The regulation, targets, and prognostic significance of miR-650. (A) Genomic organization of the IgLλ locus (chr. 22q11) and the relation of IGVL gene and J-C cluster to miR-650 (modified from Das¹⁰ ). All members of family 2 of variable (V) subgenes (V2-8, V2-14, V2-28, V2-11, V2-5, V-34, V2-33, V2-18, V2-23) for λ light chain include homologs of miR-650 (here shown in detail for V2-8). The miR-650 overlaps with the first exon of IGVL. The position of hsa-miR-650 gene (MI0003665), leader exon of IGVL, V-exon, J-C cluster, TATA box, and octamer are indicated. (B) The relation of IgL usage and the expression of miR-650. The expression of miR-650 was analyzed (TaqMan assay for mature miR-650) in CLL samples using V2 subgenes for IgLλ (n = 14: V2-14 [8×], V2-8 [2×], V2-23 [2×], V2-5 [1×], V2-18 [1×]) and compared with CLL samples using different IgLλ V family (n = 13: V3-21 [5×], V1-51 [2×], V1-44 [2×], V3-27 [1×], V3-19 [1×], V5-45 [1×], V1-96 [1×]) or expressing κ IgL (n = 20). The IgL usage (kappa vs lambda) determined by sequencing (BIOMED-2 protocol) was verified by flow cytometry and in all cases, but one biclonal case, the surface IgL expression corresponded to the PCR result. Statistical differences were tested by 2-tailed t test. Error bars represent SEM. (C-D) Survival analysis and the time to first treatment (TTFT) analysis in 80 CLL patients. Patients were divided in 2 groups (low miR-650, n = 40; high miR-650, n = 40) based on the median of miR-650 expression (median expression was used as a cut-off). Cohort characteristics are included in Table 1. Survival and TTFT analysis were done using the Kaplan-Meier survival estimator. Median survival, median TTFT, and differences between the curves were evaluated by the Log-rank test. (E) The effect of miR-650 on cell proliferation. NALM-6 cells were electroporated with a short artificial miR-650 (MIMIC miR-650) or a control short RNA (NEG. CTRL) and plated in fresh cultivation media. Cell numbers were calculated 24 hours, 48 hours, 72 hours, and 96 hours after transfection by direct cell count (Countess Automatic Cell Counter; Invitrogen). Five independent experiments were performed and statistically analyzed by 2-way ANOVA. NALM-6 cells were transfected with the efficiency of ∼ 75% (supplemental Figure 1). MOCK stands for cells treated only with the electroporation puls. Error bars represent SEM. (F) The identification of miR-650 targets. B-cell line NALM-6 was electroporated with a short RNA mimicking miR-650 (miR-650 MIMIC), or control short RNA (NEG. CTRL) and harvested 48 hours and 72 hours after transfection. NALM-6 cells were transfected with the efficiency of ∼ 75% (supplemental Figure 1). For Western blot analysis mouse-monoclonal antibodies against EBF3, ING4, CDK1 and α-tubulin (as a loading control) were used. A representative example from at least 3 transfection experiments is shown. MOCK stands for cells treated only with the electroporation puls. (G) The quantification of EBF3, CDK1, and ING4 protein levels after transfection with RNA mimicking miR-650 (harvested 72 hours after transfection). Transfection experiments were performed as at least 3 independent experiments (as described in panel F). Blot images were quantified with ImageJ 1.42q (National Institutes of Health), and the quantity of proteins is visualized as Target protein/Tubulin ratio and normalized to MOCK. miR-650 MIMIC (short RNA mimicking miR-650), NEG. CTRL (control short RNA), MOCK (cells treated only with the electroporation puls). *P < .05 (2-tailed t test). Error bars represent SEM.