Regulation of Jak2 Kinase Gene by the Mir-135 Family in Classical Hodgkin Lymphoma

Alterations of JAK/STAT signaling molecules have been reported in several lymphomas, including classical Hodgkin lymphoma (cHL). Constitutive activation of the JAK-STAT pathway is frequent in cancer and contributes to oncogenesis, with Jak kinase overexpression promoting in vitro cell transformation. JAk2 is a cytoplasmic tyrosine kinase involved in a specific subset of cytokine receptor signaling pathways. Activating mutation has been found in myeloid neoplasms but the expression of JAK2 in primary mediastinal large B-cell lymphomas and classical Hodgkin lymphomas is not due to mutations. On the other hand, miRNAs play an important role in cHL, where they regulate the expression of genes that are crucial for the transformation process and to rescue Hodgkin/Reed Sternberg cells from apoptosis. We have previously shown that miR-135a, a putative miRNA involved in JAK2 expression, is present in the miRNA signature of cHL. The aims of the study were to analyze the role of miR-135a in Hodgkin cells and to determine if Jak2 is a target gene of this miRNA. Moreover, we examined whether the expression of miR-135a in diagnostic samples predicted clinical outcome in cHL. To that aim, 50nM and 100nM of pre-miR-135 and pre-miRNA precursor Negative Control (Ambion) were transfected in the L-428 cHL cell line using nucleofection (AMAXA, Solution L and program X-001). MiR-135a levels were analyzed by stem-loop-RT-PCR and Real-time (TaqMan MicroRNA Assays, Applied Biosystems) in a 7500 Sequence Detection system. Apoptosis levels were assayed by Caspase-Glo 3/7 luminescent assay that measures caspase-3 and -7 activities. Jak2 protein levels were analyzed by Western Blot (Abcam moAb). Eighty-nine adult patients (median age, 29 yrs [range, 13–89]; males 47%) diagnosed with cHL at a single institution between September 1995 and June 2005 were studied. Seven patients (7.8%) were HIV+. Histological subtypes: nodular sclerosis (79%) and mixed cellularity (21%). Epstein-Barr Virus (EBV) was present in 28% of the samples. First-line treatment consisted of ABVD type therapies. Total RNA was extracted from formalin-fixed paraffin-embedded lymph nodes using RecoverAll Total Nucleic Acid Isolation (Ambion). Statistical analysis was performed with SPSS 14.0. Clinical outcomes analyzed were relapse rate and disease-free survival (DFS). Of 89 patients, 75 (84.3%) achieved CR, 7 (7.8%) PR, while 7 (7.8%) were chemoresistant. After a median follow-up of 43 months (range, 1–128), overall survival was 83% and DFS 74%. Results showed that miR-135a was underexpressed in the cHL L-428 cell line and in cHL samples compared to reactive lymph nodes. After pre-miR-135 transfection, the miR-135a levels in L-428 cells increased 12 Ct at 24h. Analysis at 24 and 48 hours post-transfection showed a proportion of apoptotic cells 20% and 30% higher than in control cells (pre-miRNA precursor Negative control). The analysis of Jak2 protein levels at 48 and 72 hours post-transfection showed a dose-dependent reduction of the protein expression of 28%(100nM-48h) and 58%(100nM-72h). The proliferation assay showed that pre-miR-135-transfected cells grew less than control cells transfected with pre-miRNA precursor negative control. All these results are along the same line as those found in primary tumor. Patients could be divided into two groups according to miR-135a expression: high and low expression. Patients with low miR-135a expression had a higher probability of relapse than those with high miR-135a expression (p=0.045). In the multivariate analysis, only miR-135a expression emerged as a prognostic factor for relapse (RR=6.533, p=0.021). In conclusion, miR-135a downregulation seems to play a role in the rescue of Hodgkin/Reed Sternberg cells from apoptosis and influence the transformation event through the regulation of Jak2 levels. In addition, expression levels of miR-135a may be a prognostic marker for risk of relapse in cHL patients.