miR-22 expression increases upon terminal MK maturation and drives the maturation process. (A) Bone marrow from individual wildtype 129SV mice was stained according to surface markers listed in supplemental Table 3 and DAPI for live/dead assessment. Common myeloid progenitor (CMP), MEP, and MK were sorted by FACS for analysis of miR-22-3p expression by qPCR. sno202 was used as a housekeeping gene to quantify relative expression. Expression is shown relative to CMP, with the dotted line showing Relative Expression = 1. Data represent 2 independent experiments, performed in triplicate. (B) Representative qPCR for miR-22 in K562 cells treated with 15 nM PMA over time. K562 cells are driven to megakaryocytic differentiation. sno202 was used as a housekeeping gene to quantify relative expression. Expression is shown relative to K562 treated with vehicle (Veh; DMSO) at 24 hours. For the statistical analysis, the time points were treated as replicates. (C) miR-22 overexpression promotes MK maturation. K562 cells were transiently transfected with an miR-22 overexpression vector (PIG/miR-22⁴² ) or a control (PIG/empty) and were subjected to PMA-driven megakaryocytic differentiation. GFP expression was used as a correlate for miR-22 overexpression and gated from I, no miR-22 overexpression, to IV, highest miR-22 overexpression (upper). Quantitation of percentages of CD61⁺ cells upon differentiation with 75 nM PMA or vehicle treatment and escalating empty vector or miR-22 expression (lower). (D) Utilizing CRISPR/Cas9 to knockout the miR-22 encoding stem loop from the MIR22HG in K562 cells. Schematic of the human MIR22HG on chromosome 17, specifically exon 2, which encodes the miR-22 stem loop (upper). Predicted schematics before and after locus excision and repair are shown, as well as gRNAs and genotyping primers. Agarose genotyping gel shows isolation of 6 clones each of scramble (ie, wildtype), miR-22 heterozygous, and miR-22 knockout (lower). Excision is identified by appearance of the truncated ∼480-bp band (external primers) and loss of the ∼360-bp band (internal primers). (E) qPCR for miR-22 expression in wildtype (ie, Scramble), miR-22 heterozygous, and miR-22 knockout K562 clones. sno202 was used as a housekeeping gene to quantify relative expression. Expression is shown relative to Scramble (n = 6). (F-G) K562:miR-22^KO clones were subjected to PMA-driven megakaryocytic differentiation and assayed for differentiation by CD61 expression and nuclear content (ie, increased ploidy). (F) PMA-driven megakaryocytic differentiation over 48 hours in K562:miR-22^KO clones was assessed by flow cytometry for CD61 expression and reported as fold change in median CD61 expression normalized per clone (n = 3). (G) PMA-driven megakaryocytic differentiation over 72 hours in K562:miR-22^KO clones. Frequency of high-ploidy cells was assessed by flow cytometry. Gating strategy for identifying high-ploidy cells is shown (left) and is quantified in K562:Scramble and K562:miR-22^KO clones (right) (n = 5). GFP, green fluorescent protein; SSC-A, side-scatter area. *P ≤ .05; **P < .01; ****P < .0001.