Fig. 5. FBXL6 upregulates PD-L1 and VRK2 expression via a TKT-dependent decrease in ROS accumulation and mTOR activation, leading to immune evasion and HCC metastasis.

a PD-L1 and VRK2 are upregulated in FBXL6 tumors. PD-L1 and VRK2 mRNA levels were measured by qPCR in normal tissues and FBXL6 tumors. b Huh7 and Hepa1-6 cells were transfected with the indicated plasmids or siRNA oligos for 48 h. PD-L1, VRK2, and mTOR downstream targets (p-S6K and p-4EBP1) were analyzed by Western blotting. c VRK2 and mTOR signaling pathways were assessed after 48 h of treatment with the ROS inhibitor VAS2870 in Huh7 and Hepa1-6 cells. d Huh7 or Hepa1-6 cells were treated with mTOR inhibitors (rapamycin, Rapa (100 nM); sapanisertib, Sapan (0.5 μM)) for 48 h and were then harvested for Western blotting. e–g Huh7 cells were transfected with HA-TKT in the presence or absence of mTOR inhibitors (Rapa (100 nM); Sapan (0.5 μM)) for 48 h. A portion of the cells was harvested for Western blotting (e). The other portion of the cells was seeded into Transwell plates overnight for a migration assay (f, g). Migration was quantified after crystal violet (0.5% w/v) staining. h VRK2, p-mTOR, and p-4EBP1 protein levels were analyzed in FBXL6-overexpressing tumors. i, j Huh7 cells were transfected with FBXL6 plasmids and transfected with an siRNA targeting VRK2 or treated with a VRK2 inhibitor for 48 h. The cells were reseeded into Transwell plates and incubated overnight, and migration was quantified after crystal violet (0.5% w/v) staining. Unpaired t test was used in (a). One-way ANOVA with Tukey’s multiple comparisons test was used in (i, j). Two-way ANOVA with the Bonferroni correction for multiple comparisons was used in (g). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.