Project description:To determine whether DGCR8 is required for maturation of all miRNAs, we performed miRNA microarray analysis. Using RNA from wild-type ES cells as our reference sample, we observed a global loss of miRNAs in DGCR8 knockout cells, but normal levels of expression in DGCR8 heterozygous cells. The similarity in expression levels between wild-type and heterozygous cells suggests that DGCR8 is not limiting in the maintenance of steady-state levels of miRNAs in ES cells. Of the eighty-nine miRNA array probes that showed significant signals with wild-type RNA, eighty-two were drastically reduced in the DGCR8 knockout cells. The remaining seven were not significantly altered in the DGCR8 knockout cells, but at least four of these seven miRNAs appear to be due to unavoidable contamination with RNA from the mouse embryonic fibroblast (MEF) feeder cells used for ES cell culture prior to the isolation of RNA. These miRNAs were highly expressed in the MEFs and decreased when the ES cells were temporarily passaged on gelatin-coated plates without feeders. The remaining three showed similar levels of expression in the heterozygous, knockout and MEF feeder cells. Therefore, these signals may be small RNAs that are not processed with the help of the microprocessor complex. Alternatively, these signals may result from unavoidable degradation products within the purified small RNA population. In either case, our results show that DGCR8 is broadly required for miRNA processing with little evidence for redundancy or bypass mechanisms. Keywords: cell type comparison, genetic modification MicroRNAs extracted from wild-type, DGCR8 heterozygous knockout and DGCR8 homozygous knockout were analyzed by microarray. In each array, wild-type samples serve as reference. Ratios were normalized based on positive control RNAs on the array. To determine if some of signals appeared in DGCR8 knockout ES cells are due to mice embryonic fibroblast (MEF) feeder cell contamination (this is unavoidable because these ES cells were grown on MEFs and passaged off MEF before RNA extraction). Two independent DGCR8 knockout ES cell lines were analyzed. For each cell line, array hybridization was done in duplicates. For DGCR8 heterozygous knockout ES cells, two independent batches of RNA samples were prepared and analyzed.

Project description:The microprocessor mutation DGCR8 E518K represents one of the most prevalent and stereotypical mutations in Wilms tumor (WT). It affects the dsRNA-binding domain (dsRBD) of DGCR8, which leads to a presumably disruptive charge reversal. The E518K mutation was expressed homozygously in all cases, indicative for the mutation to act in a recessive manner. Notably, this alteration has been almost exclusively reported in WT, suggesting a specific role in WT formation. To functionally characterize the DGCR8 E518K mutation in vitro, we employed DGCR8 knockout mouse embryonic stem cell (mESC) lines with inducible overexpression of wild-type (wt) or E518K-mutant DGCR8. Absence of endogenous DGCR8 mirrors the homozygous expression of mutant DGCR8 as observed in WT. Knockout of DGCR8 is known to result in a severe miRNA processing defect with a significant downregulation of all canonical miRNAs. Wild-type DGCR8 rescued this processing defect. DGCR8-E518K, in turn, was unable to fully restore miRNA expression and showed overall reduced miRNA expression levels, confirming our previous findings in bulk tumor tissue. Differentially expressed miRNAs comprised members of the embryonic stem cell specific cell cycle-regulating (ESCC) miRNAs and the let-7 miRNA family, whose antagonism is known to play a pivotal role in the regulation of critical gene expression programs in ES cells. Along with altered miRNA expression, DGCR8-E518K exhibited changes in target gene expression affecting various biological pathways. The known proliferation deficiency and cell cycle defect of DGCR8 KO mESCs could be rescued by DGCR8-E518K, albeit not to the full extent as by wild-type DGCR8. Likewise, DGCR8-E518K failed to fully block epithelial-to-mesenchymal transition (EMT). Upon embryoid body formation, however, both DGCR8-wt and -E518K were able to restore the differentiation capacity and to silence self-renewal in the knockout. Despite impaired miRNA processing and altered target gene regulation, DGCR8-E518K mESCs were still able to restore many biological processes in a DGCR8 knockout background. These findings suggest that partial reduction in activity or altered specificity might be critical in Wilms tumorigenesis.

Project description:Background: The hES-T3 cell line with normal female karyotype was used to differentiate into autogeneic fibroblast-like cells (T3HDF) as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF) for 14 passages. A feeder-free culture on Matrigel in hES medium conditioned by these autogeneic feeder cells (T3HDF) was established to maintain the undifferentiated growth of hES-T3 cells (T3/CMHDF) for 8 passages. Results: The gene expression profiles of mRNAs, microRNAs and proteins between the undifferentiated T3/HDF and T3/CMHDF cells were shown to be very similar, and their expression profiles were also found to be similar to those of T3/MEF and T3/CMMEF cells grown on MEF feeder and feeder-free Matrigel in MEF-conditioned medium, respectively. The abundantly expressed genes of T3/HDF, T3/CMHDF, T3/MEF and T3/CMMEF cells were found to play prominent roles in signaling pathways and GO process networks. The miR-302/367 cluster and miR-371/372/373 cluster were extremely abundantly expressed in undifferentiated T3/HDF and T3/CMHDF as well as T3/MEF andT3/CMMEF cells. The undifferentiated state of T3/HDF and T3/CMHDF cells was evidenced by the very high expression levels of ¡§stemness¡¨ genes and low expression levels of differentiation markers of ectoderm, mesoderm and endoderm in addition to the strong staining of OCT4 and NANOG. Conclusion: The T3HDF feeder and T3HDF-conditioned medium were able to support the undifferentiated growth of hES cells, and they would be useful for drug development and toxicity testing in addition to the reduced risks of xenogeneic pathogens when used for medical applications such as cell therapies. Keywords: genetic modification In this investigation, both miRNA and mRNA expression profiles from human embryonic stem cells (hES-T3) grown on MEF feeder (T3_MEF), feeder-free Matrigel in MEF-conditioned medium (T3_CMMEF), T3HDF (hES-T3 differentiated fibroblast-like cells) feeder and feeder-free Matrigel in T3HDF-conditioned medium were quantitatively determined. Several target genes of T3BA and T3CM cells-specific miRNAs were identified. ***This submission represents the mRNA expression component of the study only***

Dataset Information

DGCR8 is required for microRNA maturation

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure