RESUMEN
Nephrons are the functional units of the kidney. During kidney development, cells from the cap mesenchyme-a transient kidney-specific progenitor state-undergo a mesenchymal to epithelial transition (MET) and subsequently differentiate into the various epithelial cell types that create the tubular structures of the nephron. Faults in this transition can lead to a pediatric malignancy of the kidney called Wilms' tumor that mimics normal kidney development. While human kidney development has been characterized at the gene expression level, a comprehensive characterization of alternative splicing is lacking. Therefore, in this study, we performed RNA sequencing on cell populations representing early, intermediate, and late developmental stages of the human fetal kidney, as well as three blastemal-predominant Wilms' tumor patient-derived xenografts. Using this newly generated RNAseq data, we identified a set of transcripts that are alternatively spliced between the different developmental stages. Moreover, we found that cells from the earliest developmental stage have a mesenchymal splice-isoform profile that is similar to that of blastemal-predominant Wilms' tumor xenografts. RNA binding motif enrichment analysis suggests that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate alternative mRNA splicing during human kidney development. These findings illuminate new molecular mechanisms involved in human kidney development and pediatric kidney cancer.
Asunto(s)
Neoplasias Renales , Tumor de Wilms , Humanos , Niño , Empalme Alternativo , ARN Mensajero/genética , Tumor de Wilms/genética , Tumor de Wilms/patología , Neoplasias Renales/patología , Riñón/patología , Células Cultivadas , Factores de Empalme de ARN/genética , Proteínas Represoras/genéticaRESUMEN
When assembling a nephron during development a multipotent stem cell pool becomes restricted as differentiation ensues. A faulty differentiation arrest in this process leads to transformation and initiation of a Wilms' tumor. Mapping these transitions with respective surface markers affords accessibility to specific cell subpopulations. NCAM1 and CD133 have been previously suggested to mark human renal progenitor populations. Herein, using cell sorting, RNA sequencing, in vitro studies with serum-free media and in vivo xenotransplantation we demonstrate a sequential map that links human kidney development and tumorigenesis; In nephrogenesis, NCAM1(+)CD133(-) marks SIX2(+) multipotent renal stem cells transiting to NCAM1(+)CD133(+) differentiating segment-specific SIX2(-) epithelial progenitors and NCAM1(-)CD133(+) differentiated nephron cells. In tumorigenesis, NCAM1(+)CD133(-) marks SIX2(+) blastema that includes the ALDH1(+) WT cancer stem/initiating cells, while NCAM1(+)CD133(+) and NCAM1(-)CD133(+) specifying early and late epithelial differentiation, are severely restricted in tumor initiation capacity and tumor self-renewal. Thus, negative selection for CD133 is required for defining NCAM1(+) nephron stem cells in normal and malignant nephrogenesis.
Asunto(s)
Biomarcadores/metabolismo , Carcinogénesis/genética , Riñón/metabolismo , Células Madre Neoplásicas/metabolismo , Nefronas/metabolismo , Células Madre/metabolismo , Antígeno AC133/genética , Antígeno AC133/metabolismo , Animales , Antígeno CD56/genética , Antígeno CD56/metabolismo , Carcinogénesis/metabolismo , Células Cultivadas , Niño , Preescolar , Femenino , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Lactante , Riñón/embriología , Masculino , Ratones Endogámicos NOD , Células Madre Neoplásicas/patología , Nefronas/citología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Organogénesis/genética , Estudios Prospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
MicroRNAs (miRNAs) have emerged as potential cancer therapeutics; however, their clinical use is hindered by lack of effective delivery mechanisms to tumor sites. Mesenchymal stem cells (MSCs) have been shown to migrate to experimental glioma and to exert anti-tumor effects by delivering cytotoxic compounds. Here, we examined the ability of MSCs derived from bone marrow, adipose tissue, placenta and umbilical cord to deliver synthetic miRNA mimics to glioma cells and glioma stem cells (GSCs). We examined the delivery of miR-124 and miR-145 mimics as glioma cells and GSCs express very low levels of these miRNAs. Using fluorescently labeled miRNA mimics and in situ hybridization, we demonstrated that all the MSCs examined delivered miR-124 and miR-145 mimics to co-cultured glioma cells and GSCs via gap junction- dependent and independent processes. The delivered miR-124 and miR-145 mimics significantly decreased the luciferase activity of their respected reporter target genes, SCP-1 and Sox2, and decreased the migration of glioma cells and the self-renewal of GSCs. Moreover, MSCs delivered Cy3-miR-124 mimic to glioma xenografts when administered intracranially. These results suggest that MSCs can deliver synthetic exogenous miRNA mimics to glioma cells and GSCs and may provide an efficient route of therapeutic miRNA delivery in vivo.