Figure 5

Knockdown miR-33b promotes the self-renewal of MCF-10A cells.

(A) qRT-PCR analysis of the knockdown efficiency of miR-33b in MCF-10A cells. (B) Representative images of the mammospheres formed by MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b cells. (C) Quantification of primary, secondary and tertiary mammospheres formed by MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b cells. (D) A limiting dilution assay was performed on the MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b from 1 to 1,000 cells per well. The numbers of mammospheres were scored at the end of 7 days. (E) FACS analysis of the CD44⁺/CD24⁻ stem cell subpopulation in MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b cells. (F) qRT-PCR analysis of the mRNA expression of stemness-related genes Bmi-1, Nanog, Oct4 and Sox2 as well as the downstream target genes of miR-33b, HMGA2, SALL4 and Twist1 in MCF-10A/sh-ctrl, MCF-10A/sh-pre-miR-33b and MCF-10A/sh-miR-33b cells. (G) Western blot analysis of the protein expression of stemness-related genes Bmi-1, Nanog, Oct4 and Sox2 as well as the downstream target genes of miR-33b, HMGA2, SALL4 and Twist1. The full-length blots were presented in the Supplementary Figure 10. Scale bars, 100 μm. Data represent the mean ± s.d. *: P <0.05, **: P <0.01, ***: P <0.001, N.D.: not detected.