Identification of miR-486-5p target genes in CB CD34⁺ cells. (A) Strategy for identification of miR-486-5p targets in CBCD34⁺ cells. (B) Effect of miR-486-5p expression on luciferase activity in 293T cells transfected with reporter plasmids containing miR-486-5p binding sequences in 3′ UTRs of potential target genes. The data show inhibition of luciferase activity in cells expressing miR-486-5p compared with control vectors. (C) Gene expression in anti-miR-486-5p compared with scramble sequence–expressing cells determined by qPCR. (D) CB CD34⁺ cells were transduced with miRZip-anti-486-5p and scramble vector and cultured in GEMM medium for 3 days. FoxO1, PTEN, p-AKT, and AKT expression was detected by western blotting. (E) TF1 cells were transduced with pHIV7-EF1-miR-486or control vector. FoxO1, PTEN, p-AKT, and AKT expression was detected by western blotting. (F-G) CB CD34⁺ cells were cotransduced with miRZip-scramble-GFP or miRZip-anti-486-5p vectors (expressing GFP) and pLKO.1-FoxO1 shRNA and pLKO.1-scramble control vectors (expressing RFP). The CD34⁺RFP⁺GFP⁺ cells were sorted and cultured in GEMM medium for 3 days. The number of annexin V⁺ cells (F) and total cell numbers (G) were determined (n = 3). (H) CD34⁺ cells were transfected with siRNAs to potential miR-486-5p target genes, cultured in GEMM medium for 3 days and GPA⁺ cells detected by FACS. The percentage of GPA⁺ cells compared with control siRNA were determined (n = 3). Cumulative results represent the mean ± SEM. *P < .05, **P < .01, ***P < .001.