Unknown,Transcriptomics,Genomics,Proteomics

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Comparison of exosomal and cellular miRNA from patients with chronic lymphocytic leukaemia

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Simon Wagner

PROVIDER: E-GEOD-54434 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In multiple myeloma (MM), abnormal plasma cells interact with bone marrow (BM) stromal cells and vascular cells among others. A part of the BM milieu is considered highly hypoxic, and myeloma cells in situ may be influenced by circumstances other than normoxia in vitro. Hence, we attempted to confirm the role of hypoxic MM-derived exosomes in the BM milieu. We established a novel hypoxia-resistant cell line, KMS-11-HR, derived from KMS-11 cells cultured for >4 months under hypoxia (1% O2), as a model of MM cells localizing in an extensively hypoxic milieu. We used KMS-11 cells and KMS-11-HR cells, as donor cells, and HUVECs as recipient cells. Exosomes derived from KMS-11 cells (normoxia or hypoxia) and exosomes derived from KMS-11-HR cells (hypoxia-resistant sub-line) were used for validation of angiogeneic activity, such as tube formation assay. Exosomes derived from the KMS-11-HR cells significantly increased tube formation of HUVECs than those from KMS-11 cells. To identify intercellular and exosomal miRNAs specifically expressed in hypoxia-resistant cells, we assess the expression profiles of intercellular and extracellular miRNAs in KMS-11 cells and KMS-11-HR cells using Taqman MicroRNA Array v2.0 (Applied Biosystems, Bedford, MA). KMS-11 cells and KMS-11-HR cells were cultured for 24 hours under hypoxic conditions (1% O2). The exosome fraction was obtained from culture medium using Exoquick Exosome Precipitation Solution (System Biosciences, Mountain View, CA, USA). Isolation of cellular and exosomal miRNAs was performed using the miRNsasy kit (Qiagen). The expression profile of miRNAs was determined using the Human Taqman miRNA Arrays A (Applied Biosystems). RNU6B and a spike control (ath-miR159) were used as an invariant control for the cell and exosome, respectively. QRT-PCR was carried out on an Applied Biosystems 7900HT thermal cycler using the manufacturerM-bM-^@M-^Ys recommended program. Finally, all the raw data from each array was run on Data Assist Software ver.3.1 (Applied Biosystems).

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Comparison of exosomal and cellular miRNA from patients with chronic lymphocytic leukaemia

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