American Society of Hematology

Figure 1.

$Figure 1. An miR signature targets BTK in CLL. (A) Putative binding sites of 6 miRs in the 3′UTR of BTK. (B) Primary CLL cells were transiently transfected with scrambled (Scr) oligonucleotides or miR-147b, miR-210, miR-425, miR-1253, miR-4269, or miR-4667-3p for 48 hours and immunoblotted for BTK and GAPDH. The figure is representative of 7 to 10 independent CLL samples which are quantitated in (C) (P < .05 for miR-210 and miR-425, Wilcoxon signed rank test). (D) Mec2 cells transfected with mimics or inhibitors of miR-210 and miR-425 and analyzed for the levels of BTK and GAPDH (P < .05, one-sample Student t test; two-tailed P). (E-F) Reporter gene analyses using BTK 3′UTR constructs. Mec1 cells were transfected with luciferase reporter constructs expressing wt BTK or BTK with the binding sites for miR-147, miR-210, miR-425, miR-1253, miR-4269, and miR-4667-3p mutated. Cells were then transfected with an irrelevant miR (miR-181) or miR-147b, miR-210, miR-425, miR-1253, miR-4269, or miR-4667-3p, and the luciferase counts were quantitated. Data represent mean ± standard error of the mean (SEM) of 12 independent transfections (P < .05 for miR-4269 and P < .001 for miR-147b, miR-210, miR-425, miR-1253, and miR-4667-3p; paired Student t test). (G) Expression analysis of miR-210, miR-425, miR-1253, miR-4269, and miR-4667-3p in 83 CLL samples expressed as a fraction of the levels for these miRs from CD19+CD5+ B cells from healthy donors (set at 1) (P < .01 average reduction across all miRs, mixed effects model). *P ≤ .05; **P ≤ .01; ***P ≤ .001; **** P ≤ .0001.$

An miR signature targets BTK in CLL. (A) Putative binding sites of 6 miRs in the 3′UTR of BTK. (B) Primary CLL cells were transiently transfected with scrambled (Scr) oligonucleotides or miR-147b, miR-210, miR-425, miR-1253, miR-4269, or miR-4667-3p for 48 hours and immunoblotted for BTK and GAPDH. The figure is representative of 7 to 10 independent CLL samples which are quantitated in (C) (P < .05 for miR-210 and miR-425, Wilcoxon signed rank test). (D) Mec2 cells transfected with mimics or inhibitors of miR-210 and miR-425 and analyzed for the levels of BTK and GAPDH (P < .05, one-sample Student t test; two-tailed P). (E-F) Reporter gene analyses using BTK 3′UTR constructs. Mec1 cells were transfected with luciferase reporter constructs expressing wt BTK or BTK with the binding sites for miR-147, miR-210, miR-425, miR-1253, miR-4269, and miR-4667-3p mutated. Cells were then transfected with an irrelevant miR (miR-181) or miR-147b, miR-210, miR-425, miR-1253, miR-4269, or miR-4667-3p, and the luciferase counts were quantitated. Data represent mean ± standard error of the mean (SEM) of 12 independent transfections (P < .05 for miR-4269 and P < .001 for miR-147b, miR-210, miR-425, miR-1253, and miR-4667-3p; paired Student t test). (G) Expression analysis of miR-210, miR-425, miR-1253, miR-4269, and miR-4667-3p in 83 CLL samples expressed as a fraction of the levels for these miRs from CD19⁺CD5⁺ B cells from healthy donors (set at 1) (P < .01 average reduction across all miRs, mixed effects model). *P ≤ .05; **P ≤ .01; ***P ≤ .001; **** P ≤ .0001.

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