Fig 1: eIF3a regulates tumor proliferation in vitro and in vivo. (A,B) EdU assays were performed to evaluate the alterations in cell proliferation ability after eIF3a suppression. (C,D) The statistical results of EdU assays, which were calculated from three independent experiments. (E,F) Cell viability was analyzed by CCK-8 assay. (G–J) Clone formation analyses. Statistical results were calculated from three independent experiments. (K) Tumor growth and volume were measured every 3 days. (L) Tumor growth status was monitored via in vivo imaging. (M) Isolated xenograft tumors from different groups of nude mice. (N,O) The volumes and weights were measured for each tumor. (P) Representative images of eIF3A (left) and Ki67 (right) detected by IHC analysis. sieIF3a, small interfering RNA targeting eIF3a; siNC, control small interfering RNA; sheIF3a, short harpin RNA targeting eIF3a; shNC, control short harpin RNA; EdU, 5-ethynyl-2′-deoxyuridine.
Fig 2: eIF3a regulates Cdc42 and RhoA via translational activation. (A,B) q-PCR assays were used to evaluate Cdc42 (A) and RhoA (B) mRNA expression. Statistical results were obtained from three independent experiments. (C,D) eIF3a was overexpressed in 293T cells, and q-PCR analysis was performed to measure Cdc42 (C) and RhoA (D) mRNA expression. (E–H) HCT116 and HT29 cells were treated with eIF3a and control siRNAs and then subjected to luciferase reporter gene assays. The translational activation ability of eIF3a on the 5′ UTRs of Cdc42 (E,F) and RhoA (G,H) was evaluated. (I,J) RIP assays were performed to verify the direct interaction among eIF3a and the 5′ UTR of Cdc42 (I) and RhoA (J). sieIF3a, small interfering RNA targeting eIF3a; siNC, control small interfering RNA.
Fig 3: Downregulation of eIF3a affects pseudopodia formation. (A) Optical microscopy and electron microscopy were used to assess HCT116 cellular morphology after eIF3a knockdown at different magnification times. (B,C) F-actin stained with FITC-phalloidin (green) and cortactin (red) was visualized in control and eIF3a-silenced HCT116 and HT29 cells. sieIF3a, small interfering RNA targeting eIF3a; siNC, control small interfering RNA.
Fig 4: Model showing the role of the eIF3a-PPP2R1B-ERK axis in regulating BRAF inhibitor resistance. In normal BRAFV600E mutant cells, the BRAF inhibitor suppresses ERK activity and cell proliferation. When eIF3a is depleted, PPP2R1B translation is inhibited, resulting in the persistence of p-ERK and the subsequent occurrence of drug resistance.
Fig 5: eIF3a is a downstream target of miR-875-5p in HCC cells. (A) Results from TargetScan (http://www.targetscan.org) and miRanda (microRNA.org) showed the predicted miR-875-5p binding sites in 3′UTR of eIF3a mRNA. (B) Luciferase reporter assay indicated that alteration of miR-875-5p expression inversely regulated luciferase activities of wild-type (wt) but not mutant (mt) eIF3a 3′UTR plasmids. (C and D) RT-qPCR and western blotting were performed to identify the expression of eIF3a mRNA and protein in Hep3B cells transfected with miR-875-5p inhibitors. (E and F) HCCLM3 cells with miR-875-5p knockdown. (G) RT-qPCR was performed to detect eIF3a mRNA expression in 90 pairs of HCC tumor tissues and corresponding adjacent non-tumor tissues. (H) Pearson's correlation analysis disclosed the negatively correlation between eIF3a mRNA and miR-875-5p in HCC tissues. (I) Western blotting was performed to detect eIF3a expression of HCC tissues with low miR-875-5p expression and high miR-875-5p expression. (J) Western blotting was performed to determine eIF3a expression in mice xenograft tumor tissues. *P<0.05.
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