Tesi di dottorato di ricerca
BCL6 and LRF crosstalk in Dohh2 cells
Settore scientifico disciplinare
Corso di studi
tutor Dott. Rainaldi, Giuseppe
Data inizio appello
B cell Lymphoma 6 (BCL6) and Leukaemia/lymphoma related factor (LRF) are Pok-proteins over-expressed in some types of Non-Hodgkin lymphoma, such as Diffuse large B cell lymphoma (DLBCL) and Follicular lymphoma (FL).<br>BCL6 is located on the chromosome 3 in the breakpoint affecting 3q27 band which is the most frequent translocation in Non-Hodgkin’s lymphomas. This gene is a transcription repressor whose principal effect, in human B cell, is to thwart the response to DNA damage by inhibiting both a p53-dependent and a MIZ-1 dependent pathway. In the germinal centre BCL6 is constitutively expressed to suppress the p53 action. In B-cell lines BCL6 prevents apoptosis induced by DNA damage.<br>LRF, encoded by the Zbtb7a gene, is also known as Pokemon and it is a transcription repressor involved in many cellular processes as differentiation, inflammation and oncogenesis. LRF indirectly controls p53, acting through p14 and MDM2 repression, and it also plays an important role as protooncogene in Non-Hodgkin's lymphomas. Both BCL6 and LRF repression act at transcriptional level on the target genes to regulate protein expression. <br>It has been demonstrated microRNAs, a class of endogenous 22-25 nt single stranded RNA molecules, work to regulate target gene expression at post-transcriptional level. In fact, these molecules share a partial complementarity with 3’UTR region of expressed genes and act repressing translation. <br>In this project we focused our attention on the possible correlation between BCL6 and LRF over-expression in cell lines derived from Non- Hodgkin's lymphomas.<br>Since miRNAs play an important role in tumours and in regulatory circuits in which are involved, we focused our attention on the behaviour of the miRNAs belonging to the hypothetical circuit that correlates BCL6 and LRF. Using our data and the data taken from the literature demonstrated in different model systems, we reconstructed a hypothetical circuit connecting BCL6 and LRF to each other. A negative feed back loop should link BCL6 and LRF (following the interaction showed in the circuit), while in Non-Hodgkin's lymphomas these two genes are found both over-expressed. We hypothesized that this double increase could be involved in the high proliferation rate typical of tumor cells. The BCL6-LRF network is very intriguing because there are transcription factors and miRNAs: both are described as fundamental actors in the regulation of the cell cycle, often deregulated in tumours and all are fundamental in the cellular response to stimuli. We treated the Dohh2, a cell line derived from a Non-Hodgkin's lymphomas, with etoposide, a chemotherapeutic agent, that is able to reduce BCL6. We observed that in this condition the Dohh2 cells are not able to proliferate. We focused our attention on the circuit and in particular on the behaviour of the molecules belonging to it. We noticed that there is an over-expression of miR-145. It is interesting because it has been described as a tumour suppressor miRNA and it is under p53 control. This transcriptional factor is under BCL6 direct control and LRF indirect control. We decided to over-express miR-145 through transient transfection to try to reproduce the effect of etoposide. We analyzed the network 24 hours after transfection: the transcription factors and miRNAs on the pathway, that hypothetically connects BCL6 and LRF, were influenced by the transfection but we did not find any change in the proliferation rate of Dohh2 cells. We hypothesized that it was due to the fact the over-expression was not sufficiently long. So we tried to prolong the increased expression level of miR-145 in Dohh2 cells. We performed a preliminary analysis of the circuit in a population stably silenced for BCL6 in which we had a prolonged down-regulation of BCL6: in this case the molecules belonging to BCL6-LRF circuit were modified too but the proliferation did no change. We focused on the possible differences between the etoposide treatment and the others. We noticed that in the former there was not only BCL6 and LRF decrease but also miR-145 up-regulation accompanied by p53 and E2F1 increase, not present in the other treatments. We can hypothesize that the BCL6 and LRF down-regulation are necessary, together with the increase of miR-145, p53 and E2F1, to affect proliferation in Dohh2 cells. <br>We hypothesized that the alteration of the correlation between BCL6 and LRF could have been developed in lymphoma cells in order to increase their proliferation and escape from apoptosis or cell death.