Fig. 6
AnxA6 deSUMOylation promotes cell migration. (A) The binding of RHOU with AnxA6 became weakened in TPA-induced HepG2 cells. The cell lysates of wild-type HepG2 and TPA-induced HepG2 cells were performed IP to capture the AnxA6-binding complex, detected by western blot analysis with SUMO1, AnxA6 and RHOU antibodies. S-AnxA6: Flag-tagging SUMOylated AnxA6, IP: immunoprecipitation. (B) The half-life of AnxA6 was prolonged than that of AnxA6K579R. The plasmids pFlag-AnxA6 or pFlag-AnxA6K579R were respectively transfected into HepG2 cells, then incubated with 100 μg/mL CHX for 0, 2, 4 and 8 h. The cell lysates were used to detect indicated protein (up). AnxA6 levels were quantified from three independent experiments and represented as the mean ± SD (bottom). (C) Compared to wild-type AnxA6, the binding of the K579R mutant to RHOU was weaker in HepG2 cells. Co-transfection of plasmids pEGFP-RHOU, pHis-SUMO1, pFlag-AnxA6 or pFlag-AnxA6K579R in HepG2 cells was to enrich Flag-tagging AnxA6 by IP, and then EGFP-tagging RHOU expression was detected in IP precipitation. S-AnxA6: Flag-tagging SUMOylated AnxA6, IP: immunoprecipitation, IB: immunoblot, Input: same cell lysate to load. (D) DeSUMOylation attenuates AnxA6 role in suppressing the EMT process. The plasmids pFlag-AnxA6 or pFlag-AnxA6K579R were respectively transfected into HepG2 cells to measure EMT markers and p-AKT1ser473 levels (left). Quantitative results were represented as the mean ± SD of three separate experiments (right). β-actin was used as an internal control. ns, no statistical; *P < 0.05; **P < 0.01; ***P < 0.001. (E) DeSUMOylation of AnxA6 enhanced cell migration. The plasmids pFlag-AnxA6 or pFlag-AnxA6K579R were respectively transfected into HepG2 cells and performed transwell assays. (F) A mechanistic model of AnxA6 deSUMOylation contributes to cell migration and HCC progression. AnxA6 inhibits the cell migration ability of HCC that depends on its SUMOylation level. However, the SUMOylation-mediated protective mechanism is disrupted by a deSUMOylase SENP1, which leads to deSUMOylation and degradation of AnxA6 protein in EMT cells, and promotes HCC progression through RHOU/AKT1 signaling