Epigenetic Regulation of MicroRNA Expression In Hodgkin Lymphoma

In recent years, microRNAs (miRNAs) have emerged as key regulators of carcinogenesis. miRNA expression is deregulated in multiple hematological malignancies. One of the mechanisms that can affect miRNA expression is methylation in the promoter regions of miRNA genes. The main objective of the present study was to identify tumor suppressor miRNAs that are silenced by alterations in gene methylation in Hodgkin Lymphoma (HL). In addition, since demethylating agents could affect miRNA expression, we evaluated the in vitro effectiveness of 5-Aza-2-deoxycytidine (AZA), a DNA methyltransferase inhibitor, in HL cell lines.

To detect miRNAs regulated by methylation, we analyzed the expression of 670 mature miRNAs in two HL cell lines, L-428 and L-1236, before and after AZA treatment, by TaqMan Human MicroRNA Arrays V2.0 (Applied Biosystems) in an ABI 7900 HT sequence detection system. miRNA expression data was analyzed by the 2^-ΔΔCt method using RNU48 as endogenous control. To validate the methylation status, genomic DNA samples from four HL cell lines (L-428, L-1236, HDMYZ and L-540) and peripheral B-cells from healthy donors were modified by sodium bisulfite using the EZ DNA Methylation kit (Zymo Research). The DNA methylation status of these samples was analyzed by methylation specific PCR (MSP) after sodium bisulfite modification of DNA. To evaluate AZA in vitro effectiveness, we treated the four HL cell lines daily with 20 nM, 250 nM, 1 μM and 5 μM of AZA or DMSO (vehicle control), and we assayed proliferation with CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) after 72 hours of treatment.

After AZA treatment in the L-428 and L-1236 HL cell lines, 32 miRNAs (of 670 analyzed) were expressed de novo, 15 of which were present in both cell lines. miR-34a, miR-203, miR-342, miR-105, miR-490, miR-375 and the miR-500 family had well-defined CpG islands in their promoter regions. The MSP analysis showed that miR-34a, miR-203, miR-490 and miR-375 were methylated in L-428 and L-1236 HL cell lines, but not in the normal B-cells. miR-342 was methylated in L-1236 HL cell line, but not in L-428 nor normal B-cells. To further validate this we analyzed the methylation pattern in HDMYZ and L-540 HL cell lines and found that only miR-203, miR-375 and miR-490 were methylated in all 4 HL cell lines analyzed. We observed an AZA dose-dependent reduction in proliferation in all four HL cell lines. After 72 hours of treatment with AZA at 5μM, proliferation significantly decreased by 29% in L-428, 36% in L-1236, 28% in HDMYZ and 22% in L-540. Moreover, the analysis of miRNA levels during the treatment showed a re-expression of methylated miRNAs after 48 hours. The methylation analysis of these miRNAs in laser captured microdisected HL cells from patients and functional analysis are ongoing and complete data will be reported.

In summary, HL exhibits a characteristic epigenetic pattern which includes the methylation of key miRNAs during the carcinogenesis process, such as miR-34a and miR-203, which have previously been shown to act as tumor suppressors in lung, colorectal and other cancers. Moreover, we present evidence that demethylating agents allow the re-expression of these tumor suppressor miRNAs, suggesting that they could be a promising novel treatment for HL patients.

Supported by FIS grant (PS09/00547).

No relevant conflicts of interest to declare.