Fig. 5: LINC01016 promotes triple-negative breast tumor proliferation and metastasis via suppression of RFFL-mediated DHX9 ubiquitination degradation to activate the PI3K/AKT pathway.

A Knockdown of LINC01016 reduced DHX9 protein levels. B si-LINC01016 promotes more rapid DHX9 protein degradation, as demonstrated by western blotting following CHX treatment (20 μg/mL for 0–14 h). C Western blotting results show that intracellular DHX9 level is decreased in si-LINC01016-transfected cells that have not been treated with MG132 (10 μM) for 24 h. D si-NC- and si-LINC01016-transduced cells are transfected with HA-Ub and DHX9 plasmids and cultivated for 48 h, after which they are treated for 4 h with MG132 (10 μM). A DHX9-specific antibody is then used for immunoprecipitation of these cell lysates, revealing increased DHX9 ubiquitination in cells transduced with si-LINC01016. E RFFL is identified as an E3 ubiquitin ligase of DHX9 via Co-IP and MS assays, with the different band being marked with an arrow. F The results of Co-IP and subsequent western blotting using anti-DHX9, anti-RFFL, or control IgG reveal the interactions between DHX9 and RFFL. G RFFL is found by western blotting to negatively regulate DHX9 expression. H Cells overexpressing RFFL or control constructs were transfected with the HA-UB plasmid and cultivated for 48 h and then treated for 4 h with MG132 (10 μM). Increased DHX9 ubiquitination was observed in cells overexpressing RFFL relative to control cells. I Western blotting shows that RFFL overexpression is associated with reduced DHX9 protein levels, with MG132 treatment disrupting this association. J DHX9 levels were found to be reduced in cells in which LINC01016 had been knocked down, whereas RFFL knockdown reversed this effect. K Immunoprecipation assay showing that LINC01016 overexpression disrupts RFFL binding to DHX9, while LINC01016 knockdown had the opposite effect. L RFFL overexpression disrupts interactions between DHX9 and LINC01016, as confirmed via an RNA pull down assay wherein lysates of cells overexpressing RFFL or control constructs were combined with in vitro-transcribed biotinylated LINC01016 sense transcripts, followed by the assessment of DHX9 via western blotting. M PI3K/AKT pathway signaling-related proteins (p-AKT, bcl-2 and MMP-9) were upregulated in cells overexpressing LINC01016. N LINC01016 upregulation activated the PI3K/AKT pathway signaling activity, whereas DHX9 knockdown reversed this effect. Data are shown as mean ± SEM of three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001, ns. not significant.