Identify the dysregulated miRNA in human colorectal cancer tissues
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE45349 | GEO | 2013/03/20
SECONDARY ACCESSION(S): PRJNA193518
REPOSITORIES: GEO
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Project description:Exosomes are secreted into the intercellular space and carry a diverse cargo of proteins, mRNA and miRNA that have important roles in cell-to-cell communication. We found that exosomes from primary human leukaemic B-cells contain a population of miRNAs that has been selected from the general cellular pool to be enriched in miR-323-3p, miR-374b, miR-363 and miR-494. Gene targets of these miRNA are found within the TGFM-NM-2 pathway, but we noted that CD69, a protein that is essential for the regulation of normal lymphocyte egress from lymph nodes was the top predicted target of miR-363. Utilising luciferase reporter assays we demonstrate that CD69 is a target of miR-363. The interaction between miR-363 and CD69 is functionally important as demonstrated by repression of CD69 expression by a miR-363 mimic in the Jurkat T-cell line. MicroRNA expression profiles of isolated exosomes and CLL cells cultured on CD154/IL-4 feeder layers for 24 hours were determined using Taqman low-density miRNA (TLDA) arrays. Patient samples were layered on Ficoll and centrifuged for 20 minutes at 2500 rpm following which the interface layer conatining mononuclear cells was removed. The cells were washed in PBS and cultured on a feeder layer of mouse fibroblasts transfected with human CD154 in the presence of IL-4 (10 ng/ml) for 24 hours MiRNA extracts were made using a miRvana miRNA isolation kit (Applied Biosystems, Paisley, UK), and reverse transcribed using MegaPlex RT Primers and a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Equal amounts of RNA were added to each microarray plate. Human microRNA Array (A) plates (containing assays for 377 miRNA) were used (Applied Biosystems, #4398965) and reactions carried out on a 7900HT Real Time PCR system. MiRNA expression was compared in paired samples of chronic lymphocytic leukaemia cells and exosomes utilising Taqman Real-Time PCR.
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