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We sought to identify microRNAs which are deregulated in B-cell lymphoma due to epigenetic alterations and aimed to test these for subtype-specific DNA methylation patterns.

Using a microRNA expression array, we screened for microRNAs inducible by the DNA demethylating agent 5-Aza-2’-deoxycytidine (Aza) in B cell non-Hodgkin lymphoma (B-NHL) cell lines. Putative Aza-induced microRNAs were shortlisted by harboring CpG islands in their 5’-region. Array data were validated with quantitative real-time PCR on the level of primary, precursor and mature microRNAs. To determine any correlation of DNA methylation and histone acetylation cell lines were treated with suberoylanilide hydroxamic acid (SAHA) or Trichostatin A (TSA) in combination with Aza. DNA methylation was analyzed by methylation specific PCR and bisulfite sequencing of respective CpG islands in a panel of B-NHL and classical Hodgkin lymphoma (cHL) cell lines together with primary B-NHL samples and healthy controls.

Of the 872 microRNAs represented on the microarray 15 (1.7%) were induced >1.5-fold by Aza in follicular lymphoma (FL) cell line SC-1, 31 (3.6%) were activated in mantle cell lymphoma (MCL) cell line JEKO-1. Altogether 11/15 miRNAs are located downstream of a CpG island in SC-1 and 20/31 in JEKO-1, rendering them potentially susceptible to silencing via DNA methylation. Transcriptional validation of mature and precursor microRNAs identified the microRNA cluster MIR23A~27A~24-2 which was inducible by both DNA demethylating agents and histone deacetylase inhibitors. Analysis of MIR23A~27A~24-2 in a panel of B-NHL and cHL cell lines revealed that its expression was inversely correlated with the DNA methylation status of its 5’-region. Burkitt lymphoma (BL) and FL cell lines were methylated and mainly negative for miR-23a, miR-27a and miR-24 expression. In contrast, most MCL and all cHL cell lines analyzed expressed these microRNAs and were not methylated. Diffuse large B cell lymphoma (DLBCL) cell lines showed a more heterogeneous picture.

Notably, these in vitro data are in accordance with the methylation status of the MIR23A~27A~24-2 5’-region in primary samples: 23/29 BL patient samples were methylated, compared to 4/10 DLBCL samples and only 1/9 MCL samples. Peripheral blood mononuclear cells and tonsils from three healthy controls were all methylated at the MIR23A~27A~24-2 5’-region, indicating that loss of epigenetic silencing might be the aberrant event in lymphomatous entities like MCL and cHL. 

The microRNA cluster MIR23A~27A~24-2 is frequently downregulated due to DNA methylation in BL, FL, and some DLBCL, but is demethylated and highly expressed in MCL and cHL. Future investigations need to test the eligibility of this microRNA cluster as a potential diagnostic or prognostic biomarker in B-NHL and cHL.