Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation BRL-49653 by a miR-29b-dependent mechanism and induced promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells. promoter was found hypermethylated in 62.9% of patients, and its silencing was associated with greater responsiveness to cytokines, thus supporting proliferation and survival of MM cells.51 Moreover, hypermethylation of was also reported in other hematologic malignancies52,53 or solid tumors.54-56 In the present study, we investigated whether miR-29b could revert the aberrant hypermethylated status of gene promoter and affect adhesion and migratory capacities of MM and endothelial cells. Moreover, the effects of the proteasome inhibitor bortezomib, a known miR-29b-inducing agent,40 on the global DNA methylation profile and SOCS-1 expression, were analyzed. All together these findings provide a molecular rationale for developing miR-29b-based epigenetic therapies for MM. Results SOCS-1 mRNA levels are downregulated in MM cells Since it has been reported that SOCS-1 is frequently downregulated in human malignancies,52,54 first we sought to analyze SOCS-1 mRNA espression in primary cells from 55 MM patient and 29 plasma cell leukemia (PCL) patients, which were classified according to the presence of the recurrent IGH chromosomal translocations SKP1A and cyclin D expression (TC classification)57 as compared with BRL-49653 normal PCs from 4 different healthy donors. We found that SOCS-1 mRNA was indeed steadily downregulated in TC(1C4) patient groups and in PCL patients (Fig.?1A). Figure 1. SOCS-1 mRNA expression and its correlation with BRL-49653 miR-29b levels in primary MM and PCL samples.(A). Differential expression of SOCS-1 mRNA in immunoselected CD138+ cells from 4 healthy donors (N), 55 BRL-49653 MM classified according to the presence of … We next attempted to correlate SOCS-1 mRNA and miR-29b levels in our data set (34 MM and 18 pPCL patients), for which both miRNA and gene expression profiling were available. This integrated approach disclosed a positive correlation trend between SOCS-1 mRNA and miR-29b levels among all MM and PCL patients analyzed (Fig.?1B); this trend was found statistically significant within the TC2 patients group (n = 10, = 0.016; not shown), thus suggesting that miR-29b might likely act as a positive regulator of SOCS-1 in the context of a specific MM genetic background. These findings prompted us to investigate the molecular mechanisms by which miR-29b could regulate SOCS-1 expression in MM cells. Synthetic miR-29b mimics demethylate in MM cells On the basis of our previous findings indicating that miR-29b is endowed with demethylating activity,31 we investigated whether enforcing miR-29b in MM cells could modulate the expression of SOCS-1 through its promoter demethylation. To this aim, NCI-H929 or U266 cells were transfected with synthetic miR-29b mimics or scrambled control oligonucleotides (NC). The levels of miR-29b in transfected cells were assessed by quantitative real-time-PCR (qRT-PCR; Fig.?2A; Fig.?S2A). In parallel, genomic DNA was subjected to bisulfite sequencing and Sequenom MassARRAY analysis. The methylation level of each CpG site, in a total of 9 amplicons spanning the promoter, was evaluated. As shown in Figure?2B and Figure?S2B, the methylation levels widely varied, from completely unmethylated up to 100% methylated, across different CpG sites within each amplicon. Notably, miR-29b significantly reduced the methylation.