RESUMO
BACKGROUND: Recent in vitro studies using RB1+/- fibroblasts and MSCs have shown molecular and functional disruptions without the need for biallelic loss of RB1. However, this was not reflected in the recent in vitro studies employing RB1+/- retinal organoids. To gain further insights into the molecular disruptions in the RB1+/- retinal organoids, we performed a high throughput RNA sequencing analysis. METHODS AND RESULTS: iPSCs were generated from RB1+/+ and RB1+/- OAMSCs derived from retinoblastoma patients. RB1+/+ and RB1+/- iPSCs were subjected to a step-wise retinal differentiation protocol. Retinal differentiation was evaluated by Real-time PCR and flow cytometry analysis of the retinal markers. To gain further insights into the molecular differences in RB1+/- retinal organoids, a high throughput RNA sequencing followed by differential gene expression analysis and gene set enrichment analysis (GSEA) was performed. The analysis revealed a shift from the regular metabolic process of glycolysis to oxidative phosphorylation in the RB1+/- retinal organoids. To investigate further, we performed assays to determine the levels of pyruvate, lactate and ATP in the retinal organoids. The results revealed significant increase in ATP and pyruvate levels in RB1+/- retinal organoids of day 120 compared to that of the RB1+/+. The results thus revealed enhanced ATP production in the RB1+/- retinal organoids. CONCLUSION: The study provides novel insights into the metabolic phenotype of heterozygous RB1 mutant suggesting dysregulation of energy metabolism and glycolytic pathways to be first step even before the changes in cellular proliferation or other phenotypic consequences ensue.
Assuntos
Trifosfato de Adenosina , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Organoides , Retina , Proteínas de Ligação a Retinoblastoma , Retinoblastoma , Ubiquitina-Proteína Ligases , Humanos , Trifosfato de Adenosina/metabolismo , Diferenciação Celular/genética , Glicólise/genética , Heterozigoto , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Mutação/genética , Organoides/metabolismo , Retina/metabolismo , Retina/citologia , Retinoblastoma/genética , Retinoblastoma/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Retinoblastoma (RB1) protein is a multifunctional protein that plays an important role in cell cycle regulation and cell differentiation, including adipogenesis. A detailed literature search to understand the role of RB1 in adipogenesis revealed that the nature of the RB1 inactivation (in vivo/in vitro) led to differences in adipogenesis. The majority of these studies were animal-based, and the only study in humans employed an in vitro mode of RB1 inactivation. To overcome these differences and lack of human studies, we sought to explore the role of RB1 in adipogenesis using orbital adipose mesenchymal stem cells (OAMSCs) from patients with retinoblastoma that innately carry a heterozygous RB1 mutation. We hypothesized that these patient-derived RB1 mutant OAMSCs can model in vivo RB1 inactivation in humans. Our study revealed increased adipogenesis with a bias toward brown adipogenesis in the RB1 mutant in addition to an increased number of adipocytes in the mitotic phase.
Assuntos
Adipogenia , Proteínas de Ligação a Retinoblastoma , Retinoblastoma , Células-Tronco , Ubiquitina-Proteína Ligases , Adipócitos , Adipogenia/genética , Diferenciação Celular , Humanos , Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Osteosarcoma (OS) is a bone malignancy affecting children and adolescents. Retinoblastoma (RB) patients with germline RB1 mutations are susceptible to osteosarcoma in the second decade of their life. Several studies, particularly in mice, have revealed a role for RB1 in osteogenesis. Since, there is species specific difference attributed in retinoblastoma tumorigenesis between mice and human, we assumed, it is worthwhile exploring the role of RB1 in osteogenesis and thus onset of osteosarcoma. In this study, we analyzed the temporal gene expression of the osteogenic markers, tumor suppressor genes and hormone receptors associated with growth spurt during in vitro osteogenesis of mesenchymal stem cells derived from orbital adipose tissue of germline RB patients and compared it with those with wild type RB1 gene. Mesenchymal stem cells with the heterozygous RB1 mutation showed reduced expression of RB1 and other tumor suppressor genes and showed deregulation of osteogenic markers which could be an initial step for the onset of osteosarcoma.
Assuntos
Neoplasias Ósseas/genética , Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , Mutação , Osteossarcoma/genética , Neoplasias da Retina/genética , Proteínas de Ligação a Retinoblastoma/genética , Ubiquitina-Proteína Ligases/genética , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Adulto , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Diferenciação Celular , Condrócitos/metabolismo , Condrócitos/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Perfilação da Expressão Gênica , Heterozigoto , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Órbita/metabolismo , Órbita/patologia , Osteogênese/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Cultura Primária de Células , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Neoplasias da Retina/metabolismo , Neoplasias da Retina/patologia , Proteínas de Ligação a Retinoblastoma/metabolismo , Transdução de Sinais , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Retinoblastoma (RB) is a pediatric intraocular tumor caused by mutations in retinoblastoma (RB1) gene. We have generated induced pluripotent stem cell line VRFi001-A from a bilateral retinoblastoma patient with heterozygous RB1 gene deletion. The iPSC line VRFi001-A retained the mutation and expressed pluripotency markers, had a normal karyotype and was capable of trilineage differentiation.
