Figure 3
miRNA targeting EGFR is upregulated in spheroids and affects the response to EGFR TKI. (a) Cells were cultured in 2D for four days; half of the cells were then plated in 3D and the other half were stored at -80C. After four additional days, half of the spheroids were stored at -80C and the other half were returned to 2D culture for an additional four days and then stored at -80C. RNA isolation was performed in parallel. (b) miR-146a-5p shows a significant increase of expression in 3D culture, and a significant return to 2D levels in 3D-2D. (c). Transfection of spheroids with a miR-146a-5p inhibitor reduced expression by more than 50% compared to non-transfected cells. Expression measured by RT-qPCR, relative to non-transfected control. Error bars represent standard deviation. (d) Spheroids transfected with miR-146a-5p inhibitor show increased sensitivity to 150 nM erlotinib compared to non-transfected cells. Survival normalized to untreated controls; plot shows mean ± standard deviation. Kruskal–Wallis test with Dunn’s correction for multiple comparisons; n = 6. (e) Three additional miRNA that target EGFR show significant upregulation in 3D compared to 2D. (b, e). miRNA expression measured by miRNA microarray. Two-way ANOVA with Tukey’s test for multiple comparisons; n = 3; error bars represent standard deviation.
