MiR-221 and MiR-222 Patterns Characterize Burkitt Lymphoma in Human and Mouse Model

Abstract 1304

Recently, a class of noncoding RNAs called microRNA (miRNAs) has emerged as critical gene regulators in cell growth, differentiation, disease and development. MiRNAs are 18–24 nucleotide long noncoding RNAs, which regulate gene expression by pairing with 3′ untranslated region (UTR) of target mRNA and inhibiting protein translation and/or inducing mRNA degradation. Deregulated miRNA expression is reported in various human diseases including lymphomas, suggesting an important role in their pathogenesis.

According to WHO classification, Burkitt lymphoma (BL) is a rare, highly aggressive NHL composed of monomorphic medium-sized B cells with multiple nucleoli and numerous mitotic figures and is more common in children than in adults. The molecular feature of BL is the translocation that places MYC under the control of immunoglobulin gene regulatory elements. High levels of c-MYC have been clearly shown to have a tumour-promoting effect. However, there is recent evidence that infrequent cases may lack an identifiable MYC translocation, the explanation for which is still uncertain, though suggesting the existence of pathogenetic mechanisms alternative to genetic alterations.

Over the past years miRNA signatures have been described to characterize and classify different types of BL or to investigate the expression of miRNAs possibly regulated by c-Myc in BL cases positive or negative for Myc translocation. However, it remained unclear the functional role of differentially expressed miRNAs and no further studies have been conducted. We performed miRNA expression profile to gain further insights into the molecular pathology of BL. We conducted array analysis on a set of 5 sporadic BL patients, 3 endemic BL patients, 9 reactive tissues and 11 cases of mononucleosis. Our profile is the first one that shows the different expression between BL cases and normal B cells whereas recent miRNA profiles have been conducted in BL compared to other B-NHL (B-CLL, MCL & FL). A common trend of miRNAs altered expression was also observed by NanoString analysis in 10 BL cell lines compared to 5 normal CD-19+ B cells. Among several miRNAs previously described be deregulated in BL we identified a severe down-regulation of miR-221, miR-222 in all classes of comparisons we analyzed. The down-regulation of miR-221 and miR-222 associated to BL has been also confirmed by q-RT-PCR method in a different cohort of BL patients (20) compared to the healthy controls (6). We found that interesting considering the up-regulation of miR-221 and miR-222 previously confirmed in a lot of solid tumors by multiple studies. We are investigating a different role of the cluster miR-222 and miR-221 in lymphomas that have a different process in carcinogenesis than solid tumors.

In vivo models to study the lymphomagenesis of BL have been created but until now no one studied the importance of the miRNAs in vivo. We analyzed the expression of miR-221 and miR-222 in a Myc transgenic mouse model. The transgene construct consists of the Myc oncogene (c-myc) in association with the Emu immunoglobulin heavy chain enhancer and Myc promoter. Expression of the mouse Myc transgene is restricted to the B cell lineage. Previously it has been shown an increase of pre-B cells in the bone marrow throughout life of hemizygotes and a transient increase in large pre-B cells in the blood at 3–4 weeks of age; moreover spontaneous pre-B and B cell lymphomas reach an incidence of 50% at 15–20 weeks in hemizygous progeny of a wildtype female mated with a hemizygous male. We observed the development of Burkitt lymphoma within 10 weeks of birth in 14 out of 25 Eu-Myc transgenic mice and a premature death in 5 out or 25 transgenic mice within 6–8 weeks of birth without showing any enlarged lymph nodes. Transgenic mice with masses showed the same phenotype characterized by enlarged spleen (3 fold), lymphosarcomas associated with BL and enlarged lymph nodes around the neck area. B-cells have been negatively selected from enlarged lymph nodes and enlarged spleen. A qRT-PCR has been conducted to evaluate the miR-221 and miR-222 expression. The miRNA levels showed a down-regulation in B cells collected from the masses when compared to normal B cells derived from the spleen of WT mice.

In conclusion, our study reveals new insights into the functional significance in loss of miR-221 and miR-222 expression in BL pathogenesis.