MiRNA Profile Selectively Expressed In K562 Cells-derived Exosomes

Objective:Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the expansion of a clone of hematopoietic cells that carry the Philadelphia (Ph) chromosome. Tyrosine kinase inhibitors (TKI) imatinib has significantly changed the clinical management of patients with CML. However, up to 33% of CML patients will not achieve optimal response. This is associated with the complex mechanisms of CML progression. Recently, one of the core components of the exosome was identified as a protein associated with CML; it showed that exosome was associated with CML.In recent years, increasing studies have been conducted to investigate the exosomes. Exosomes originate from the inward budding of the endosomal membrane, forming the multivesicular bodies (MVBs). They are released into the extracellular space by fusion of the MVBs with the plasma membrane. Exosomes are small vesicles (30-100 nm in diameter) that carry proteins、RNA、miRNA, and have been found to mediate the intercellular signal transduction. The release of exosomes from K562 CML cells has been reported, and the exosomes can promote angiogenesis in a src-dependent fashion. Aberrantly release of miRNA from exosomes may cause significant alterations in biological signaling pathways that may affect the disease development, such as cardiovascular disorders, inflammatory diseases, gynecological diseases and cancers. The present study was conducted to investigate miRNA profile selectively expressed in K562 cell-derived exosomes, and the role of selectively expressed miRNA.Method:Exosomes were isolated from conditioned media from K562 cells by ultracentrifugation and inspected by scanning electron microscopy. To confirm that the exosomes were harvested, Western blot assay were performed. MiRNA microarray was performed on K562 cells and K562 cell-derived exosomes with the 6th generation of miRCURYTM LNA Array (v.16.0). To confirm the result of miRNA microarray, miRNA were analyzed by real time RT-PCR. The potential targets of the up-regulated miRNA were predicted. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to determine the up-regulated miRNA.Results:(1) MiRNA expression profiles between K562 cells and K562 cells derived exosomes were significant differences. (2) In exosomes,49 miRNA were up-regulated as compared to K562 cells. (3) These up-regulated miRNA were further analyzed by quantitative real-time PCR, and results were similar to those in miRNA array. (4) Bioinformatical analysis (including Gene Ontology and KEGG pathway analysis) was performed. The results revealed that 66 GO terms and 26 signaling pathways were associated with the potential targets of 49 up-regulated miRNA. (5) Networks of 9 GO terms and 5 pathways were built between up-regulated miRNA and their target genes. The result showed that a total of 25 up-regulated miRNA and their potential targets might be involved in 9 GO terms, and a total of 23 up-regulated miRNA and their potential targets were associated with 5 pathways. (6) The result of our cell to cell adhesion assay showed that exosome-treated group had less adherent K562 cells than the control group. It suggested that exosomes may lead to reducing or defect of adhesion function.Conclusion:In conclusion, in this study, we detected and analyzed miRNA profile selectively expressed in K562 cell-derived exosomes. With complex pathway analysis, we preliminarily infered that these selectively expressed miRNA could regulate intracellular signal pathway through altering gene expression. These results help us to understand the potential roles of K562 cell-derived exosomes in CML-related processes.