Alterations in the mir-15a/16-1 Loci Impairs Its Processing and Augments B-1 Expansion in De Novo Mouse Model of Chronic Lymphocytic Leukemia (CLL)

Abstract

New Zealand Black (NZB) mice, a de novo model of CLL, share multiple characteristics with CLL patients, including decreased expression of miR-15a/16-1. We previously discovered a point mutation and deletion in the 3' flanking region of mir-16-1 of NZB and a similar mutation has been found in a small number of CLL patients. However, it was unknown whether the mutation is the cause for the reduced miR-15a/16-1 expression and CLL development. Using PCR and in vitro microRNA processing assays, we found that the NZB sequence alterations in the mir-15a/16-1 loci result in deficient processing of the precursor forms of miR-15a/16-1, in particular, we observe impaired conversion of pri-miR-15a/16-1 to pre-miR-15a/16-1. The in vitro data was further supported by derivation of congenic strains with replaced mir-15a/16-1 loci at one or both alleles: NZB congenic mice (NmiR+/-) and DBA congenic mice (DmiR-/-). The level of miR-15a/16-1 reflected the configuration of the mir-15a/16-1 loci with DBA congenic mice (DmiR-/-) showing reduced miR-15a levels compared to homozygous wild-type allele, while the NZB congenic mice (NmiR+/-) showed an increase in miR-15a levels relative to homozygous mutant allele. Similar to Monoclonal B-cell Lymphocytosis (MBL), the precursor stage of the human disease, an overall expansion of the B-1 population was observed in DBA congenic mice (DmiR-/-) relative to wild-type (DmiR+/+). These studies support our hypothesis that the mutations in the mir-15a/16-1 loci are responsible for decreased expression of this regulatory microRNA leading to B-1 expansion and CLL development.

Role of mir-15a/16-1 in early B cell development in a mouse model of chronic lymphocytic leukemia

“…In order to study the effects of the miR-15a/16-1 deficiency on the mouse B cell development the congenic mice were generated by consecutive crossbreeding wild type DBA and CLL mouse model NZB mice with mutant mir-15a/16-1 selection. The resultant strain termed DBA −/− was used for aging followed by further analysis [ 9 ]. First, we found that the level of miR-15a was decreased in both B1 and B2 cells derived from NZB and DBA congenics (DBA −/− ) when compared to the wild-type DBA (Figure 1A ), which positively correlated with Dleu2 levels in splenocytes.…”

“…Furthermore, no difference in B1P levels was observed in younger animals suggesting that this is an age-associated phenomenon (data not shown). Recently, we have shown that DBA congenic animals exhibit an increased percentage of lineage − sca1 + c-kit + (LSK) cells in DBA −/− congenic mice [ 9 ]. To test their ability to give rise to B lineage cells, LSK progenitors derived from the DBA, NZB and DBA −/− mice bone marrow were differentiated in vitro under conditions favoring B cell development.…”

“…It possesses several genetic defects other than mir-15a/16-1 mutation hence it is not an ideal model to study effects of individual mutations. Recently, we reported generation of a DBA congenic mouse model with an NZB-derived mutated mir-15a/16 locus, and showed that this mutation is responsible for reduced levels of mature miR-15a due to the defects in microRNA processing [ 9 ]. Several approaches have been exploited in this work to further understand the role of miR-15a in B cell development using this mouse model coupled with both in vitro and in vivo assays.…”

“…This then is being transported to a cytoplasm by Exportin 5 protein and cleaved into a mature micro-RNA molecule by Dicer enzyme [ 8 ]. Recently, we have demonstrated that mir-15a mutation and deletion in NZB mouse are responsible for its decreased expression levels and this is due to a blockage of Drosha-mediated cleavage of primary transcript [ 9 ].…”

MicroRNAs in Autoimmunity and Hematological Malignancies

“…Moreover, in our subset of patients, the RPIII-mediated transcription of miR-15a and miR-16-1 was associated with a poor prognostic marker of CLL [ 115 ]. The 13q14 deletion is not the only cause of the miR-15a and miR-16-1 down-regulation: for instance, a point mutation downstream the miR-16-1 in the New Zealand Black (NZB) CLL mouse models affects the processing of primiR-15a/16-1 to pre-miR-15a/16-1 , thus leading to decreased expression of those miRNAs [ 116 ]. A common pathogenic pathway that link miR-15a/16-1 cluster, miR-34b/c cluster and tumor protein p53 with 13q, 11q, and 17p deletion has been reported in CLL.…”