Abstract
Background: Mature B cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells are common and constitute the majority of leukemias and lymphomas. MicroRNAs are known to play a role in oncogenesis, lineage-selection, and immune cell function, including early B cell differentiation. However, the full extent and function of microRNA expression during mature B cell differentiation and in B cell malignancies are not known.
Methods: From normal young patients undergoing tonsillectomies, we sorted the mature B cell subsets (naive, germinal center, memory and plasma) using FACS, based on their expression of CD19, CD38, IgD and CD27. These sorted B cells were profiled for microRNA expression using a highly sensitive multiplexed real-time PCR assay, as well as for gene expression at the whole genome level using Affymetrix U133plus microarrays. miRNA targets can be predicted based on seed sequence matching of their 2–8 nt to the 3′UTR of gene transcripts. For each B cell stage, we experimentally validated microRNA regulation of predicted target genes of interest, LMO2, MYBL1 and PRDM1, by microRNA over-expression experiments and luciferase assays.
Results: We found that microRNAs have a characteristic expression pattern that defines each mature B cell stage. Examination of both microRNA and mRNA expression showed that in each B cell population, the target genes predicted based on seed matching were expressed at lower levels, results that were highly significant (P<1E-10). We found that differential microRNA expression is important at every B cell stage transition, and differentially expressed microRNAs frequently target differentially expressed transcription factors. In the naive to germinal center B cell and germinal center B cell to memory cell transitions, we found that miR-223 had an inverse relationship with its predicted target genes LMO2 and MYBL1. To test this relationship predicted based on seed pairing, in Germinal Center-derived BJAB cells, we over-expressed miR-223 by introducing its precursor, and saw a subsequent knockdown of LMO2 and MYBL1 at both the mRNA and protein level. We confirmed seed sequence specificity by comparing miR-223 knockdown of luciferase reporter activity on the LMO2 3′UTR compared to its seed sequence mutant. We further found that miR-9 and miR-30 family members directly regulate PRDM1 (blimp1), a master regulator of the GC to PC transition. In U266 cells (PC-derived), introduction of miR-9 and miR-30 family precursor resulted in decreased PRDM1 protein expression, although transcript levels were not changed, consistent with previous evidence that miRNA can regulate at the post-transcriptional steps. We further profiled over 50 tumors derived from various B cell malignancies (small lymphocytic lymphoma, Burkitt lymphoma, and the molecular subsets of diffuse large B cell lymphoma) and found that these malignancies maintain the expression patterns of their respective lineage; microRNA expression profiles of normal B cells could correctly classify the lineage of these tumors in over 80% of the cases. In contrast to other malignancies, common lymphomas do not down-regulate microRNAs, but rather maintain the microRNA-expression patterns of their normal B-cell counterparts.
Conclusion: Through concomitant microRNA and mRNA-profiling, we demonstrate a regulatory role for microRNAs at every stage in mature B-cell differentiation. Further, we have experimentally identified a direct role for the microRNA-regulation of key transcription factors in B-cell differentiation: LMO2, MYBL1 and PRDM1 (Blimp1). Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B-cells.
Restriction of memory B cell differentiation at the germinal center B cell positive selection stage