Expression of miR-29a and miR-142-3p in AML BM blasts and their function in promoting myelopoiesis. (A) Expression levels of miR-29a and miR-142-3p were determined by real-time PCR analysis of BM CD34⁺ cells from AML patients and healthy donors using TaqMan probes. The differences were shown to be significant by one-tailed unpaired Mann-Whitney U nonparametric t test analysis. (B-E) BM CD34⁺ cells from 3 AML patients (patients 11, 5, and 6 were diagnosed as French-American-British M5, M2, and M4, respectively; all demonstrated low expression levels of miR-29a and miR-142-3p; supplemental Table 1) were infected with Lenti-miR-29a, Lenti-miR-142-3p, or Lenti-control. After infection for 24 hours, the cells were cultured in monocytic or granulocytic induction medium for the indicated number of days, after which the cells were collected and the GFP-positive cells were analyzed for myeloid marker expression. The expression levels of CD14 (B) and CD11b (C) in lentivirus-infected blast cells (#11 and #5 of supplemental Table 1) were analyzed by FACS analysis compared with their expression levels in CD34⁺ BM cells from normal donors (N13 and N12). Red histogram represents unstained CD34⁺ cells; and black histogram, cells induced for the indicated number of days. The monocytic (D) and granulocytic (E) differentiation of CD34⁺ cells from AML patient 6 were determined by FACS analysis. (F) Western blot analysis of the target proteins, CCNT2, CDK6, and TAB2. The protein extracts were obtained from the cells at day 6 of granulocytic and monocytic induction cultures of BM CD34⁺ cells derived from AML 6, and infected with Lenti-29a, Lenti-142-3p, or Lenti-control. GAPDH was detected as the loading control.