MiR-130b and miR-128a are upregulated in patient blasts and have tumorigenic functions in MLL-AF4⁺ B-ALL cells. (A) Experimental approach and sorting strategy of BM cells from patients. (B) Differential expression analysis of miRNAs in blasts at diagnosis (CD19⁺CD10⁻NG2⁺CD34⁺, CD19⁺CD10⁻NG2⁻CD34⁺ and CD19⁺CD10⁻CD34⁻) vs nonblasts at remission (CD19⁻CD10⁻). Data were compared using a limma test. Expression of (C) miR-130b and (D) miR-128a in patients’ leukemic blasts (diagnosis), nonblasts (remission), and human FL (huFL) hematopoietic cells (10-20 weeks postconception [wpc]). (E) ATAC-Seq profiles of human FL LMPP derived from 10 to 20 wpc (n = 2) and SEM cells (GSE117865) alongside FLAG-MLL-Af4 chromatin immunoprecipitation (ChIP) sequencing (ChIP-Seq) profiles of miR-130b and miR-128a loci in MLL-Af4 human leukemic blasts (GSE84116). The gray box highlights the promoter that regulates each miRNA. Arrows indicate an open chromatin region or a chromatin region with MLL-AF4 binding. Expression of MLL-AF4, MEIS1, HOXA9, CDK6, BCL2, miR-130b, and miR-128a in SEM cells transfected with siRNA against MLL-AF4 (siMA6) after (F) 48 hours and (G) 72 hours. The log2-fold change (LOG2FC) is calculated using SEM sicontrol as a reference. (H) Proliferation after 72 hours in culture (initial cell concentration, 10⁵ cells per milliliter) and (I) apoptosis assay in SEM cells when the activity of miR-130b and/or miR-128a is inhibited (pmiRZip-130b/128a). pmiRZip-scramble was used as a control. (J) Survival curve of SEM pmiRZip-scramble–, pmiRZip-130b– and pmiRZip-128a–engrafted NSG recipients. MiRNA inhibition in leukemic cells was achieved through lentiviral transduction and monitored with GFP. Unless stated otherwise, data were compared using a Mann-Whitney U test: *P < .05; **P < .01; ***P < .001; ****P < .0001. Experiments were conducted in triplicate or more. Graphs are presented as mean ± standard error of the mean. NS, not significant; snRNA, small nuclear RNA.