Microrna Mir-21 Is Upregulated after Different Microenvironmental Stimuli and Controls Proliferation, Chemotaxis and Chemoresistance in Chronic Lymphocytic Leukemia
Authors: Crespo Marta , Carabia Júlia , Purroy Noelia , Carpio Cecilia , Abrisqueta Pau , Palacio Carles , Bosch Francesc 
Published: October 2019

Abstract
Aberrant expression of diverse microRNAs (miRNAs) has been related to pathogenesis and clinical outcome in patients with CLL. miR-21 is overexpressed in a wide variety of neoplasms where it participates in oncogenic events such as proliferation, resistance to treatment and metastasis. In CLL, miR-21 expression has been associated to refractoriness to fludarabine and to shorter overall survival. In addition, high expression of ZAP-70 protein in CLL is related to shorter overall survival and higher probability of progression. Several biological mechanisms have been described explaining the adverse prognosis associated with high ZAP-70 expression. In this sense, ZAP-70 protein increases the capability of CLL cells to respond to different survival and migration signals provided by the microenvironment. In a previous work, we found that transfected B-cell lines with a ZAP-70 expressing vector (Calpe at al, Blood 2011) had an increased expression of several molecules, including miR-21. Against this background, we studied the relationship between ZAP-70 protein and miR-21. For this, we firstly 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) in conditions mimicking the microenvironment in the proliferation centers (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 compared to cells in suspension. Interestingly, this increase was only significant in patients with high expression of ZAP-70 (N=8; p= 0.0379). To define the role of ZAP-70 in miR-21 regulation, we stably transfected Ramos B-cells with ZAP-70 protein and found that both MAPK and STAT3 participate in the induction of miR-21 expression after ZAP-70 activation upon BCR crosslinking. Moreover, using this system we observed downregulation of the tumor suppressors PTEN, PDCD4 and PIAS3, all of which have been found to be targeted by miR-21 in malignant cells. Next, we aimed to study the functional role of miR-21 in primary CLL cells co-cultured in conditions mimicking the microenvironment from the proliferation centers. For this, we analyzed survival, proliferation and response to fludarabine after inhibition of miR-21 expression by transfecting primary CLL cells with antisense miR-21 inhibitor. The co-culture of primary CLL cells significantly increased their survival after 48 hours regardless the inhibition of miR-21. However, the induction of proliferation (measured as percentage of Ki-67 positive cells) was significantly inhibited by the suppression of miR-21 (N=13; p=0.022). Next, the analysis of a small pilot cohort showed a consistent but not yet significant overcoming of the chemoresistance induced by the co-culture after the inhibition of miR-21 Interestingly, the sensitivity to fludarabine of primary CLL cells cultured in suspension was also increased by the inhibition of miR-21. Finally, we examined the ability of primary CLL cells to migrate towards CXCL12 and observed that inhibition of miR-21 resulted in a 2.62-fold reduction of the migration index (ratio of cells migrating towards culture media to cells migrating towards media with 200ng/mL CXCL12). In conclusion, we have showed the correlation and participation of ZAP-70 protein in the regulation of miR-21. We have also observed that miR-21 contributes to CLL proliferation, resistance to fludarabine, and chemotaxis towards CXCL12. Although further experiments are warranted in order to fully elucidate the regulation and functional role of miR-21 in CLL, these results help to enlighten the biology behind the adverse clinical outcome of patients with CLL and high expression of ZAP-70, and could potentially be exploited for the development of new treatments in a near future.

Disclosures
Bosch: Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.