help uncover the mechanism of RUNX1. METHODS: Genes associated with RUNX1 were screened by CHIP-seq, among which the target gene was verified by Dual Luciferase experiment. Then the efficacy of the target gene on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS was explored in vivo as well as in vitro. Finally, whether the regulatory effects of RUNX1 on alveolar hypercoagulation and fibrinolytic in ARDS would be related with the screened target gene was also sufficiently explored. RESULTS: Among these screened genes, AKT3 was verified to be the direct target gene of RUNX1. Results showed that AKT3 was highly expressed either in lung tissues of LPS-induced rat ARDS or in LPS-treated alveolar epithelia cell type II (AECII). Tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) were increasingly expressed both in lung tissues of ARDS and in LPS-induced AECII, which were all significantly attenuated by down-regulation of AKT3. Inhibition of AKT3 gene obviously ameliorated the LPS-induced lung injury as well as the collagen I expression in ARDS. RUNX1 overexpression not only promoted the expressions of TF, PAI-1, but also boosted AKT3 expression in vitro. More importantly, the efficacy of RUNX1 on TF, PAI-1 were all effectively reversed by down-regulation of AKT3 gene. CONCLUSION: AKT3 is an important target gene of RUNX1, through which RUNX1 exerted its regulatory role on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS. RUNX1/ATK3 signaling axis is expected to be a new target for the exploration of ARDS genesis and treatment.
Asunto(s)
Lipopolisacáridos , Síndrome de Dificultad Respiratoria , Animales , Ratas , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Regulación hacia Abajo , Lipopolisacáridos/toxicidad , Inhibidor 1 de Activador Plasminogénico/genética , Síndrome de Dificultad Respiratoria/inducido químicamente , Síndrome de Dificultad Respiratoria/genéticaRESUMEN
ObjectiveTo observe the effects of Fuzitang (FZT) on the proliferation of MH7A cells, the human rheumatoid arthritis synovial fibroblasts, and the expression of miR-155 and explore its anti-rheumatoid arthritis mechanism. MethodMH7A cells were cultured in vitro and divided into a blank group, high- (25 g·L-1) and low-dose (12.5 g·L-1) FZT groups, and a positive drug group (hydroxychloroquine, 0.006 25 g·L-1). The cell proliferation was detected by cell counting kit-8(CCK-8) method, and the change in the MH7A cell cycle was detected by flow cytometry. The mRNA expression of miR-155 and its downstream genes, including SH2 domain-containing inositol 5-phosphatase-1(SHIP-1), protein kinase B 3(Akt3), and mammalian target of rapamycin(mTOR), was detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR), and the protein expression of phosphatidylinositol 3-kinase (PI3K), Akt3, and mTOR was detected by Western blot. ResultFZT in vitro in a concentration of 6.25 g·L-1 above could inhibit the proliferation of MH7A cells in the significant dose- and time-effect manner. Compared with the blank group, the FZT groups showed increased proportions of cells in the G2/M phase (P<0.05), and the high-dose FZT group showed a decreased proportion of cells in the G0/G1 phase (P<0.05). The arresting effect of FZT on the cell cycle was in a significant dose-effect manner. Compared with the blank group, the FZT groups showed down-regulated miR-155 and mTOR mRNA expression (P<0.05), and the high-dose FZT group showed up-regulated SHIP1 mRNA expression and down-regulated Akt3 mRNA expression (P<0.05). Compared with the blank group, the FZT groups showed reduced protein expression of PI3K, Akt3, and mTOR (P<0.05). ConclusionFZT can significantly inhibit the proliferation of MH7A cells, and the mechanism is related to the promotion of the expression of SHIP-1 and down-regulation of the gene expression of the PI3K/Akt3/mTOR signaling pathway by down-regulating the expression of miR-155.
RESUMEN
This study examined the expression and potential mechanism of microRNA (miRNA)-424-5p in nasopharyngeal carcinoma (NPC). NPC tissues were collected from 40 patients who were enrolled in the study, and skin samples were collected from 26 healthy subjects during plastic surgery as controls. We performed various in vitro assays using miR-424-5p to examine its function in primary NPC-1 cells. Bioinformatics was employed to analyze potential target genes and signaling pathways of miR-424-5p. We found that miR-424-5p expression in NPC tissues is downregulated and negatively correlated with lymph node metastasis and clinical staging. Expression of miR-424-5p in NPC cells was also downregulated, and transfection with miR-424-5p mimics inhibited proliferation, migration, and invasion of NPC-1 cells. Bioinformatics identified the AKT3 gene as a potential target of miR-424-5p and dual luciferase assays confirmed this finding. Upregulation of AKT3 expression rescued the inhibitory effect of miR-424-5p on the proliferation, migration, and invasion. Our results suggest that miR-424-5p inhibited the proliferation, migration, and invasion of NPC cells by decreasing AKT3 expression.
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Anciano , Regulación Neoplásica de la Expresión Génica , Neoplasias Nasofaríngeas/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Transducción de Señal , Movimiento Celular , Neoplasias Nasofaríngeas/genética , Western Blotting , MicroARNs/genética , Línea Celular Tumoral , Proliferación Celular , Proteínas Proto-Oncogénicas c-akt/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Carcinoma Nasofaríngeo/genética , Invasividad NeoplásicaRESUMEN
Objective To investigate the prognostic value of AKT3 expression in gastric cancer. Methods AKT3 expression data in The Cancer Genome Atlas(TCGA)datasets and its clinical information were downloaded. Statistically assessed was performed for relationship with clinicopatho?logical factors and prognosis. Gene set enrichment analysis(GSEA)was used to predict the gene sets modulated by AKT3. Results The expres?sion of AKT3 was associated with T stage(P=0.001),TNM stage(P=0.049)and differentiation(P<0.001).High level of AKT3 expression indi?cates poor prognosis(P=0.001). AKT3 could regulate gene sets involving cell adhesion molecule,cytoskeleton regulation,focal adhesion and TGF?βsignaling pathway. Conclusion AKT3 could be used as a potential prognostic marker and a therapeutic target in gastric cancer.
RESUMEN
ObjectiveTo investigate the effects of Akt3 gene knockout on neuropathic pain behaviors induced by chronic constriction injury of sciatic nerve (CCI).MethodsExperiment was divided into two groups:Akt3 knockout group (Akt3-/-,n =12),wild type group (WT,n =12 ).Randomly numbered,the right sciatic nerve of mice were received the operation of chronic constriction injury.Paw withdrawal mechanical threshold (PWMT)and paw withdrawal thermal latency (PWTL) were tested on day 1 before operation and day 1,3,5,7,10,14,17,21 afterCCI.ResultsThe basic values of PWMT(right:(1.09±0.20)g,(1.17±0.22)g;left:(1.17±0.15)g,(1.22±0.23)g,P>0.05) andPWTL(right:(6.18±1.11)s,(6.20±1.25)s;left:(5.82±0.91)s,(5.92± 1.71 ) s,P > 0.05 ) had no statistically significant differences between two groups.On day 1 after operation,compared with basic values,the PWMT and PWTL of the right paw in both Akt3-/- group and WT group decreased significantly (P < 0.05 ),and at least lasted up to day 21.The PWMT( 3d:(0.42 ± 0.22 ) g,(0.72 ± 0.36) g ; 17d:(0.29 ±0.15)g,(0.49 ±0.19) g;21d:(0.27 ±0.18)g,(0.56 ±0.15)g,P<0.05) and PWTL(5d:(2.43 ±0.68)s,(3.13±0.52)s;17d:(2.43±1.26)s,(3.84±1.29)s ;21d:(2.14±1.23)s,(4.07±1.26)s,P<0.05 ) of the right paw in Akt3-/- group was significantly lower than those in WT group.The PWMT and PWTL of the left paw in Akt3-/- group and WT group had no obvious differences (P > 0.05 ). However.compared to left paw,the PWMT and PWTL of the right paw of the two groups were obviously lower (P < 0.05 ).ConclusionThe neuropathic pain induced by CCI increased in Akt3 gene knockout mice.
RESUMEN
Objective To study the expression of protein kinase B (Akt/PKB) isoforms in the gastrocnemius muscle of rats at different ages. Methods The expression levels of mRNA and protein of three Akt isoforms in the gastrocnemius muscle of 30-month-old and 6-month-old rats were detected respectively by RT-PCR and Western blotting. Data were analyzed statistically. Results (1) The levels of Akt1 and Akt2 mRNA in the 30-month-old rats was significantly higher than those in the 6-month-old rats (t=7.124, P=0.000; t=2.598, P=0.021), however, no significant difference was found in the level of Akt3 mRNA between the two groups (t=0.460, P=0.653) . (2) The level of Akt-Thr308 phosphorylation in the 30-month-old rats was significantly lower than that in the 6-month-old rats (t=-9.861, P=0.000), while the level of Akt2 protein in the 30-month group was significantly higher than that in the younger rats (t=7.522, P=0.000). No significant differences were detected in the levels of Akt1 and Akt3 proteins between the two groups (t=0.469, P=0.646; t=0.058, P=0.955). Conclusion The expression levels of three Akt isoforms in the gastrocnemius muscle of rats change with age, suggesting that the three isoforms of Akt have different functions in the gastrocnemius muscle metabolism of rats at different ages.