MiR-29 silencing by DNA methylation in Burkitt lymphoma cells: Crosstalk between MYC and DNMT3B

Abstract

Background: In the last years increased evidences have strongly suggested that altered miRNA expression could play a significant role in the cancer development and progression depending on the tissue type and specific targets. MicroRNAs are small non-coding regulatory RNAs that bind to specific sites of their target genes and regulate post-transcriptional gene expression. MicroRNA genes may be regulated through epigenetic mechanisms, such as specific histone modifications and/or DNA methylation of CpG islands on promoter regions, or on those that are localized next to miRNA genes. MYC plays an important role in microRNA regulation including the miR-29 family of microRNAs. We have previously shown that miR-29a/b/c expression is down-regulated in Burkitt lymphoma (BL) tumor samples and associated to DNA methyltransferase (DNMT) 1 and 3B overexpression. Moreover, the ectopic expression of miR-29a/b/c in BL cell lines inhibited DNMT3B expression. Objectives: Given the regulatory role of MYC in the miR-29 and DNMT1/ 3B expression, the aim of the study was to investigate the contribution of DNA methylation on miR-29 silencing in BL cells, targeting by both methylation on promoters and enhancers regions. Methods: We investigated methylation on promoters and enhancers regions of miR-29a/b/c in BL cell lines (BL41 and Raji) using bisulfite pyrosequencing assays. Next, BL cells were treated with 5-aza-2’-deoxicitidine (decitabine) and evaluated miR29 a/b/c expression and methylation status. The expression of MYC, DNMT1 and DNMT3B at varying times and decitabine concentrations were accessed by Western blot. Results: BL41 and Raji cell line presented methylation in CpG sites located in both promoter and enhancer regions. After 24h of treatment with 1.0 uM decitabine , miR-29s promoter and enhancers regions were demethylated in both BL cell lines. Similar results were observed at 72h even using lower decitabine concentrations. Additionally, the expression of miR-29s was upregulated by decitabine treatment at 24 h (1.0 uM) in both cell lines, and at 72h (0.5, 0.25 and 0.125 uM) in BL41cells. Notably, lower decitabine concentrations (0.5, 0.25 and 0.125 uM) down regulated DNMT1 and DNMT3B protein expression levels, but no effect in the MYC protein was observed. Conclusion: In summary, the miR-29a/b1 and miR-29b2/c genes have methylated CpG sequences that may contribute to the regulation of miR-29s expression in BL cells. The findings suggest an interplay among MYC/miR-29/ DNMT3B pointing to miR-29 methylation as shut-off mechanism mediated by MYC overexpression in BL pathogenesis.

Background: In the last years increased evidences have strongly suggested that altered miRNA expression could play a significant role in the cancer development and progression depending on the tissue type and specific targets. MicroRNAs are small non-coding regulatory RNAs that bind to specific sites of their target genes and regulate post-transcriptional gene expression. MicroRNA genes may be regulated through epigenetic mechanisms, such as specific histone modifications and/or DNA methylation of CpG islands on promoter regions, or on those that are localized next to miRNA genes. MYC plays an important role in microRNA regulation including the miR-29 family of microRNAs. We have previously shown that miR-29a/b/c expression is down-regulated in Burkitt lymphoma (BL) tumor samples and associated to DNA methyltransferase (DNMT) 1 and 3B overexpression. Moreover, the ectopic expression of miR-29a/b/c in BL cell lines inhibited DNMT3B expression. Objectives: Given the regulatory role of MYC in the miR-29 and DNMT1/ 3B expression, the aim of the study was to investigate the contribution of DNA methylation on miR-29 silencing in BL cells, targeting by both methylation on promoters and enhancers regions. Methods: We investigated methylation on promoters and enhancers regions of miR-29a/b/c in BL cell lines (BL41 and Raji) using bisulfite pyrosequencing assays. Next, BL cells were treated with 5-aza-2’-deoxicitidine (decitabine) and evaluated miR29 a/b/c expression and methylation status. The expression of MYC, DNMT1 and DNMT3B at varying times and decitabine concentrations were accessed by Western blot. Results: BL41 and Raji cell line presented methylation in CpG sites located in both promoter and enhancer regions. After 24h of treatment with 1.0 uM decitabine , miR-29s promoter and enhancers regions were demethylated in both BL cell lines. Similar results were observed at 72h even using lower decitabine concentrations. Additionally, the expression of miR-29s was upregulated by decitabine treatment at 24 h (1.0 uM) in both cell lines, and at 72h (0.5, 0.25 and 0.125 uM) in BL41cells. Notably, lower decitabine concentrations (0.5, 0.25 and 0.125 uM) down regulated DNMT1 and DNMT3B protein expression levels, but no effect in the MYC protein was observed. Conclusion: In summary, the miR-29a/b1 and miR-29b2/c genes have methylated CpG sequences that may contribute to the regulation of miR-29s expression in BL cells. The findings suggest an interplay among MYC/miR-29/ DNMT3B pointing to miR-29 methylation as shut-off mechanism mediated by MYC overexpression in BL pathogenesis.