Activation of STAT3, which was negatively regulated by SH3BGR, contributes to miR-K6-3p-induced endothelial cell migration and angiogenesis.

<p><b>(A)</b>. Western blotting analysis of phosphorylated STAT3 and VEGFA in HUVEC transduced with lentivirus-mediated empty vector (<b>mpCDH</b>) or miR-K6-3p (<b>miR-K6-3p</b>), and further transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting STAT3 (<b>shSTAT3</b>). Results shown were from a representative experiment of three independent experiments with similar results. The values of density of protein bands after normalization to housekeeping were shown. <b>(B)</b>. Transwell migration assay for HUVEC treated as in (<b>A</b>). The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing five technical replicates. * <i>P</i> < 0.05, ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test. (C). Microtubule formation assay for HUVEC treated as in (A). The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing five technical replicates. ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test. <b>(D)</b>. Western blotting analysis of phosphorylated STAT3, STAT3 and VEGFA in HUVEC transduced with lentivirus-mediated empty vector (<b>mpCDH</b>) or miR-K6-3p (<b>miR-K6-3p</b>) and further treated with the JAK2 inhibitor, AG490 (<b>AG490</b>) or its control (<b>DMSO</b>). Results shown were from a representative experiment of three independent experiments with similar results. The values of density of protein bands after normalization to housekeeping were shown. <b>(E)</b>. Transwell migration assay for HUVEC treated as in (<b>D</b>). The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing five technical replicates. * <i>P</i> < 0.05, ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test. <b>(F)</b>. Microtubule formation assay for HUVEC treated as in (D). The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing five technical replicates. * <i>P</i> < 0.05, ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test. <b>(G)</b>. HUVEC treated as in (<b>A</b>) 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 five tumors (n = 5). Three independent experiments were performed and similar results were obtained. <b>(H)</b>. The mRNA expression of MMP1, MMP13, VEGFA and VEGFR2 in the Matrigel plugs treated as in (G) were determined by qPCR. The quantified results represent the mean ± SD. Three independent experiments were performed and similar results were obtained, each experiment containing four technical replicates. * <i>P</i> < 0.05, ** <i>P</i> < 0.01 and *** <i>P</i> < 0.001 for Student’s <i>t</i>-test. <i>n</i>.<i>s</i>., not significant.