RESUMEN
Emerging evidence indicates that circRNAs are broadly expressed in osteosarcoma (OS) cells and play a crucial role in OS progression. Recently, cancer-specific circRNA circPRKAR1B has been identified by high-throughput sequencing and is recorded in publicly available databases. Nevertheless, the detailed functions and underlying mechanisms of circPRKAR1B in OS remains poorly understood. By functional experiments, we found that circPRKAR1B enhanced OS cell proliferation, migration, and promotes OS epithelial-mesenchymal transition (EMT). Mechanistic investigations suggested that circPRKAR1B promotes OS progression through sponging miR-361-3p to modulate the expression of FZD4. Subsequently, we identified that EIF4A3 promoted cirPRKAR1B formation through binding to the downstream target of circPRKAR1B on PRKAR1B mRNA. Further rescue study revealed that overexpression of the Wnt signalling could impair the onco-suppressor activities of the silencing of circPRKAR1B. Interestingly, further experiments indicated that circPRKAR1B is involved in the sensitivity of chemoresistance in OS. On the whole, our results demonstrated that circPRKAR1B exerted oncogenic roles in OS and suggested the circPRKAR1B/miR-361-3p/FZD4 axis plays an important role in OS progression and might be a potential therapeutic target.
Asunto(s)
Neoplasias Óseas/metabolismo , Carcinogénesis/metabolismo , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , ARN Helicasas DEAD-box/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Receptores Frizzled/metabolismo , MicroARNs/metabolismo , Osteosarcoma/metabolismo , ARN Circular/metabolismo , Transducción de Señal/genética , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Carcinogénesis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Transición Epitelial-Mesenquimal/genética , Silenciador del Gen , Humanos , Masculino , Ratones , Ratones Desnudos , MicroARNs/genética , Osteosarcoma/genética , Osteosarcoma/patología , ARN Circular/genética , Transfección , Carga Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.
Asunto(s)
Corteza Suprarrenal/enzimología , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Transducción de Señal , Corteza Suprarrenal/metabolismo , Animales , Diferenciación Celular , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 7/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Esteroides/metabolismo , Zona Fascicular/citología , Zona Fascicular/enzimología , Zona Fascicular/metabolismo , Zona Glomerular/citología , Zona Glomerular/enzimología , Zona Glomerular/metabolismoRESUMEN
The cyclic AMP/protein kinase A signaling cascade is one of the main pathways involved in the pathogenesis of adrenocortical tumors. The PKA R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. Inactivating mutations of PRKAR1A are associated with Carney complex and a subset of sporadic tumors and the abundance of R2B protein is low in a subset of secreting adrenocortical adenomas. We previously showed that PRKAR1A and PRKAR2B inactivation have anti-apoptotic effects on the adrenocortical carcinoma cell line H295R. The aim of this study was to compare the effects of PRKAR1A and PRKAR2B depletion on cell proliferation, apoptosis, cell signaling pathways, and cell cycle regulation. We found that PRKAR2B depletion is compensated by an upregulation of R1A protein, whereas PRKAR1A depletion has no effect on the production of R2B. The depletion of either PRKAR1A or PRKAR2B promotes the expression of Bcl-xL and resistance to apoptosis; and is associated with a high percentage of cells in S and G2 phase, activates PKA and MEK/ERK pathways, and impairs the expression of IkB leading to activate the NF-κB pathway. However, we observed differences in the regulation of cyclins. The depletion of PRKAR1A leads to the accumulation of cyclin D1 and p27kip, whereas the depletion of PRKAR2B promotes the accumulation of cyclin A, B, cdk1, cdc2, and p21Cip. In conclusion, although the depletion of PRKAR1A and PRKAR2B in adrenocortical cells has similar effects on cell proliferation and apoptosis; loss of these PKA subunits differentially affects cyclin expression.
Asunto(s)
Glándulas Suprarrenales/citología , Glándulas Suprarrenales/enzimología , Puntos de Control del Ciclo Celular , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Subunidad RIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Transducción de Señal , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , FN-kappa B/metabolismo , Subunidades de Proteína/metabolismoRESUMEN
Prkar1b gene encodes regulatory type I, beta subunit (RIß) of cAMP dependent protein kinase A in mouse. Among the various isoforms of regulatory and catalytic subunits that comprise mammalian PKA, RIß subunit is considered to be one of the important subunits for neuronal functions. This is involved in multiple forms of synaptic plasticity, and influences memory and learning by maintaining hippocampal long-term potentiation (LTP). Deficient expression of this gene has been implicated in autoimmune disease systemic lupus erythematosus (SLE). We have identified two novel non-coding exons of the Prkar1b gene (designated as exon 1A and exon 1B), which are spliced to the canonical exon 2 and constitute the 5' untranslated region giving rise to three alternative transcript isoforms. We have also confirmed the expression of the previously known first exon (designated as exon 1C) with known transcript published earlier. The transcripts containing exons 1A, 1B and 1C are differentially regulated during the development and tissue types. In silico study of more than 20 kb nucleotide sequence upstream of known translational initiation codon revealed three distinct promoter regions named as PA, PB, and PC upstream of the exon 1A, exon 1B and exon 1C respectively. PB is non-CpG related promoter but PA and PC are CpG related promoters, however all three promoters are TATA less. Further analysis showed that these promoters possess potential signature sequences for common as well as different transcription factors suggesting complex regulation of Prkar1b gene.