Epigenetic silencing of miR-342-3p in B cell lymphoma and its impact on autophagy

Background miR-342-3p, localized to 14q32, is a tumor suppressor miRNA implicated in carcinogenesis. Given the presence of a promotor-associated CpG island for its host gene,EVL, we hypothesized that intronic miR-342-3p is a tumor suppressor co-regulated with host gene by promoter DNA methylation in B cell lymphoma. Results By bisulfite pyrosequencing-verified methylation-specific PCR (MSP),EVL/MIR342methylation was detected in five (50%) lymphoma cell lines but not normal peripheral blood and tonsils.EVL/MIR342methylation correlated with repression of both miR-342-3p and EVL in cell lines. In completely methylated SU-DHL-16 cells, 5-AzadC treatment resulted in promoter demethylation and re-expression of miR-342-3p and EVL. In 132 primary lymphoma samples,EVL/MIR342was preferentially methylated in B cell lymphomas (N= 68; 68.7%) than T cell lymphoma (N= 8; 24.2%) by MSP (P< 0.0001). Moreover,EVL/MIR342methylation was associated with lower miR-342-3p expression in 79 primary NHL (P= 0.0443). In SU-DHL-16 cells, the tumor suppressor function of miR-342-3p was demonstrated by the inhibition of cellular proliferation and increase of cell death upon over-expression of miR-342-3p. Mechanistically, overexpression of miR-342-3p resulted in a decrease of LC3-II, a biomarker of autophagy, which was pro-survival for SU-DHL-16. Pre-treatment with 3-methyladenine, an autophagy inhibitor, abrogated tumor suppression associated with miR-342-3p overexpression. By luciferase assay, MAP1LC3B, a precursor of LC3-II, was confirmed as a direct target of miR-342-3p. Finally, in SU-DHL-16 cells, overexpression of miR-342-3p downregulated the known target DNMT1, with promoter demethylation and re-expression of tumor suppressor E-cadherin. Conclusions Intronic miR-342-3p is co-regulated with its host gene EVL by tumor-specific promoter DNA methylation in B cell lymphoma. The tumor suppressor function of miR-342-3p was mediated via inhibition of pro-survival autophagy by targeting MAP1LC3B and downregulation of DNMT1 with demethylation and re-expression of tumor suppressor genes.