Epigenetic modifications including changes in DNA methylation lead to inhibited gene expressions and consequent phenotypic alterations. MicroRNAs (miRNAs) are a class of small non-coding RNAs (19∼25 nucleotides) which functions as endogenous silencers of target genes. In general, most miRNAs are downregulated in many cancers and specifically in multiple myeloma (MM). We hypothesized that the mechanism of low expression of tumor suppressor miRNAs in MM is through epigenetic silencing, specifically through CpG island hypermethylation. The aim of this study is to identify methylation-silenced miRNAs and clarify their contribution to MM development and progression.
MicroRNA microarray analysis was performed in a panel of six MM cell lines (MM1S, RPMI 8266, OPM2, U266, H929 and IMI9) with or without the treatment of 5 μM of 5'-aza-2'-deoxycytidine (5-Aza-CdR, DNA demethylating agent) to screen for the most commonly upregulated miRNAs in response to DNA demethylation. These results were compared to a global methylation data of patients with MM (GEO GSE21304). Of these, we further examined the role of miR-152 and miR-10b-5p in MM. Induced expression of miR-152 and miR-10b-5p with 5-Aza-CdR-treatment was validated with real-time PCR. The DNA methylation status of CpG islands of these two candidates was further evaluated by methylation specific PCR (MSP) and bisulfate sequencing PCR (BSP). The expression levels of miR-152 and miR-10b-5p in both newly diagnosed MM patients (N = 60) and normal healthy donor (N = 5) were further analyzed (GEO GSE16558). MiR-152 and miR-10b-5p mimics were transiently transfected into H929 cells, respectively and in vitro functional validation including cell proliferation, cell apoptosis and adhesion assays.
We identified miR-152 as the most common upregulated miRNA (in all of the applied six cell lines); 7 miRNAs (miR-10b-5p, -320b, -4521, -548b-3p, -584-5p, -616-3p and -497-5p) and 77 miRNAs were upregulated by 1.5-fold or more in any four or three applied cell lines. Among them, the methylation of miR-152 and miR-10b-5p was reported in other hematologic malignancies, but little is known in MM. We first focused our attention on miR-152 and miR-10b-5p; and validated their significant upregulation in MM cell lines with 5-Aza-CdR treatment by real-time PCR. With specific methylated- or unmethylated primers for miR-152 or miR-10b-5p, MSP results indicated that miR-152 methylated PCR products had high levels in all control cells, whereas the unmethylated PCR products were significantly increased in the 5-Aza-CdR-treated cells. The BSP results of the promoter region of miR-152 showed extensive methylation throughout its promoter region (∼1000 bp upstream) in both H929 and IM9 cells, which was reversible following 5-Aza-CdR treatment. The average methylation levels of miR-152 were 77% and 84% in H929 and IM9 cells, however, after 5-Aza-CdR treatment, the methylation level decreased to 19% and 10%, respectively. Similar results were found for miR-10b-5p in both cell lines. Together, these findings confirmed that miR-152 and miR-10b-5p were suppressed through CGI methylation in MM cell lines. Compared with healthy donor, the expression of miR-152 and miR-10b-5p were significantly decreased in newly diagnosed MM patients. Moreover, overexpression experiments demonstrated that the cell proliferation of H929 cells, as detected by MTT assay, was significantly reduced after the restoration of miR-152 or miR-10b-5p (p< 0.05). Western blot results showed that cleaved PARP, caspase 9 and caspase 3 proteins were all significantly increased after miR-152 or miR-10b-5p mimics transfection. Adhesion assays showed that the adhesion capacity to stroma or fibronectin of H929 cells with miR-152- or miR-10b-5p-overexpression was significantly reduced compared with the negative control cells (p< 0.05).
As methylation-sensitive miRNAs, miR-152 and miR-10b-5p may play an important role as tumor suppressors in MM, targeting methylation of miRNAs could be a promising approach for the treatment of MM.
Ghobrial:BMS: Advisory board Other, Research Funding; ONYX: Advisory board, Advisory board Other; NOXXON: Research Funding; Sanofi: Research Funding.
Asterisk with author names denotes non-ASH members.