Mir-485-3p and Mir-654-3p Expression in Bone Marrow Mesenchymal Stromal Cells in Patients with Monoclonal Gammopathies Is Related to the Status of the Disease
Authors: Fernandez de Larrea Carlos , Diaz Tania , Navarro Alfons , Lozano Ester , Mena Mari-Pau , Isola Ignacio , Tovar Natalia , Cibeira Maria Teresa , Rosinol Laura , Bladé Joan 
Published: October 2019

Abstract
Background: Crosstalk between malignant plasma cells and surrounding cells in the bone marrow (BM), such as mesenchymal stromal cells (MSCs), endothelial cells and immune cells, is crucial for pathogenesis of multiple myeloma (MM) and in asymptomatic monoclonal gammopathies. In these diseases, microRNAs (miRNAs) could be useful as biomarkers for diagnosis, prognosis and evaluation of treatment response. miRNAs can be released to the serum and transferred among MM cells and BM-MSCs as cell-cell communication. Previously, we have showed a serum 14-miRNA signature associated with complete remission (CR) after autologous stem-cell transplantation (ASCT). In this sense, patients in CR with partial recovery of two normal serum miRNA levels, similar to those with monoclonal gammopathy of undetermined significance (MGUS), was associated with better prognosis. The aim of this study was to analyze the miRNAs profile in mesenchymal stromal cells derived from bone marrow of patients with multiple myeloma in different status of the disease, comparing with MGUS controls.
Methods: We analyzed samples from 95 patients with MGUS (N=23), MM at diagnosis (N=14), relapsed/refractory MM (N=14), MM in partial response (PR) or very good partial response (VGPR) (N=15), MM in CR (N=24) and healthy donors (N=5). Mononuclear cells from BM samples were cultured in DMEM containing 10% FBS. After a week, non-adherent cells were removed, whereas BM-MSCs were selected by their adherence to the plastic and their phenotype was confirmed by multiparametric flow cytometry. In a first screening phase, we analyzed 670 microRNAs in 20 primary BM-MSC from patients with MGUS (N=4), symptomatic MM (N=8) and MM in CR (N=8). miRNAs differentially expressed were identified according to a supervised analysis using significance analysis of microarrays (SAM) and Student's t-test based on multivariate permutation (with random variance model). miRNAs differentially expressed between groups of patients were validated in the whole cohort of BM-MSC from patients. Paired malignant plasma cells (CD38+) miRNA expression from patients with symptomatic MM as well as miRNA in serum samples paired with BM-MSC samples were also compared. RmiR package was used to identify miRNA targets, cross-correlating the miRNA expression data from the present study with our findings on the gene expression signature (Affymetrix Human Genome U219 array) in 12 BM-MSCs from patients (4 MGUS, 4 symptomatic MM and 4 in CR), based on the predicted targets from TargetScan and miRBase databases.
Results: In the screening phase, we identified a miRNA profile of 10 miRNAs (miR-663b, miR-654-3p, miR-206, miR-411*, miR-885-5p, miR-668, miR-638, miR-485-3p, miR-744* and miR-199a) differentially expressed between patients with symptomatic MM and MM in CR (adjusted p-value <0.0001). In the validation phase, miR-485-3p and miR-654-3p resulted differentially expressed in the three groups of patients: MGUS, symptomatic MM and patients in CR (ANOVA test: p=0.0101 and p=0.0228, respectively). The levels of these miRNAs were significantly decreased in patients with MM than in those with MGUS, and these levels seemed to recover when patients achieved CR. These two miRNAs (miR-485-3p and miR-654-3p) were also correlated with all degrees of response in MM and with asymptomatic gammopathies (ANOVA test: p=0.0154 and p=0.0487, respectively). Moreover, paired cross-correlation among these two miRNAs expression with our results in mRNA gene expression profile data showed 324 for miR-485-3p and 265 for miR-654-3p genes (correlation index < -0.8) (Figure 1A and 1B). miR-485-3p and miR-654-3p showed a higher expression in BM-MSC than in MM CD38+ cells, suggesting MSC as cell of origin for these miRNAs. Serum expression of these two miRNAs was concordant with the observed in BM-MSC, with higher in patients in CR and MGUS than in those with symptomatic MM (Figure 1C and 1D). miRNA expression in BM-MSC supernatant as well as the identification of the biological role and validation of the miRNA targets are ongoing.
Conclusion: miR-485-3p and miR-654-3p expression in mesenchymal stromal cells from bone marrow in patients with multiple myeloma and asymptomatic monoclonal gammopathies is related to the status of the disease and the response to treatment. These miRNAs are also expressed in serum, resulting in potential biomarkers for disease activity and risk of progression.

Disclosures
Rosinol: Janssen, Celgene, Amgen, Takeda: Honoraria. Bladé:Janssen: Honoraria.