Fig. 3
miR-449a inhibits cell proliferation and migration of BTSC in vitro. a Analysis of the stem cell frequency of Pten/p53 and Rb/p53 BTSCs using the extreme limiting dilution assay. Negative wells were counted 7 days post-seeding. Frequency of sphere-forming cells: Pten/p53: 1/4.96; Rb/p53: 1/8.32, n = 12, p = 0.065, indicating lower rate of self-renewal of Rb/p53 cells. b High levels of miR-449a in Rb/p53 mBTSC compared to Pten/p53 BTSC (p < 0.001), normalized for Pten/p53. c miR-449a reduces cell proliferation (confluence assay): Pten/p53 (miR-449alow) BTSC grow faster than Rb/p53 (miR-449ahigh) mBTSC. Treatment of Pten/p53 BTSCs with miR-449a mimic reduces their proliferation, whilst antagomir-treated Rb/p53 (Rb/p53ant) BTSC show increased proliferation, compared to scrambled-treated controls, respectively. d The gap closure assay shows the cell migration over 12 h into an artificially generated cell-free space. miR-449alow cells (i.e. Rb/p53ant and Pten/p53scramble) migrate faster and almost close the gap after 12 h; whereas miR-449ahigh cells (i.e., Rb/p53scramble and Pten/p53mimics) hardly advance into the gap. Recording of multiple time points during the 12 h period visualizes the dynamics of migration and confirms a statistically significant difference in the live cell imaging assay (right figure part). e Quantification of the invasion of a mass of 5000 cells into 3D collagen. The left figure part shows an example of the outgrowth of processes from spheres. miR-449alow cells (i.e., Rb/p53ant (top right) and Pten/p53scramble (bottom left)) show substantial outgrowth into the matrix. In contrast, miR-449ahigh cells (i.e., Rb/p53scramble (top left) and Pten/p53mimics (bottom right)) hardly advance into the matrix (left panel). The right figure part shows quantification with Image J, confirming the statistically significant difference (n = 12). f RT-qPCR analysis of differentiation markers in BTSC shows a proneural gene expression pattern in Pten/p53 and in antagomir-treated Rb/p53 BTSCs compared to the baseline of untreated Rb/p53 BTSC (orange error bars). Inhibition of miR-449a (i.e., Rb/p53ant, red bars) resulted in the shift of basal expression profile of Rb/p53 cells towards that of Pten/p53 BTSCs (grey bars)
