Fig 2: The expression and survival analysis of UBE2O in HCC. (A) The levels of UBE2O mRNA were detected in 80 pairs of HCC and nontumor (NT) tissues. (B) Left: The presentative result of UBE2O staining in a sample containing both HCC and adjacent NT tissues. Right: The IHC score of UBE2O in HCC (n=40) was remarkably higher than that in adjacent NT tissues (n=40). (C) UBE2O expression was assessed in MIHA, HCCLM3, Huh7, HepG2, Hep3B, and MHCC97H cells. n=three independent repeats, *P<0.05. (D) We compared the survival of HCC patients with low (n=39) or highly (n=41) expressed UBE2O.

Fig 3: (A) Nucleocytoplasmic separation assays were used to confirm the cytoplasmic and nuclear distribution of components in BC cells. (B) Schematic illustration showing the overlapping miRNAs targeting both HCG18 and UBE2O using the TargetScan, miRWalk, and starBase databases. (C) qRT-PCR was used to determine the expression of predicted miRNAs in MDA-MB-231 NC /MDA-MB-231 HCG18-sh and MCF-7 NC /MCF-7 HCG18-sh cells. (D,E) MS2-RIP followed by miRNA qRT-PCR to detect the interactions between miRNAs and HCG18. (F) MS2-RIP followed by miRNA qRT-PCR to detect the interactions between miRNAs and UBE2O mRNA 3'-UTR. (G) HCG18-Wt/HCG18-mut and miR-103a-3p mimics were cotransfected into HEK-293T cells, and then dual-luciferase reporter assays were performed. The data are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. The data represent at least three independent experiments.

Fig 4: UBE2O induced proliferation, EMT and CSPs in a manner dependent on the regulation of the UBE2O/AMPKa2/mTORC1 axis in BC.a Western blotting revealed that p-Raptor expression was increased in MDA-MB-231sh-UBE2O#1cells, which indicated mTORC1 pathway inhibition. As a downstream effector of mTORC1, p-S6K expression was also decreased. The opposite results occurred in MCF-7OE-UBE2O cells. b–f AMPKa2 was knocked down in MDA-MB-231sh-NC/MDA-MB-231sh-UBE2O#1cells. Then, b scratch assays, c invasion assays, d colony formation assays, e CCK-8 assays and f sphere formation assays were performed (upper: magnification × 100, Scale bar, 100 µm; lower: magnification × 400, Scale bar, 20 µm). g Western blot assays were performed to detect the expression of EMT markers (CDH1, CDH2, vimentin and slug), CS markers (CD44, ABCG2, OCT3/4 and MYC) and mTORC1 pathway-related markers (p-Raptor and p-S6K) after AMPKa2 suppression in MDA-MB-231sh-NC/MDA-MB-231sh-UBE2O#1cells. The data are shown as the mean ± s.d. Student’s t test was used for statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001. The data represent at least three independent experiments.

Fig 5: RAPA treatment reverses the promoting role of UBE2O in HCCLM3 cells. (A) UBE2O overexpressing HCCLM3 cells were treated with RAPA (10 ng/mL) for 48 h, a specific mTOR inhibitor. UBE2O-induced p-mTOR upregulation was markedly reduced by RAPA treatment. (B) MTT, (C) EdU, and (D) transwell analyses demonstrated that RAPA treatment reversed the tumor-promoting role of UBE2O in HCCLM3 cells. n=three independent repeats, *P<0.05.