Identification of putative targets of miR-22 that may mediate its effect on megakaryopoiesis. (A) Venn diagram showing the strategy for selecting putative targets of miR-22 from RNA-sequencing data. K562:scramble and K562:miR-22^KO were subjected to total RNA isolation, library preparation, and RNA-sequencing and analysis. To identify putative direct targets of miR-22, all targets needed to contain miR-22 seed sequences conserved in human and mouse; the yellow circle represents the top 30% of predicted TargetScan⁵¹  targets from the multimiR package (R). The blue circle represents genes upregulated in K562:miR-22^KO cells, as would be expected of direct targets of miR-22. Four putative miR-22 targets were identified. The 2 chosen for further study are outlined in red. (B) Schematic showing the locations of predicted miR-22 seed sequences in human and mouse in the 3′-UTRs of the genes chosen for further study. (C-D) qPCR to confirm dysregulation of putative miR-22 targets chosen for future study in K562:miR-22^KO cells (n = 3) (C). Expression is shown relative to scramble clone SCR3. (D) qPCR to confirm dysregulation of putative miR-22 targets chosen for future study miR-22 overexpressing cells. Expression is shown relative to p1501:Empty. GUSB was used as a housekeeping gene to quantify relative expression. **P < .01; ***P < .001. CDS, coding sequence.