Figure 6

Elevated levels of miR-145 leads to reduced microvasular cell migration. (a) Migration of BJ-hTERT cells was evaluated using a microfluidic chemotaxis chamber. Individual cells, cultured in a stable PDGF-BB gradient (0 to 20 ng/ml over a distance of 400 μm), were tracked using time-lapse microscopy. Cells were transfected with control dsRNA or Pre-miR-145 and average migrated distances toward the gradient and perpendicular to the gradient were calculated. The bar graphs show average values from three independent experiments ± standard error of the mean (P-value obtained using the two-tail t-test). The polar plots illustrate the direction of migration for individual cells in the control experiments (top) and in the Pre-miR-145 transfected cultures (bottom). The radius of each 15 degree sector indicates the number of cells that migrated in this direction. A total of 285 and 239 cells were tracked for the negative control and Pre-miR-145, respectively. (b) Migration of Bj-hTERT cells transfected with control single-stranded RNA or Anti-miR-145 in a PDGF-BB gradient, as described above for Pre-miR-145. The bar graphs show average results from five independent experiments, and a total of 701 and 622 cells were tracked for the negative control and Anti-miR-145, respectively. (c) Migration of HUVECs in response to a VEGFA-165 gradient (0 to 50 ng/ml), as described above for PDGF-BB. Results are average values from three independent experiments, and a total of 185 and 191 cells were tracked for the negative control and Pre-miR-145, respectively. (d) Migration of VAECs was evaluated using scratch wound assays. Cells were electroporated with either a negative control dsRNA or a synthetic miR-145 dsRNA (Pre-miR-145) and cultured for 48 hours. A scratch wound was generated in the cell monolayer and the degree of wound closure determined 24 hours later. The graph shows the mean migrated distance (difference in wound width after 24 hours ± standard error of the mean, n = 3). Proliferative activity of VAECs 48 hours post-transfection was assessed by quantification of BrdU incorporation. Cells were pulsed for 4 hours and incorporated BrdU was measured using a colorimetric ELISA (mean absorbance ± standard error of the mean; n = 4).