DNA methylation of tumour suppressive microRNA in mantle cell lymphoma

Abstract

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin’s lymphoma (NHL) due to neoplastic proliferation of CD5+ve naïve B-cells within the lymph node. It is a rare subtype of NHL accounting for approximately 10% of NHL worldwide and 3% in Hong Kong. MCL is characterized by the primary translocation t(11;14)(q13;q32) which leads to juxtaposition of cyclinD1 (CCND1) to the IgH gene hence overexpression of CCND1 protein. Epigenetic aberrations such as DNA hypermethylation has been implicated in lymphomagenesis which represses expression of proteincoding genes and non-coding RNA. Among different classes of non-coding RNA, microRNA is a class of non-coding RNA characterized by the short, single-stranded RNA sequences of 19-25 nucleotides, which represses translation of target protein-coding genes by sequence-specific binding to the 3’-untranslated region of the messenger RNA. microRNA has been shown to be important in regulating a wide range of gene functions. Depending on the protein-coding gene target, microRNA can be oncogenic if a tumour suppressive gene is targeted while microRNA can be tumour suppressive if an oncogene is repressed. However, very little is known about DNA methylation in mantle cell lymphoma due to the rarity of this disease.

This thesis aims to investigate the role of DNA methylation of tumour suppressive microRNA in MCL using the candidate microRNA approach and microarray approach. Furthermore, the tumour suppressive functions of these microRNAs are verified in vitro.

With the candidate approach, microRNA reported silenced by DNA methylation in other haematological or solid cancers are studied in MCL, and compared with other B-cell lymphomas. In particular, tumour-specific methylation of miR-203, miR-124-1, miR-34 family, miR-129-2 and miR-340 is detected in MCL patients, and their tumour suppressive function verified in cell lines. In spite of less frequent methylation of these microRNAs in MCL patients compared with other B-cell lymphomas, methylation of three genomic loci (miR-124-1, miR-34b/c and miR-129-2) is associated with inferior survivals in MCL patients. With the microarray approach, expression profiling of 754 mature microRNAs is performed in two MCL cell lines before and after treatment with demethylating agent, 5AzadC, with a view to restore expression of hypermethylated, hence silenced microRNAs in untreated cells. Among the up-regulated microRNAs, miR-155-3p has been identified as a novel tumour suppressive microRNA silenced in MCL, targeting lymphotoxin-β which is important in development and maintenance of the lymph node immune-architecture. Methylation of miR-155-3p in MCL is comparable with other Bcell and NK cell lymphoma but is less frequent than T-cell neoplasms. Despite absence of survival impact in MCL patients, methylation of miR-155-3p is associated with lower microRNA expression levels in MCL and other NHLs.

In conclusion, based on these two approaches, epigenetic silencing of six microRNAs (miR-203, miR-124-1, miR-34 family, miR-129-2, miR-340 and miR-155-3p) by DNA methylation have been identified in MCL, and is implicated in lymphomagenesis.