MiR-28 Silencing In Germinal Center-Derived Lymphomas

Abstract 703

In order to gain insights into the role of microRNA (miRNAs) in mature B cell function and lymphomagenesis, we performed a comprehensive miRNA expression profiling of normal mature B cells and of germinal center (GC)-derived lymphomas. miRNA expression profiles were generated using a commercial array platform designed to interrogate approximately 700 miRNAs. Normal GC B cells were isolated from tonsil tissue of 5 pediatric healthy donors and subjected to miRNA profiling in parallel with tumor specimens obtained from 10 Burkitt Lymphoma (BL), 16 Follicular Lymphoma (FL) and 20 Diffuse Large B Cell Lymphoma (DLBCL) patients. Each tumor type displayed a distinct miRNA profile and appeared to be clearly separated from the normal counterpart. Interestingly, a set of miRNAs was expressed in normal GC cells, but not in lymphoma samples, suggesting that structural and/or functional loss of miRNAs occur during lymphomagenesis. Among these, miR-28 was found to be up-regulated in GC B cells, while it was completely silenced in BL and significantly reduced in a large fraction of DLBCL and FL. Quantitative RT-PCR analysis confirmed miR-28 reduced expression in these GC-derived lymphoma subtypes including both primary biopsies and cell lines. MiR-28 is an intragenic miRNA encoded by the LPP gene locus, located on chromosome 3q28. Deletions affecting miR-28 and LPP were previously reported in FL (Schwaenen et al. Genes Chromosomes Cancer 2009; 48:39-54) and similarly we identified LPP deletions in about 9% of DLBCL investigated for copy number alterations by SNP arrays. Further FISH analyses performed in cell lines lacking miR-28 expression (12 DLBCL and 27 BL) failed to identify chromosomal aberrations in the LPP locus, suggesting that mechanisms other than genetic losses, possibly of epigenetic nature, are involved in miR-28 silencing in lymphomas. In order to investigate the effects of miR-28 in lymphoma cells, we generated stable lymphoma cell lines displaying inducible expression of miR-28. Re-expression of miR-28 in lymphoma cells led to a retarded growth due to a combination of G1-cell-cycle arrest and increased apoptosis. Furthermore, lymphoma cells expressing miR-28 lost their clonogenic properties as shown by their inability to form colonies in soft agar. Taken together these results suggest a tumor suppressor function for miR-28 in lymphoma cells. Toward the identification of the direct miR-28 target genes, we implemented computational target prediction methods with gene expression profiling data obtained from the miR-28 engineered cell lines. Approximately two thousand miR-28 direct candidate targets were computationally predicted, 88 of which displayed transcriptional down-regulation upon miR-28 induction, suggesting that miR-28 may affect the stability of the target transcript. The candidate targets included genes involved in the control of cell proliferation and apoptosis and in cell signaling, consistent with the phenotypic changes induced by miR-28 expression. In 4 out of 5 tested candidate targets, 3′-UTR reporter gene assay confirmed the direct effect of miR-28 on the target gene. Finally, we have generated a conditional, GC B cell-specific miR-28 knock-out mouse model, which will provide critical insights on the physiologic as well as the tumor suppressor role of miR-28 in vivo.