Regulation of pri-miRNA BIC Transcription and Processing in Burkitt Lymphoma.

BIC a primary microRNA (pri-miR-155) transcript is highly expressed in the vast majority of Hodgkin lymphoma and diffuse large B cell lymphoma cases. High miR-155 levels are also observed in these B cell malignancies. In contrast, only very low or no expression of BIC and miR-155 are observed in Burkitt lymphoma cases. In the normal lymphoid system, BIC expression is observed in a proportion of the germinal center B cells and we have previously shown induction of BIC expression by BcR stimulation.

In this study we show the crucial involvement of PKC and NF-κB in the regulation of BIC expression upon BcR triggering of the Burkitt lymphoma cell line, Ramos, which is commonly used as a model system for germinal centre B cell stimulation. Surprisingly, Northern blot analysis did not reveal any miR-155 expression upon induction of BIC expression whereas other microRNAs were clearly detectable. Ectopic expression of BIC in Ramos and HEK293 cells resulted in miR-155 expression in HEK293 but not in Ramos cells suggesting a specific block of BIC to miR-155 processing in Ramos. In line with the results obtained with Ramos, lack of miR-155 expression after induction of BIC expression was also observed in other Burkitt lymphoma cell lines, indicating a generic and specific blockade in the processing of BIC in Burkitt lymphoma. In contrast, induction of BIC expression in normal tonsillar B cells resulted in very high levels of miR-155 expression and induction of BIC expression in Hodgkin lymphoma cell lines also resulted in elevated levels of miR-155. More recent reports have indicated that miRNA processing may be regulated by editing of pri-miRNA sequences by adenosine deaminases acting on RNA. ADARs can convert adenosines to inosines in stemloop sequences which may interfere with proper recognition and cleavage of pri-miRNA by the microprocessor complex. To evaluate whether ADAR editing might explain the observed differences in BIC to miR-155 processing, we analyzed the miR-155 sequence including 100nt up- and downstream of BIC RT-PCR products from stably transfected Ramos cells and PMA/Ionomycin stimulated CA-46 Burkitt lymphoma cells and compared those with sequences obtained from stably transfected HEK293 cells and L1236 cells. Analysis of 30 individual clones per cell line revealed no or very few conversions in the BIC transcripts of the four cell lines. This indicates that ADAR editing is not responsible for the observed BIC processing blockade in BL.

Our data provide evidence for two levels of regulation for mature miR-155 expression: one at the transcriptional level involving PKC and NF-κB, and one at the processing level. Burkitt lymphoma cells not only express low levels of BIC but also prevent processing of BIC via an, as yet, unknown mechanism.