Microenvironment-Mediated Regulation of Micrornas In B-Lymphocytes as a Novel Mechanism for Terminal B-Cell Differentiation and Lymphomagenesis.

Abstract 3853

B-cell differentiation is tightly regulated by synchronized suppression and/or induction of specific transcription factors. Among them, B-cell lymphoma 6 (BCL6) and PRDM1 are considered to be master regulators for germinal center formation and terminal B-cell differentiation. Dysregulation of BCL6 and PRDM1 also have been associated with lymphomagenesis. Their regulation still need further study especially at the posttranscriptional level. Here, by using co-culture system and whole genomic microRNA microarray profiling, we show for the first time that direct B lymphoma cell-stroma cell contact between follicular dendritic cells and B-lymphocytes could induce upregulation of miR-30 family and downregulation of miR-9 and let-7 family. In silico analysis showed that miR-30s can target genes BCL6 and miR-9/let7 can target PRDM1 with direct binding sites in 3`UTR region of their mRNAs. The microarray data can be proved by microRNA specific Q-RT-PCR. Specifically, by both gain of function and loss of function studies, we functionally verified that FDCs Regulate Expression of BCL6 and PRDM1 via Cell-Cell Direct Contact induced correlated microRNA dysregulation. To further validate the direct interaction between BCL6 and miR-30, we constructed luciferase reporters containing the BCL6 3`-UTR that included miR-30 binding sites and a mutant 3`-UTR harboring mutations in the “seed pairing” sequences of the miR-30 binding site. Co-transfection of miR-30 and reporter construct into cells significantly decreased luciferase activity in wild-type but not in mutant BCL6-3`-UTR transfected cells, supporting the role of miR-30 family in the regulation of BCL6 expression. BCL6 and PRDM1 and their regulation miRNAs, let-7 and miR-30, also can be validated in primary normal B-lymphocytes and lymphoma cells by using our co-culture system. Dysregulation of BCL6 and PRDM1 is often associated with lymphomagenesis. We firstly identified that BCL6 is the direct target of miR-30 family and also verified PRDM1 is the target of miR-9, and let-7 in our system. Our studies provide a novel mechanism of post-transcriptional regulation of BCL6 and PRDM1 by several microRNAs. In the context of micro-environment, it provides a clue for germinal center B-cell differentiation as well as B-cell lymphomas progression regulated by lymphocyte cell-stroma cell contact through microRNAs.