Regulation Of Microrna Mir-21 Expression By ZAP-70 Protein In Chronic Lymphocytic Leukemia

Aberrant expression of diverse microRNAs (miRNAs) has been related to pathogenesis and clinical outcome in patients with CLL. In this regard, miR-21 expression has been associated to refractoriness to fludarabine and to shorter overall survival. MicroRNA miR-21 has been found to be overexpressed in a wide variety of neoplasms where it participates in oncogenic events such as proliferation, resistance to treatment, and metastasis. In CLL, high expression of ZAP-70 protein is related to shorter overall survival and higher probability of progression. Moreover, ZAP-70 protein participates in the signaling of the B cell receptor (BCR) and different chemokine receptors, therefore increasing the capability of CLL cells to respond to survival and migration signals provided by the microenvironment. In order to further elucidate the molecular mechanisms defining bad prognosis CLL we studied the relationship between ZAP-70 protein and miR-21. Firstly, we analyzed the correlation of ZAP-70 with miR-21 in primary CLL cells from 32 patients. In this series we observed that miR-21 expression analyzed by QRT-PCR was significantly higher in patients with high expression of ZAP-70 compared to patients with low expression of ZAP-70 (mean miR-21 in high ZAP-70 (N=17) = 5.781 ± 1.517; mean miR-21 in low ZAP-70 (N=15) = 0.6783 ± 0.2730; p=0.0082). In order to further analyze the molecular mechanisms regulating the induction of miR-21 and the potential role of ZAP-70 protein in this process, we co-cultured primary CLL cells (N=16) with bone marrow stromal cells with concomitant stimulation of CD40 and TLR9. In these conditions, besides ZAP-70 activation, we observed a 3.6 ± 0.78 mean fold induction in miR-21 expression after 48 hours of co-culture compared to cells in suspension. Interestingly, when we independently analyzed patients with low or high expression of ZAP-70 (N=8 for each group) we observed that this increase was only significant in patients with high expression of ZAP-70 (p= 0.0379). We therefore decided to specifically study the role of ZAP-70 in the regulation of miR-21. For this, we stably transfected Ramos cells with a vector expressing a ZAP-70-GFP fusion protein or GFP only as a control. The stimulation of the BCR in these cells caused the activation of ZAP-70 protein and the subsequent activation of the MAPK and AKT pathways, as evidenced by western blot analysis. Interestingly, BCR stimulation for 12 hours in Ramos-GFP cells significantly induced the expression miR-21 (19.55 ± 1.49 vs. 65.03 ±12.98; 3.32 fold change) whereas in those cells transfected with ZAP-70 the increase was greater (13.26 ± 2.13 vs. 138.77 ± 11.72; 10.4 fold change), suggesting that ZAP-70 is directly participating in the pathway leading to miR-21 upregulation. In addition, only after 4 hours of BCR stimulation we already observed upregulation of primary-miR21, specially in cells with expression of ZAP-70 (2.1 fold in Ramos GFP vs. 7.3 fold in Ramos GFP-ZAP-70), indicating that the regulation of miR-21 is at the transcriptional level. Furthermore, inhibition of the MAPK pathway, but not of AKT, impaired the upregulation of miR-21 in both cell lines. STAT-3 transcription factor has been shown to participate in the induction of miR-21 expression in different cell types. In this system, we observed that BCR stimulation induced the activation of STAT-3 and that its biochemical inhibition impaired the upregulation of miR-21, indicating that STAT-3 is probably participating in miR-21 induction in malignant B-lymphocytes. Finally, we analyzed the downregulation of potential miR-21 targets. For this, we studied the downregulation of the tumor suppressors PTEN, PDCD4 and PIAS3, all of which have been found to be regulated by miR-21 in malignant cells. We observed that, after 48 hours of BCR stimulation, the protein levels of all three genes were downregulated, indicating that these proteins may be targeted by miR-21 in this cellular system. In conclusion, we have showed the participation of ZAP-70 protein in the regulation of miR-21, an oncomiR that has been related to short survival and resistance to treatment with fludarabine in CLL. Although further experiments are warranted in order to fully elucidate the role of ZAP-70 in miR-21 induction in CLL, these results enlighten the biology behind the adverse clinical outcome of patients with CLL and high expression of ZAP-70, and can potentially be exploited for the development of new treatments.