Epigenetic silencing of EVL/miR-342 in multiple myeloma

Abstract

miR-342-3p, localized to 14q32, is a tumor suppressor miRNA implicated in multiple cancers. As the promoter region of its host gene, EVL, is embedded in a CpG island, we postulated that miR-342-3p is an intronic miRNA co-regulated with its host gene by promoter DNA methylation in multiple myeloma (MM). By methylation-specific polymerase chain reaction, verified by quantitative bisulfite pyrosequencing, methylation of EVL/miR-342 was absent in all healthy controls (n = 10) and 12 of 15 (80%) human myeloma cell lines (HMCLs), but partially methylated in 3 of 15 (20%) HMCLs, including KMS-12-PE, OCI-MY5, and RPMI-8226R. In HMCLs, by real-time quantitative reverse transcription-polymerase chain reaction, methylation of EVL/miR-342 correlated with lower expression of both EVL (P = 0.013) and miR-342-3p (P = 0.023). Moreover, in KMS-12-PE and RPMI-8226R cells, both partially methylated for EVL/miR-342, 5-AzadC treatment led to demethylation of EVL/miR-342 and re-expression of miR-342-3p. Upon removal of 5-AzadC, continuous culture resulted in restoration of EVL/miR-342 methylation and downregulation of miR-342-3p. In primary samples, methylation of EVL/miR-342 was detected in 1 of 18 (5.6%) monoclonal gammopathy of undetermined significance (MGUS), 8 of 63 (12.7%) diagnostic MM, and 5 of 30 (16.7%) relapsed MM. EVL/miR-342 methylation was preferentially detected in IgD MM but not found to impact survival. Collectively, in MM, miR-342-3p is an intronic miRNA regulated by promoter DNA methylation of its host gene, EVL, in a tumor-specific manner. Methylation of EVL/miR-342 was present in consecutive stages of myelomagenesis including MGUS, diagnostic MM, and relapsed MM.