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SRX388831: Identification of distinct miRNA target regulation between breast cancer molecular subtypes using AGO2-PAR-CLIP and patient datasets
1 ILLUMINA (Illumina Genome Analyzer IIx) run: 6.2M spots, 224.1M bases, 161.4Mb downloads

Design: AGO2-PAR-CLIP. MCF7 cells were obtained from ATCC and grown at 37ºC in an atmosphere containing 5% CO2 in Dulbecco's modified Eagle's medium (1X D-MEM/high-glucose/L-glutamine/sodium pyruvate) supplemented with 10% heat inactivated fetal bovine serum, 100 unit/ml penicillin, 100 mg/ml streptomycin (Invitrogen, Sigma and Gibco). Cells were grown in the presence of 100 µM 4-thiouridine (4SU) for 24 h and AGO2 complexes were immunoprecipitated using a monoclonal antibody against AGO2 (Millipore clone 9E8.2), according to Hafner et al. 2010 (PMID: 20371350). We used lysis buffer in lieu of high-salt wash buffer to not disrupt the monoclonal antibody-bead interaction. Crosslinked RNA of 20-40 nt in length was recovered from the 100 kDa AGO2 immunoprecipitated protein complexes separated on SDS gel, confirmed by Western blot probing with a polyclonal antibody recognizing AGO2 (Millipore 07-590). The isolated RNA was converted into cDNA libraries, and sequenced by Illumina at the Rockefeller University Genomics Center.
Study: Homo sapiens PAR-CLIP
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Background Various microRNAs (miRNAs) are up- or downregulated in tumors. However, the repression of cognate miRNA targets responsible for the phenotypic effects of this dysregulation in patients remains largely unexplored. To define miRNA targets and associated pathways, together with their relationship to outcome in breast cancer, we integrated patient-paired miRNA-mRNA expression data with a set of validated miRNA targets and pathway inference. Results To generate a biochemically-validated set of miRNA-binding sites, we performed argonaute-2 photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (AGO2-PAR-CLIP) in MCF7 cells. We then defined putative miRNA-target interactions using a computational model, which ranked and selected additional TargetScan-predicted interactions based on features of our AGO2-PAR-CLIP binding-site data. We sub-selected modeled interactions according to the abundance of their constituent miRNA and mRNA transcripts in tumors, and we took advantage of the variability of miRNA expression within molecular subtypes to detect miRNA repression. Interestingly, our data suggest that miRNA families control subtype-specific pathways; for example, miR-17, miR-19a, miR-25 and miR-200b show high miRNA regulatory activity in the triple-negative, basal-like subtype, whereas miR-22 and miR-24 do so in the HER2 subtype. An independent dataset validated our findings for miR-17 and miR-25, and showed a correlation between the expression levels of miR-182 targets and overall patient survival. Pathway analysis associated miR-17, miR-19a and miR-200b with leukocyte transendothelial migration. Conclusions We combined PAR-CLIP data with patient expression data to predict regulatory miRNAs, revealing potential therapeutic targets and prognostic markers in breast cancer.
Sample: General Sample for Homo sapiens (MCF7 cell line)
SAMN02438370 • SRS512686 • All experiments • All runs
Organism: Homo sapiens
Name: MCF7B_TAF11
Instrument: Illumina Genome Analyzer IIx
Strategy: OTHER
Source: OTHER
Selection: size fractionation
Layout: SINGLE
Spot descriptor:

Runs: 1 run, 6.2M spots, 224.1M bases, 161.4Mb
Run# of Spots# of BasesSizePublished


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