The MicroRNA (miR) Profile in B-Cell Chronic Lymphocytic Leukemia (CLL) Reveals a Differential Expression of miR-21, miR-155 and miR-150 between Leukemic and Normal B Lymphocytes, and of miR-150, miR-29bc and miR-223 between IgVH Mutated and Unmutated Patients.
Authors: Chiaretti Sabina , Fulci Valerio , Goldoni Marina , Azzalin Gianluca , Tavolaro Simona , Messina Monica , Maggio Roberta , Peragine Nadia , Miss Simona Santangelo , Mauro Francesca R. , Guarini Anna , Macino Giuseppe , Foa Robert 
Published: October 2019

Abstract
Background. CLL is the most common leukemia in the western world and is characterized by the accumulation of relatively mature B cells. From a clinical point of view, CLL patients display a variable course. This clinical heterogeneity is sustained by different biologic parameters, such as the immunoglobulin variable gene (IgVH) mutational status, ZAP-70 expression and specific cytogenetic alterations. Even though extensive molecular characterizations of CLL cells have been performed, a candidate gene responsible for the disease is still lacking. Thus, there is a compelling need to identify other mechanisms that may play a role in CLL. miRs are an abundant class of small non-coding RNAs which modulate the expression of their target mRNAs at the post-transcriptional and/or translational level. So far, 462 miRs have been identified in the human genome: many are involved in cancer, acting either as oncogenes or tumor suppressors.
Methods. In order to define the potential role of miRs in CLL, the expression of these small RNAs was evaluated by cloning, as well as by quantitative Real-Time PCR (qRT-PCR) in a total of 56 CLL patients: 9 cases were evaluated by cloning, 46 by qRT-PCR and 4 by both methods; peripheral blood B lymphocytes and cord blood cells enriched for CD19+ were used as controls.
Results. By cloning, it was possible to identify roughly 20 miRs that contribute to more than 90% of the expressed miRs in CLL and healthy donors. Furthermore, we observed a significant upregulation (at least 3 fold) of miR-21, miR-155 and miR-150 in CLL patients compared to healthy donors. To validate these findings, we next measured the expression of 22 of the most expressed miRNAs in 51 CLL patients and in 7 healthy donors (5 CD19+ peripheral blood lymphocytes and 2 CD5+ cord blood cells) by qRT-PCR: this approach confirmed the differential expression of miR-21, miR-155 and miR-150 expression between leukemic and normal cells, and identified 2 additional miRs, namely miR-92 and miR-222, that are downregulated in the controls. Unsupervised clustering based on qRT-PCR results allowed to subdivide the cases analyzed into 3 major subgroups: the first comprised all the 5 controls, the second a set of patients that had a strong downregulation of miR-15 and miR-16, and the third was inclusive of all the remaining cases. It is of worth noting that the patients who showed a downregulation of miR-15 and miR-16 had a del13q14 in homozygosis, in line with previous reports. Finally, a supervised approach highlighted a differential expression of miR-150, miR-29bc and miR-223 between IgVH mutated and unmutated; consistently with this finding, miR-150 was also differentially expressed between ZAP-70+ and ZAP-70− cases.
Conclusions. Our study indicates that 3 miRs are strongly and homogeneously upregulated in CLL patients, suggesting that they may play an important role in disease initiation and/or progression. Finally, a small set of miRs is distinctive of prognostic subgroups. Further investigations on these small RNAs is ongoing, to specifically define their role in CLL.