Figure 4.

Effect of Forced Modulation of miR-532-5p Expression

(A) Confirmation of effective transfection by miR modulators, with anti-miR causing miR inhibition (i) and miR mimic causing increased miR expression intracellularly and in conditioned medium (ii and iii); n = 8 biological replicates. *p < 0.05, **p < 0.01, ****p < 0.0001 versus scrambled. (B) Inhibition of miR-532-5p does not affect APC functions. (C) Forced expression of miR-532-5p induces a decrease in metabolic activity and apoptosis; n = 6 biological replicates, *p < 0.05 versus scrambled. (D and E) The effect of miR-532-5p modulation on network formation was investigated in a Matrigel assay. (D) (i) Co-culture of miR-532-5p-inhibited APCs with HUVECs significantly decreased the ability of the latter to form networks on Matrigel compared to scrambled-treated APCs; n = 9 biological replicates, **p < 0.01 versus HUVECs alone, ⁺⁺p < 0.01 versus scrambled-APCs. (ii) DiL (red) staining of APCs shows miR-532-5p inhibition resulted in no change in the number of APCs found on the network branches or in the nodes (representative images, magnification ×10). (E) (i) Overexpression of miR-532-5p produced a significant increase in branch formation compared to control, but no change in the number of cells on branches or nodes was observed (ii), Representative images, magnification ×10; n = 8 biological replicates, *p < 0.05 and **p < 0.01 versus HUVECs, ⁺p < 0.05 versus scrambled-APCs. (F) (i) Inhibition of miR-532-5p decreased HUVEC monolayer resistance, while (ii) miR-mimic increased resistance; n = 5–8 biological replicates. *p < 0.05 versus scrambled. Values are means ± SE.