Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Single cell genotyping of Myeloid Leukaemia of Down Syndrome cells

ABSTRACT: Single cells from ML-DS sample 186-2 (bone marrow) were analysed to determine whether mutations in JAK2 and CSF2RB ocurred in the same or different cells in this patient. Single live, CD19neg, CD3neg, CD45mid and CD117 positive cells were sorted into 96-well plates on the BD Fusion with antibodies as described above. Cord blood single cells were sorted similarly to serve as a control for the single cell Sequencing.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Homo sapiens

SUBMITTER: Catherine Garnett

PROVIDER: E-MTAB-7935 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Although mutations in Kras are present in 21% of lung tumors, there is a high level of heterogeneity in phenotype and outcomes amongst lung cancer patients suggesting the importance of other pathways. Wnt/β-catenin signaling is a known oncogenic pathway that plays a well defined role in colon and skin cancer but its role in lung cancer remains unclear. We show that activation of Wnt/β-catenin in the bronchiolar epithelium of the adult lung does not promote tumor development by itself. However, activation of Wnt/β- catenin signaling leads to a dramatic increase in tumor formation both in overall tumor number and size compared to KrasG12D alone. We show that activation of Wnt/β- catenin signaling significantly alters the KrasG12D tumor phenotype resulting in a phenotypic switch from bronchiolar epithelium to the highly proliferative distal progenitors found in the embryonic lung. This is associated with a decrease in E- cadherin expression at the cell surface which may increase metastasis in Wnt/β-catenin signaling positive tumors. Together, these data suggest that activation of Wnt/β-catenin signaling in combination with other oncogenic pathways in lung epithelium may lead to a more aggressive phenotype due to the imposition of an embryonic distal progenitor phenotype accompanied by decreased E-cadherin expression. We performed microarray analysis of control murine lung, CC10-cre:KrasG12D, and CC10-cre:Ctnnb1ex3flox:LSL-KrasG12D double mutant micro-dissected murine lung tumors to determine their transcriptional phenotype. Lungs of five-month-old mice were PBS inflated and all the tumors in each lobe were dissected. The total number of tumors obtained from three out of the 5 pulmonar lobes of each animal was called a sample the other two lobes were saved in case there were problems and the array needed to be repeated. Trizol was used to isolate RNA for microarray analysis. Samples & Genotypes: control murine lung n=2 animals, CC10-cre:KrasG12D n=2 animals, and CC10-cre:Ctnnb1ex3flox:LSL-KrasG12D n=2 animals.

Project description:Modification of microRNA sequences by the 3' addition of nucleotides to generate so-called M-bM-^@M-^\isomiRsM-bM-^@M-^] adds to the complexity of miRNA function, with recent reports showing that 3' modifications can influence miRNA stability and efficiency of target repression. Here we show that the 3' modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications result predominantly from adenylation and uridylation, and are seen across tissue types, disease states, and developmental stages. To quantitatively profile 3' nucleotide additions, we developed and validated a novel assay based on NanoString Technologies' nCounter platform. For certain miRNAs, the frequency of modification was altered by processes such as cell differentiation, indicating that 3' modification is a biologically regulated process. To investigate the mechanism of 3' nucleotide additions, we used RNA interference to screen a panel of eight candidate miRNA nucleotidyl transferases for 3' miRNA modification activity in human cells. Multiple enzymes, including PAPD1, PAPD4, PAPD5, ZCCHC6, ZCCHC11, and TUT1, were found to govern 3' nucleotide addition to miRNAs in a miRNA-specific manner. Three of these enzymesM-bM-^@M-^SPAPD1, ZCCHC6 and TUT1M-bM-^@M-^Shave not previously been known to modify miRNAs. Collectively, our results indicate that 3' modification observed in next generation small RNA sequencing data is a biologically relevant process, and identify enzymatic mechanisms that may lead to new approaches for modulating miRNA activity in vivo. We validated the specificity of the platform by assaying three pools of synthetic RNA oligonucleotides representing canonical, variant 1, and variant 2 versions of 5 miRNAs (miR-15a, miR-15b, miR-125a-5p, miR-143 and miR-221).Each bridge pool (i.e., specific to canonical, variant 1 or variant 2) was used to assay the three mixtures of synthetic miRNA oligonucleotides. In order to assess accuracy, we also assayed mixtures of 5 synthetic miRNAs containing 60% canonical miRNA, 30% variant 1, and 10% variant 2. Triplicate technical replicates of each sample were performed.

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Single cell genotyping of Myeloid Leukaemia of Down Syndrome cells

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