Loss of expression of both miR-15/16 loci in CML transition to blast crisis

Abstract

Despite advances that have improved the treatment of chronic myeloid leukemia (CML) patients in chronic phase, the mechanisms of the transition from chronic phase CML to blast crisis (BC) are not fully understood. Considering the key role of miR-15/16 loci in the pathogenesis of myeloid and lymphocytic leukemia, here we aimed to correlate the expression of miR-15a/16 and miR-15b/16 to progression of CML from chronic phase to BC. We analyzed the expression of the two miR-15/16 clusters in 17 CML patients in chronic phase and 22 patients in BC and in 11 paired chronic phase and BC CML patients. BC CMLs show a significant reduction of the expression of miR-15a/-15b/16 compared to CMLs in chronic phase. Moreover, BC CMLs showed an overexpression of miR-15/16 direct targets such as Bmi-1, ROR1, and Bcl-2 compared to CMLs in chronic phase. This study highlights the loss of both miR-15/16 clusters as a potential oncogenic driver in the transition from chronic phase to BC in CML patients.

Keywords: blast crisis; chronic myeloid leukemia; miR-15/16 cluster.

Fig. 1.

MiR-15/16 cluster expression in CML patients. MicroRNAs expression by qRT-PCR in CD34+ healthy controls and CML patients’ cells in CP (n = 17) and in BC (n = 22). Box plots of miR-15a (statistical analysis for all groups Kruskal–Wallis P value = 0.0005431) (A), miR-15b (statistical analysis for all groups Kruskal–Wallis P value = 8.292 × 10⁻⁵) (B), miR-16 (statistical analysis for all groups Kruskal–Wallis P value = 0.0003215) (C). (D–F) MicroRNAs expression by qRT-PCR of paired CML patients’ cells (n = 11) in CP and BC. Bipartite graph of miR-15a (one-way Wilcoxon signed-rank P value = 0.021) (D), miR-15b (one-way Wilcoxon signed-rank P value = 0.02686) (E), miR-16 (one-way Wilcoxon signed-rank P value = 0.01611) (F). n.s., not significant. *, 0.01< P value ≤ 0.05; **, 0.001 < P value ≤ 0.01; ***, 0.0001 < P value ≤ 0.001; ****P value ≤ 0.0001.

Fig. 2.

MiR-15/16 targets expression in CML patients. (A) Immunoblotting of ROR1, Bmi-1, and Bcl-2 performed on five CML patients’ cells in CP and five CMLs cells in BC. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalizer. (B) Densitometry relative quantification of ROR1, Bmi-1, and Bcl-2 expression respect to GAPDH loading control. CML patients’ cells in CP and in BC were grouped. One-way Wilcoxon rank-sum test was applied. (C and D) Gene expression by qRT-PCR in CML patients’ cells in CP (n = 17), in BC (n = 20) and CD34+ cells from healthy donors (n = 4). Box plots expression of BMI1 (statistical analysis for all groups Kruskal–Wallis P value = 0.00027) (C) and BCL2 (statistical analysis for all groups Kruskal–Wallis P value = 4.37 × 10⁻⁵) (D). (E) Western blot analysis of ROR1, Bmi-1, and Bcl-2 performed on three paired CML patients’ cells in CP, BC, and CD34+ from two healthy donors. β-actin was used as a normalizer. *, 0.01 < P value ≤ 0.05; **, 0.001 < P value ≤ 0.01; ***, 0.0001 < P value ≤ 0.001; ****P value ≤ 0.0001.