Methylation of miR-34a, miR-34b/c, miR-124-1 and miR-203 in Ph-negative myeloproliferative neoplasms

BACKGROUND: MicroRNA (miR) miR-34a, -34b/c, -124-1 and -203 are tumor suppressor miRs implicated in carcinogenesis. METHODS: We studied DNA methylation of these miRs in Philadelphia-negative (Ph-ve) myeloproliferative neoplasms (MPNs). Methylation-specific PCR (MSP), verified by direct sequencing of the methylated MSP products, was performed in cell lines, normal controls and diagnostic marrow samples of patients with MPNs. RESULTS: Methylation of these miRs was absent in the normal controls. miR-34b/c were homozygously methylated in HEL cells but heterozygously in MEG-01. In HEL cells, homozygous miR-34b/c methylation was associated with miR silencing, and 5-aza-2'-deoxycytidine treatment led to re-expression of both miR-34b and miR-34c, consistent with that both miRs are under the regulation of the same promoter CpG island. miR-34a was heterozygously methylated in MEG-01 and K-562. miR-203 was completely unmethylated in K-562 and SET-2 but no MSP amplification was found in both HEL and MEG-01, suggestive of miR deletion. In primary samples, four each had miR-34b/c and -203 methylation, in which two had concomitant methylation of miR-34b/c and -203. miR-34a was methylated in one patient and none had methylation of miR-124-1. Seven patients (15.6%) had methylation of at least one of the four miRs. miR methylation did not correlate with clinical parameters, disease complications or JAK2 V617F mutation. CONCLUSION: This is the first report of miR hypermethylation in MPNs. miR-203 hypermethylation is not specific to Ph+ve leukemias but also present in Ph-ve MPNs. miR-34b/c methylation was associated with reversible miR silencing. There was no correlation of miR methylation with clinical demographic data or outcome.