Abstract
BackgroundmiR-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.ResultsBy bisulfite pyrosequencing-verified methylation-specific PCR (MSP), EVL/MIR342 methylation was detected in five (50%) lymphoma cell lines but not normal peripheral blood and tonsils. EVL/MIR342 methylation 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/MIR342 was 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/MIR342 methylation 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.ConclusionsIntronic 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.
Highlights
MiR-342-3p, localized to 14q32, is a tumor suppressor miRNA implicated in carcinogenesis
Methylation of EVL/MIR342 in normal healthy controls and Non-Hodgkin’s lymphoma (NHL) cell lines By methylation-specific PCR (MSP), promoter DNA methylation of EVL/MIR342 was studied in the bisulfite-converted DNA of normal healthy controls, including peripheral blood buffy coats (n = 10) and tonsil tissues (n = 11), and NHL cell lines (n = 10)
By quantitative bisulfite pyrosequencing, NHL cell lines showing MIR342 was completely methylated (MM), Partially methylated (MU), and Completely unmethylated (UU) had a mean methylation level of 96.62, 44.20, and 5.30%, respectively (Additional file 1: Figure S1), confirming the methylation status derived by MSP
Summary
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. Based on the lineage and maturity of neoplastic cells, NHL can be classified as precursor and mature form of B-, T- or NK-cell lymphoma [2]. Zhang et al Clinical Epigenetics (2020) 12:150 hypomethylation and locus-specific DNA hypermethylation of promoter-associated CpG islands of tumor suppressor genes (TSGs) [6], leading to reversible silencing of TSGs [5]. Promoter DNA methylation-mediated silence of TSGs, such as p16INK4a, SOCS3 and SHP1 [7,8,9], has been implicated in the pathogenesis of B-cell lymphoma
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