Specific Exosomal microRNA Are Differentially Expressed Between High and Low-Risk Myeloma Suggesting They Are Pathogenically Important

Introduction

Treating High Risk myeloma (HRMM) is an important clinical challenge and understanding the basis of its pathogenesis could offer new therapeutic options.  At present very few clues exist as to what is crucially deregulated between HR and LR and micro RNA (miR) are prominent candidates.  Since the first miRNA was discovered over 20 years ago, the important role of these small noncoding RNA molecules in multiple myeloma pathogenesis has been recognized. In addition to their function in regulating gene expression within their cell of origin, miR molecules encased in exosomes are also secreted into the circulation. In this setting exosomes can deliver their content to other cells throughout the body, and have been suggested to be a key factor mediating the interaction of the MM cells and their microenvironment. We have tested the hypothesis that miR content of exosomes in bone marrow serum of patients with myeloma have an impact on biological behavior and as such can distinguish between patients with GEP 70-defined high- and low-risk disease.

Methods

Exosomes were isolated from bone marrow serum using Invitrogen kits according to manufacturer's instructions.  Aliquots from the resulting samples were analyzed using electron microscopy to confirm the presence of exosomes. The exosome preparations were spiked with the Caenorhabditis elegance miR mimetic Ce_miRNA39_1 as a control, and miR was subsequently isolated using miRNeasy Kit (Qiagen). The miR were converted to cDNA with polyA tailing, pre-amplified 10 cycles. Aliquots of the preamplified cDNA were loaded into the samples wells and primer pairs of 68 miR, reported to be differentially expressed in myeloma or other cancers, were loaded into the reagent wells of Fluidigm's 96x96 Dynamic Array IFCs (integrated fluidic circuit) and the arrays processed on a Fluidigm BioMark. Results were analyzed using the company's Real-Time PCR analysis software. The level of the 68 miRs were analyzed in preamplified exosomal miR cDNA from 13 healthy donors, and 76 untreated NDMM patients (54 low-risk, 22 high-risk as categorized by GEP 70 gene model).

Results

Electron microscopy analysis confirmed the presence of exosomes, sized between 50 and 150nm in all of the sample preparations. With a cutoff ratio of 1.5, 3 miRs were differentially expressed between HRMM and LRMM: miR-192, and 215 were present at a higher level in exosomes from LRMM (1.6, and 1.8 fold, respectively) than in HRMM, while miR 720 and 1308 were higher in HR MM (2.9 and 3.3 fold, respectively). Of the 4 differentially expressed miRs, 2 ( 192, and 1308) were 2-82 fold higher in MM bone marrow serum exosomes than in exosomes from the healthy donors, one (720) had equal levels, and one (miR-215) was not detected in healthy donor samples.

Among the 68 miRs analyzed, 4 were differentially expressed between HR- and LRMM. miRNA 192 and 215 both target the MDM2/TP53 axis, and are lower in bone marrow serum exosomes from HRMM in comparison LRMM. In contrast, miR-720 and 1308 are higher in HRMM.  In this context miR-720 inhibits tumor invasion in breast cancer and modulates proliferation in esophageal cancer; miR-1308 is a fragment of t-RNA, targets the apoptotic pathway, and has anti-apoptotic function. It has been reported that miR-137 is deregulated in solid tumors, and that overexpression can induce apoptosis in MM cell lines. It is interesting that it was present in bone marrow serum exosomes from only 2 low-risk myeloma patients and not in exosomes isolated from purified myeloma plasma cells from 12 high-risk patients.

Conclusion

These results indicate that exosomal microRNA are associated with the risk status of myeloma patients at presentation, either as a reflection of risk or as effectors. Their presence in protecting vesicles in the circulation indicates that miR have the capacity to modulate the properties of the microenvironment and myeloma cells in remote loci. To better elucidate the role of exosomal miR in the interaction of myeloma cells with the microenvironment it is important to determine the source of the exosomes.