Expression profiling basal and luminal prostate epithelial cells
Ontology highlight
ABSTRACT: Immuno-stained (keratin 14+ basal marker) frozen prostate sections were subjected to laser-guided microdissection to isolate basal and luminal epithelial prostate cells for expression profiling. RNA was amplified using the AmpTec TRinucleotide kit (AmpTec GmBH). Expression profiling performed using the Invitrogen post-labelling kit and the CRUK whole genome array (WGA) gene set. Keywords: repeat sample
Project description:Epithelial cell cultures derived from benign and HRPC tissue biopsies were expanded in culture for 2-3 weeks. CD133+ and CD133- cells were isolated using the miltenyi magnetic bead system after adherence to collagen. CD133+ and CD133- RNA was isolated and amplified using the RiboAmp HS kit and expression profiling performed using the CRUK WGA gene set. Keywords: Tumour vs normal comparison
Project description:ETS transcription factor ERG is aberrantly activated in ~50% of human prostate cancers. We found that ERG shows a preference of basal over luminal context to drive prostate cancer. However, the molecular basis of the cell context dependency remains elusive. Here we use ERG+/+;Pten-/- (EP) mouse prostate organoids with dual basal/luminal lineage reporters to profile ERG-driven gene expression programs from sorted basal vs luminal cells.
Project description:Custom oligonucleotide array CGH. We designed two fine-tiling oligonucleotide microarrays to cover the specific amplicons observed at chromosome 2p22.1 and 10q11.21 This was undertaken using the Agilent custom array design tool e-Array (Agilent, Santa Clara, CA, USA; https://earray.chem.agilent.com/), and comprised 700 probes covering 43.56Ð43.70 Mb on chromosome 10 and 5000 probes covering 39Ð40 Mb on chromosome 10 with a median probe interval of 200 bp on 2x105K microarray. Due to limited amount of material, DNA was whole genome amplified (WGA) using the GenomePlex Complete Whole Genome Amplification Kit (Sigma, Gillingham, UK) starting with 10 ng of sample and control DNA, and following the manufacturers protocol. WGA DNA was labelled using the Agilent Genomic DNA ULS labelling kit, hybridised as per manufacturers instructions, and scanned on the Agilent 2505B Microarray Scanner System.
Project description:Recent studies demonstrate both basal and luminal cells of the prostate gland can initiate tumorigenesis upon oncogenic transformation. However, it remains unclear how molecular mechanisms operating within each cell lineage contribute to the initiation and progression of the prostate cancer. Here we investigate functions of individual miRNAs using genetically engineered mouse models. By both quantitative miR-Seq and in situ hybridization, we identify microRNA-205 (miR-205) as the most highly expressed miRNA and specific to the basal cells in the prostate. MicroRNA-205 expression is further elevated in the basal cells in the well-established Pten null tumorigenic mouse model. To investigate the role of miR-205 in Pten-deletion mediated tumorigenesis, we generated a Pten/miR-205 double knockout mouse model. Concurrent deletion of both miR-205 and Pten significantly compromised tumor progression in both basal and luminal compartments. We observed significantly reduced tumor size and compromised proliferation in both basal and luminal cells. We have previously demonstrated a critical requirement of miR-205 for maintaining the PI(3)K signaling and pAkt levels in skin stem cells. Consistent with this role, we observed strong reduction of pAkt and significantly increased cellular senescence in the basal cells of the dKO, compared to the Pten KO alone. These results suggest that miR-205 is cell-autonomously required for the tumorigenesis of the basal cells. Taken together, we have identified miR-205 as an important regulator in prostate cancer. Our study also reveals an essential and unexpected role of the basal cells for promoting prostate tumorigenesis.
Project description:To investigate the molecular reprogramming of epithelial cells of the prostate during basal to luminal differentiation in vivo, we took advantage of the spatial restriction of multipotent basal cells at the distal region (tip - 100μm) of the developing prostate at postnatal day (P)10 (Tika et al., Development, 2019). The distal tips (100μm) of P10-P13 prostate glands were manually dissected from the main ducts under the stereoscope in order to enrich for multipotent basal cells and basal-derived luminal cells. Cell populations were isolated for RNA sequencing based on EpCAM and Cd49f expression via FACS.
Project description:6 benign TURP samples taken directly from patients were subjected to collagen digestion overnight. Epithelial cells isolated were adhered to collagen for 15 minutes. Adherent cells were passed through a magnetic bead column and cells positive for CD133 antibody were double selected. RNA was extracted from CD133+ and CD133- cells from each patient and amplified using the RiboAmp HS RNA amplification system. CD133+ (Cy5) and CD133- (Cy5) cDNA was hybridised to 30K cDNA array (CRUK facility) using the Invitrogen post labelling system. In each case that RNA was co-hybridised to a human pooled universal reference cDNA (Cy3). Keywords: repeat sample
Project description:We set out to determine if prostate epithelial cell-types exhibited age-related differences in their gene expression profiles. We used fluorescence activated cell sorting to isolate basal and luminal cells from dissociated preparations of prostate tissue taken from young adult (3 months) and old (24 months of age) mice. RNA was extracted from sorted cells to evaluate differences in gene expression. We found significant enrichment for progenitor genes in old luminal cells including an immune/inflammatory profile that overlapped with a previously identified CD38-lo human prostate luminal progenitor signature. This study demonstrates a luminal progenitor signature in old mouse prostate.
Project description:Mammary epithelium is hierarchically organized, with multipotent basal mammary stem cells (MaSCs) producing both luminal and basal cells during development or upon transplantation. Recent studies suggested that most breast cancers, including Basal-Like breast cancer (BLBC), might originate from luminal cells, and oncogenic events, such as ectopic expression of PIK3CA(H1047R), could induce multipotency in committed luminal cells. p53 is the most commonly mutated gene in human breast cancer; in particular, its inactivating mutations are found in most BLBCs, raising a question as to whether p53-loss plays a key role in acquisition of multipotent MaSC-like properties by luminal cells. By in situ lineage-tracing, we found that induced loss of p53 in Keratin 8 (K8)+ luminal cells led to their clonal expansion, due to increased cell cycle activity and attenuated apoptosis control, but did not directly affect their luminal identity. All induced mice eventually developed either Claudin-Low mammary tumors with 9qA1 (Yap1) amplification or Basal-Like tumors with 6qA1-A2 (Met) amplification. These data suggest that although p53 does not directly control the luminal fate, its loss facilitates acquisition of MaSC-like properties by luminal cells and predisposes them to development of mammary tumors with loss of luminal identity.
Project description:Performed scRNA-seq of an autochthonous mouse model at an early stage of disease initiation. Despite broad expression of ERG in all prostate epithelial cells, proliferation was enriched in a small, stem-like population of cells with mixed-luminal basal identity (intermediate cells).
Project description:High quality RNA was extracted from the whole seedlings (Combined root and leaf samples) using TRI Reagent (Ambion, Inc. USA) and pooled from 12 independent stressed and non-stressed plant samples separately, and treated with DNase-I (QIAGEN GmbH, Germany). Subsequently, RNA cleanup was carried out using RNeasy Plant Mini Kit (QIAGEN GmbH, Germany) and 5 ug of total RNA from each sample in triplicates were reverse-transcribed to double stranded cDNA using the GeneChipï¾® One-Cycle cDNA Synthesis Kit. The biotin-labelled cRNA was made using the GeneChipï¾® IVT Labelling Kit (Affymetrix, CA, USA). Twenty microgram of cRNA samples was fragmented and out of which which 7.5 ug cRNA were hybridized for 16 hours at 45C to the Affymetrix GeneChipï¾® Rice Genome Array (Santa Clara, CA, USA). After washing and staining with R-phycoerythrin streptavidin in a Fluidics Station, using the Genechipï¾® Fluidics Station 450, the arrays were scanned by the Genechipï¾® 3000 Scanner. The chip images were scanned and extracted using default settings and the CEL files were produced with the Affymetrix GeneChip Operating Software (GCOS 1.2). The resulting .CEL files were imported into the GeneSpring GX 10 (Agilent Technologies Inc, Santa Clara CA) and normalized with the PLIER16 algorithm. The resulting expression values were log2-transformed. Average log signal intensity values of three technical replicates for each sample were used for advance analysis.