Overexpression of miR-126 exhibited a more potent effect than knockout of miR-126 on long-term LSC/LIC self-renewal and frequency. (A) The tertiary BMT recipients were transplanted with leukemic BM cells isolated from the secondary BMT recipient mice of the AE9a, AE9a+miR-126, and miR-126KO+AE9a groups. Kaplan-Meier curves and P values (log-rank test) are shown. (B) Expression of miR-126 was detected by qRT-PCR assay in leukemic BM cells (CD45.2⁺) of tertiary BMT recipients. **P < .01. Survival curves (C) and relative miR-126 expression levels (D) of the 3 groups in quaternary BMT are shown. (E) Mouse BM leukemic cells from secondary BMT recipient mice were used as donor cells for BMT in the limiting dilution assays. The estimated LSC/LIC frequencies of the AE9a, AE9a+miR-126, and miR-126KO+AE9a groups are 1/166 619 (95% CI, 1/529 517-1/52 429), 1/2476 (95% CI, 1/7237-1/847), and 1/27 399 (95% CI, 1/66 212-1/11 338), respectively. Significance of the frequency difference: AE9a vs AE9a+miR-126, P = 5.35 × 10⁻⁹ (χ²-test); AE9a vs miR-126KO+AE9a, P = .0092; AE9a+miR-126 vs miR-126KO+AE9a, P =.00072. (F) Mouse spleen leukemic cells from secondary BMT recipient mice were used as donor cells. The estimated LSC/LIC frequency of the AE9a+miR-126 group is 1/95 040 (95% CI, 1/215 902-1/41 837), significantly greater (P = .00088) than that of the miR-126KO+AE9a group at 1/653 224 (95% CI, 1/1 380 618-1/309 066). CI, confidence interval; Dose, number of donor cells; Tested, total number of mice used as BMT recipients in the limiting dilution assay; Response, mice that developed leukemia within 15 weeks post BMT.