Fig 2: PHIP and ACTN4 are direct targets of miR-142-5p(A) Schematic of the complementary sequence between miR-142-5p and PHIP and ACTN4. Mut nucleotides in the PHIP and ACTN4 3' UTRs are in lowercase. (B) HOKs were cotransfected with the miR-142-5p or NC mimics and luciferase reporter constructs containing WT or Mut of PHIP or ACTN4 3' UTRs. The luciferase activities were detected. (C) qRT-PCR was used to detect ACTN4 and PHIP expression in OSCC tissues. (D) qRT-PCR was used to compare ACTN4 and PHIP expression in OSCC and healthy cell lines. (E) PHIP and ACTN4 expression in SCC15 and CAL27 cells transfected with NC or sh-circPHIP was determined via qRT-PCR. Relative mRNA levels are shown. (F) OSCC cells were transfected with the miR-142-5p inhibitor or mimic and analyzed via western blotting. (G) SCC15 and CAL27 cells were transfected with sh-circPHIP, with or without the miR-142-5p inhibitor, and the indicated protein levels were detected via western blotting. All data are presented as mean ± SEM of three independent experiments. Student’s t test, **p < 0.01, ***p < 0.001. Representative images are shown.

Fig 3: Characterization of RAW 264.7 macrophages-derived DEX-packaging MVs. (A) Size distribution analysis of MV-DEX by nanoparticle tracking analysis. (B) Transmission electron microscopy images of MV-DEX showed double-membrane vesicles. Boxed area is enlarged in the right. (C) Markers of macrophage (CD68, F4/80 and CD206) and microvesicles (actinin 4) were confirmed in MV-DEX by Western blot. (D) HPLC analysis revealed the concentrations of DEX in MVs were dependent on the applied drug doses. (E) RAW cells were incubated with 30 μmol/L fluorescein-DEX. MVs were isolated and observed under confocal microscopy. DEX was shown as the green fluorescent (E1) (scale bar, 1 μm). GECs were incubated with fluorescein-DEX packaging MVs for 12 h and then observed under confocal microscopy (E2) (scale bar, 10 μm). n=3. Data are presented as mean ± SD.

Fig 4: PHIP and ACTN4 function as driver genes in OSCC cells(A) Overexpression of PHIP and ACTN4 in SCC15 and CAL27 cells with stable depletion of circPHIP, and the levels of indicated proteins were determined via western blotting. (B) Transwell migration and invasion assays were performed in SCC15 and CAL27 cells with stable depletion of circPHIP and PHIP or ACTN4 overexpression. (C) Wound-healing assays were performed in SCC15 and CAL27 cells with stable depletion of circPHIP and PHIP or ACTN4 overexpression. (D) CCK-8 assays were performed in SCC15 and CAL27 cells with stable depletion of circPHIP and PHIP or ACTN4 overexpression. (E) Colony-formation assays were performed in SCC15 and CAL27 cells with stable depletion of circPHIP and PHIP/ACTN4 overexpression. (F) Apoptosis assays were performed in SCC15 and CAL27 cells stably depleting circPHIP and PHIP or ACTN4 overexpression. All data are presented as mean ± SEM of three independent experiments. Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. Representative images are shown. Scale bars, 20 µm.

Fig 5: OTUD3 enhances the stability of ACTN4 through deubiquitylation. (A, B) co-IP experiments between endogenous OTUD3 and ACTN4 in HCCLM3 and Huh7 cells. ACTN4 was detected in the immunoprecipitation when the anti-OTUD3 antibody was used as bait. (C) Colocalization of OTUD3 and ACTN4 in HCCLM3 and Huh7 cells (Scale bar: 14μm). (D) HCCLM3 and Huh7 cells were treated with 15μM proteasomal inhibitor MG132 for the indicated time, and the levels of ACTN4 were then detected. (E) HCCLM3 cell transfected with OTUD3 shRNA or shNC together with stably OTUD3 overexpressing Huh7 cells and negative control were treated with 20μM cycloheximide (CHX). Cells were collected at different time points and were detected ACTN4 protein expression. (F) HCCLM3 cells with OTUD3 knockdown or OTUD3 overexpression were treated with MG132 (15μM). Cells were collected at 6 h and immunoblotted with the antibodies indicated. (G, H) the knockdown or upregulation of OTUD3 altered the ubiquitination of ACTN4 in both HCCLM3 and Huh7 cells. The cells in each group were treated with MG132 (15μM). The levels of ubiquitin-attached ACTN4 were detected by western blot analysis with ubiquitin (Ub) antibody.