Figure 6. Activation of c-Met, which is negatively regulated by CD82, contributes to miR-K6-5p-induced endothelial cell invasion and angiogenesis.

(a). Western-blotting analysis of phosphorylated c-Met in HUVECs transduced with lentivirus-mediated empty vector (mpCDH) or miR-K6-5p (miR-K6-5p), and further transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting c-Met (shc-Met). Results shown were from a representative experiment of three independent experiments with similar results.

(b). Matrigel invasion assay for HUVECs treated as in (a). The quantified results represent the mean ± SD. Three independent experiments, each with five technical replicates, were performed. * P < 0.05 and ** P < 0.01 for Student’s t-test.

(c). Microtubule formation assay for HUVECs treated as in (a). The quantified results represent the mean ± SD. Three independent experiments, each with five technical replicates, were performed. * P < 0.05, ** P < 0.01 and *** P < 0.001 for Student’s t-test.

(d). The mRNA expression levels of MMP10 and VEGFA in HUVECs treated as in (a) were determined by qPCR. The quantified results represent the mean ± SD. Three independent experiments, each with three technical replicates, were performed. * P < 0.05 and ** P < 0.01 for Student’s t-test.

(e). HUVECs treated as in (a) were examined for their proangiogenic effects in Matrigel plug assay in nude mice as described in the “Materials and Methods” section. The hemoglobin levels of the Matrigel plugs were determined with hemoglobin content calculated based on the standard curve. Data represent mean ± SD, each group with at least six tumors. Three independent experiments were performed and similar results were obtained.

(f). The mRNA expression levels of MMP10 and VEGFA in the Matrigel plugs treated as in (e) were determined by qPCR. The quantified results represent the mean ± SD. Three independent experiments, each with four technical replicates, were performed. * P < 0.05 and ** P < 0.01 for Student’s t-test.

(g). Western-blotting analysis of phosphorylated c-Met and total c-Met in HUVECs transduced with lentivirus-mediated empty vector (mpCDH) or miR-K6-5p (miR-K6-5p) and further treated with the c-Met inhibitor, PF-2341066 (PF-2341066) or its control (DMSO). Results shown were from a representative experiment of three independent experiments with similar results.

(h). Matrigel invasion assay for HUVECs treated as in (g). The quantified results represent the mean ± SD. Three independent experiments, each with five technical replicates, were performed. * P < 0.05 and ** P < 0.01 for Student’s t-test.

(i). Microtubule formation assay for HUVECs treated as in (g). The quantified results represent the mean ± SD. Three independent experiments, each with five technical replicates, were performed. * P < 0.05 and ** P < 0.01 for Student’s t-test.