Epstein Barr Virus Micrornas Repress BCL6 Expression in Diffuse Large B Cell Lymphoma.

Epstein Barr Virus (EBV) is able to transform B-cells by disrupting the normal B-cell differentiation programme, leading to the development of different types of B-cell lymphoma. BCL6 is a key transcriptional repressor during normal B-cell differentiation that is required for germinal centre reaction and it has been shown to repress NF-kB in Diffuse Large B-Cell Lymphoma (DLBCL). In some B-cell lymphomas the expression of BCL6 and infection with EBV are mutual exclusive occurrences, although the causal mechanism of this phenomenon and its biological significance remain elusive.

A microRNA expression profile was investigated using Agilent's Human miRNA microarray kit. microRNA targets were predicted with the miRanda algorithm (http://www.microrna.org/). Immunohistochemical and in situ hybridization analyses were performed on 149 DLBCL samples to investigate the expression of BCL6 and EBV status, respectively. Luciferase assays were carried out by cloning the BCL6 3'-UTR into the pGL3-Control vector.

22 viral microRNAs were found to be upregulated specifically in EBV-positive cases of DLBCL. By applying the miRanda algorithm, 10 of these 22 microRNAs were predicted potentially to target BCL6. To explore this possibility, immunohistochemical analysis and in situ hybridization were carried out on 149 cases of DLBCL. The results showed an almost perfect inverse correlation between BCL6 protein expression and EBV infection, even in the absence of LMP1 protein expression (p<0.001). Only one out of 34 EBV-positive cases expressed BCL6, although 87 out of 115 EBV-negative cases expressed BCL6. This phenomenon was confirmed in DLBCL cell lines. ebv-miR-BART3, ebv-miR-BART7, ebv-miR-BART9 and ebv-miR-BART17-5p were selected from the list of ten microRNAs for further functional validation, on the basis of the score calculated by miRanda. Thus, we transfected synthetic microRNAs and measured the luciferase activity in our reporter system. At least three of the assayed microRNAs were able to reduce the luciferase activity of the reporter. The effect of these microRNAs on the endogenous BCL6 protein was investigated in lymphoid BCL6-expressing cell lines by Western blot. The four microRNAs were able to downregulate the levels of endogenous BCL6.

DLBCL cases infected by EBV express a restricted set of viral microRNAs. Several of these microRNAs can potentially target the BCL6 gene, highlighting the importance of BCL6 downregulation in the context of EBV infection. Functional studies demonstrate that at least three of these microRNAs are able to downregulate BCL6 expression. The reason why EBV downregulates BCL6 is still unknown, but we propose that it might be required for DLBCL cells to survive in the context of EBV-induced NF-κB pathway activation.

No relevant conflicts of interest to declare.