SH3BGR/STAT3 signaling partially mediates miR-K6-induced endothelial cell migration and angiogenesis.

(A). Western blotting analysis of expression of SH3BGR, phosphorylated STAT3 and STAT3 in HUVEC infected with BAC16 KSHV wide type virus (KSHV_WT) or BAC16 KSHV miR-K6 deletion mutant virus (miR-K6_Mut) and further transduced with lentivirus-mediated a mixture of short hairpin RNAs targeting SH3BGR (shSH3BGR). 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). Transwell migration 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. * P < 0.05 and ** P < 0.01 for Student’s t-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. * P < 0.05 and *** P < 0.001 for Student’s t-test. (D). Western blotting analysis of expression of phosphorylated STAT3 and STAT3 in HUVEC infected with BAC16 KSHV wide type virus (KSHV_WT) or BAC16 KSHV miR-K6 deletion mutant virus (miR-K6_Mut) and further tranfected with pCMV3-Flag-STAT3 construct (STAT3) or its control pCMV3-C-Flag (pCMV). The antibody against Flag-tag was used to detect the exogenous expression of STAT3. 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. (E). Transwell migration 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. * P < 0.05 and ** P < 0.01 for Student’s t-test. (F). 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. * P < 0.05, ** P < 0.01, and *** P < 0.001 for Student’s t-test. (G). Schematic representation of the mechanism by which miR-K6-3p facilitates endothelial cell migration and angiogenesis. In normal endothelial cells, SH3BGR binds STAT3 to inhibit STAT3 activation. During KSHV infection, reduction of SH3BGR as a result of miR-K6-3p targeting releases STAT3 to activate STAT3, leading to the higher expression level of matrix metalloproteinases (MMPs) and angiogenic factors, and ultimately promotes endothelial cell migration and angiogenesis.