The over expression of miR-34a in B lymphoma cells neutralizes the proapoptotic/antiproliferative activity of miR-34a through the down regulation of cMYC and the consequent repression of p53

The follicular lymphoma cell line DoHH2, which over expresses BCL6 and has a wild type p53, was used to test the tumor suppressor activity of miR-34a. We transiently transfected miR-34a into DoHH2 cells and found that miR-34a failed to either induce apoptosis or reduce cell proliferation. To explain the lack of effect of miR-34a on cell proliferation we determined the expression of p53 and its direct target p21. Interestingly, we found that p53 strongly diminished in miR-34a transfected cells both at transcriptional and post-transcriptional levels and that the down regulation of p53 was accompanied by a reduced transcription of p21. As BCL6 is a transcriptional repressor of p53 and p21 we cannot exclude that their down regulation following miR-34a over expression was due to an enhanced expression of BCL6. On the contrary we found that BCL6 decreased at both the mRNA and the protein levels in miR-34a transfected cells. We also found that miR-34 over expression determined a decrease in cMYC level. As it has been shown that cMYC controls p53 expression and that the over expression of miR-34a caused a reduction of cMYC level we concluded that the down regulation of BCL6 was unable to displace the control of cMYC on p53 and that the expected proapoptotic/antiproliferative activity of miR-34a was probably neutralized through the down regulation of cMYC and the consequent repression of p53. These data suggest that cMYC has a pivotal role in driving DoHH2 cell proliferation by controlling p53. On this assumption we attempted to modify p53 without affecting cMYC levels. For that we silenced BCL6 in DoHH2 cells using a previously reported effective siRNA and found that a 50% reduction of BCL6 protein induced the up regulation of p53 resulting in increased apoptosis and a partial inhibition of cell proliferation. The activation of p53 was confirmed by the up regulation of p21 and miR-34a but surprisingly cMYC was unaffected. The opposite results obtained by either BCL6 silencing or over expressing miR-34a from one side suggest that acting on p53 without affecting cMYC expression is an effective strategy to hit the proliferation of DoHH2 cells and from the other side that the use of tumor suppressor miRNAs as cancer therapeutic molecules should take into account the gene expression network of target cells.