FIG. 2.
Gain of function of the miR-106b family promotes cell cycle progression; knockdown reverses the phenotype. (A) miR-106b promotes cell division. A growth curve measuring cell numbers following transfection of a control duplex or miR-106b into HMECs shows that miR-106b promotes cell division. Error bars represent coefficients of variation. (B) miR-106b and miR-106a gain of function led to an increase in S-phase cells. HMECs were transfected with the indicated microRNA or control duplex (luciferase). BrdU incorporation was analyzed using flow cytometry. Shown are scatter plots of fluorescence intensities of BrdU incorporation (y axis) against DNA content (x axis). Magenta gates capture the S-phase populations (positive for BrdU incorporation), and the numbers depict percentages of cells in S phase. 7-AAD, 7-amino-actinomycin D. (C) The miR-106b family is required for the G1-to-S transition. HMECs were transfected with control duplex (luciferase, top left), microRNAs (middle), or anti-miRs (bottom) and treated with nocodazole for 16 h. “miR-106b mutant” has mutations in positions 2 and 3 of the seed region (top right). Cell cycle profiles were analyzed using flow cytometry. Shown are histograms of cell numbers (y axis) against DNA content (determined by measuring fluorescence intensity; x axis).