Fig. 4

USP15 as a direct target of miR-202-5p. (a) Heat map showing the differential expression (fold changes) of miRNA between K562 and K562G cells from RNA-seq analysis. (b) Venn diagram performed that miRNA upregulation in K562G (green panel) cells overlapped the predicted miRNA ((http://www.targetscan.org/vert_72/) (red panel) which may target USP15. (c) K562 cells were transfected with miR-202-5p mimic, mimic-NC, miR-202-5p inhibitor or inhibitor-NC, respectively. Protein level of USP15 was measured by Western blot analysis. (d) Potential binding site of miR-202-5p at the 3′ UTR of USP15. (e) K562 cells co-transfected cells with miR-202-5p mimic and wild-type (WT) or mutant (mut) USP15 3′-UTR-luciferase reporter. Luciferase reporter assays were used to detect luciferase activity. **P < 0.01 vs. mimic-NC. (f) USP15 3′-UTR and control RNA with biotin-labeled uridine triphosphate were transfected into K562 cells for 24 h. The miRNAs were extracted after a pull-down assay, and miR-202-5p expression was detected by qRT-PCR. Multiple miRNAs could be pulled down by RBM5 3′-UTR. **P < 0.01 vs. control RNA. (g) qRT-PCR was used to test miR-202-5p level in CML cell lines (K562 and KCL22) and PBMCs of healthy donors. Normalized to U6. ***P < 0.001 vs. normal (h) qRT-PCR detected the expression of miR-202-5p expression in PBMCs of CML-CP (n = 30) patients compared with PBMCs of healthy honors (n = 30). **P < 0.01 vs. normal. (i) Fluorescence in situ hybridization (FISH) detected the miR-202-5p in CML cell lines and PBMCs of healthy donors. Blue staining represents the nucleus and red staining indicates miR-202-5p. Scale bar = 20 μm. (j) FISH detected the miR-202-5p in PBMCs of CML-CP patients and PBMCs of healthy donors. Scale bar = 20 μm