Fig. 4. Let-7 miRNAs inhibit invasion of invasive breast cancer cells by reducing MZF1 expression.

a Invasion of MCF7-p95ΔNErbB2 cells in 3D Matrigel upon transfection with 20 nM control, let-7d, let-7e, and let-7g mimic and MZF1 siRNA. Images of the invasive spheres were taken at 10× magnification. Quantification of the average extend of outgrowth in (a) was done using ImageJ. For each of 5 spheres per treatment, the 10 greatest distances traveled by invading cells were estimated and a mean outgrowth was calculated for each treatment. Data are presented as % of control mimic and the effect of let-7e and MZF1kd is assigned with *, **, and *** indicating p < 0.05, p < 0.01, and p < 0.001, respectively, in a Welch’s t-test. b Invasion of MCF7-p95ΔNErbB2 cells in 3D Matrigel upon transfection with a control and 20 nM let-7e mimic in the absence and presence of transiently expressed WT MZF1 lacking the 3′-UTR. Images of the invasive spheres were taken with 10× magnification. Quantification is done as described in a. Data are presented as % of control and the effect of let-7e is assigned with * indicating p < 0.05 in a Welch’s t-test. c Immunoblot analysis of MZF1 protein expression in MCF7-p95ΔNErbB2 cells transfected with 20 nM of control and let-7e mimic in the absence and presence of transient overexpression of WT MZF1. β-actin was used to control for equal loading. The immunoblot was quantified using Image Studio Lite. Data are presented as relative to control mimic treatment in the absence of transient WT MZF1 overexpression. d Immunoblot analysis of ErbB2 protein expression in MCF7-p95ΔNErbB2 cells response to forced let-7e expression (20 nM). β-actin was used to control the equal loading. The immunoblot is a representative of three independent experiments that were quantified using Image Studio Lite. Data are presented as the mean of experiments ± SD relative to control. The effect was non-significant according to a Welch’s t-test