DNA methylation of microRNA and long non-coding RNA in mantle cell lymphoma

Abstract

Mantle cell lymphoma (MCL), arising from malignant transformation of CD5+ naïve B-cell population in the mantle zone of lymph node, is a rare but aggressive subtype of B-cell non-Hodgkin’s lymphoma (NHL), accounting for 4-6% of all NHL patients. Genetically, MCL is characterized by the presence of t(11;14)(q13;q32) translocation, thereby cyclin D1 overexpression and consequently accelerated G1/S cell cycle transition. microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are two important classes of non-coding RNAs. Mature miRNAs, measuring about 22 nucleotides, downregulate target genes via sequence-specific binding to 3’-UTR of the target genes, resulting in degradation of mRNA or translational block. LncRNAs, generally > 200 nucleotides, may regulate gene expressions by multiple mechanisms operating at epigenetic, transcriptional or post-transcriptional levels. Dysregulation of miRNAs and lncRNAs has been shown in a variety of tumors, hence important in carcinogenesis. DNA methylation is an epigenetic modification by which a methyl group (-CH3) is added to the C5 position of cytosine in a CpG dinucleotide. Cancer cells are characterized by global DNA hypomethylation and gene-specific promoter DNA hypermethylation. Moreover, promoter DNA hypermethylation emerges to be an important mechanism mediating silencing of both protein-coding and non-coding tumor suppressor genes. However, apart from intergenic tumor suppressor miRNAs, the effect of DNA methylation-mediated silencing of intronic miRNAs and lncRNAs on lymphomagenesis remains largely unexplored. Herein, it was hypothesized that reversible silencing of intronic miRNAs and lncRNAs was mediated by promoter DNA methylation in MCL and other subtypes of NHL. By a candidate gene approach, promoter methylation of intronic miRNAs (miR-342-3p and miR-1250-5p) and lncRNAs (NKILA and HOTTIP) were investigated. Results showed that both the promoter CpG islands of host genes of both intronic miR-342-3p and miR-1250-5p, and that of lncRNAs (NKILA and HOTTIP) were methylated in a tumor-specific manner. Moreover, intronic miR-342-3p and miR-1250-5p were reversibly silenced and co-regulated by promoter DNA methylation of their host genes, EVL and AATK respectively. Furthermore, in NHL cells, restoration of miR-342-3p or miR-1250-5p resulted in increased cell death and decreased cellular proliferation, hence inherent tumor suppressor functions. Mechanistically, miR-342-3p was shown to inhibit pro-survival autophagy via directly targeting MAP1LC3B, precursor of LC3-II that is a biomarker of autophagy. Moreover, as miR-342-3p was known to target DNMT1, promoter demethylation led to re-expression of a hypermethylated and silenced tumor suppressor, E-cadherin. On the other hand, miR-1250-5p was demonstrated to inhibit NHL cell proliferation by targeting MAPK1 hence repression of MAPK/ERK signaling, and SDF-1-dependent migration by targeting WDR1. LncRNAs, NKILA and HOTTIP, were reversibly silenced via promoter DNA methylation in NHL cells. Moreover, knockdown of NKILA led to decreased cell death and enhanced cellular proliferation, consistent with a tumor suppressive function in NHL cells. In primary samples, HOTTIP methylation was frequently detected in MCL. Conversely, miR-342-3p, miR-1250-5p, or NKILA were preferentially methylated in B-NHL other than MCL, implicating an epigenetic heterogeneity among diverse NHL subtypes. In conclusion, intronic miR-342-3p and miR-1250-5p, as well as lncRNAs HOTTIP and NKILA were regulated by reversible methylation-mediated silencing in MCL and other subtypes of NHL, which implicated in lymphomagenesis.