Fig 1: USP4 Overexpression Increases the Deubiquitination and Translocation of SMAD4 into Nuclei in Cells(A) SMAD4 mRNA expression in EC tissues and adjacent normal tissues detected by qRT-PCR (n = 30). (B) Quantitation of SMAD4 protein in EC tissues and adjacent normal tissues by immunohistochemical analysis (n = 30). (C) Pearson correlation analysis of USP4 expression with SMAD4 expression. (D) Immunofluorescence detection of SMAD4 and USP4 localization in TE-1 cells (×400). (E) GST pull-down detection of SMAD4 and USP4 interaction. (F) coIP detection of the effect of USP4 on SMAD4 ubiquitination. (G) Immunoblotting detection of ubiquitination of SMAD4 after 30-min co-incubation of different concentrations of USP4 with purified Flag-SMAD4. (H) Western blot analysis of the effect of the optimal concentration (20 nm) of recombinant USP4 on the deubiquitination of SMAD4. (I) The effect of the optimal concentration (20 nm) of recombinant USP4 on the deubiquitination of SMAD4. (J) Immunoblotting analysis of whole-cell lysate and immunoprecipitate of HEK293T cells with stable expression of HA-Ub after transfection of Flag SMAD4 or Flag-SMAD4 K519R. (K) Ubiquitination status of SMAD4 after incubation of 508-529 covalent ubiquitin peptide with 100 nM USP4 on K519 for different times. (L) SMAD4 ubiquitination status was observed by immunoblotting after affinity purification of the tagged SMAD4 protein in TE-1 cells. (M) Western blot analysis of ubiquitination of SMAD4 in cells stably expressing HA-Ub after transfection with Myc-USP4-WT or Myc-USP4-CS, respectively. (N) Western blot analysis of the effect of USP4 inhibition on the ubiquitination of SMAD4 in cells stably expressing HA-Ub. (O) Immunofluorescence detection of SMAD4 expression in cytoplasm and nucleus (×400). (P) Western blot analysis of SMAD4 protein in cytoplasm and nucleus of TE-1 cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Paired t test was used for comparison between data of EC tissues and adjacent normal tissues, while unpaired data between the other two groups were compared by unpaired t test. Comparisons among multiple groups were performed using one-way ANOVA. Data are shown as mean ± standard deviation of three technical replicates.
Fig 2: miR-30e Promotes Cell Proliferation, Migration, Invasion, and EMT in Eca109 Cells by Targeting USP4 In Vitro(A) Putative miR-30e binding sites in the 3' UTR of USP4 mRNA in the Starbase database. (B) A heatmap of DEGs in the EC gene expression profiling GSE89102, in which each row represents a DEG, and each column represents a sample. (C) Expression of USP4 analyzed in the EC gene expression profiling GSE89102. (D) Expression of USP4 in EC analyzed by the GEPIA website, where red represents cancer tissues and black represents normal control tissues. (E) mRNA expression of USP4 examined by qRT-PCR in EC tissues and adjacent normal tissues (n = 30). (F) Quantitation of USP4 protein in EC tissues and adjacent normal tissues by immunohistochemistry analysis. (G) Expression of USP4 examined by qRT-PCR in Eca109, TE-1, and HEEC cells. (H) Quantitative western blot analysis of USP4 protein in Eca109, TE-1, and HEEC cells. (I) Pearson correlation analysis of USP4 expression with miR-30e expression. (J) miR-30e binding to USP4 confirmed by dual luciferase reporter assay in 293T cells. (K) Silencing efficiency of USP4 expression in Eca109 cells measured by qRT-PCR. (L) Silencing efficiency of USP4 expression in Eca109 cells measured by quantitative western blot analysis. Eca109 cells were transfected with mimic-NC + vector, miR-30e mimic + vector, and miR-30e mimic + USP4. (M) Expression of USP4 and miR-30e determined by qRT-PCR in Eca109 cells. (N) Quantitation of EdU-positive Eca109 cells. (O) Quantitation of migration of Eca109 cells detected by scratch test. (P) Quantitation of invasion of Eca109 cells detected by Transwell assay. (Q) Quantitative western blot analysis of EMT-related proteins (E-cadherin, N-cadherin, and Vimentin) in Eca109 cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Paired t test was used for comparison between data of EC tissues and adjacent normal tissues, while unpaired data between the other two groups were compared by unpaired t test. Comparisons among multiple groups were performed using one-way ANOVA. Data are shown as mean ± standard deviation of three technical replicates.
Fig 3: A Schematic for the SOX2/miR-30e/USP4/SMAD4/CK2 Regulatory Network in ECSOX2-upregulated miR-30e targets USP4 and decreases its expression, thus reducing SMAD4 expression to activate CK2, which ultimately leads to EC cell proliferation, invasion, and migration.
Fig 4: SOX2 Modulates the USP4/SMAD4/CK2 Axis via miR-30e Upregulation to Induce the Tumor Growth of EC in Nude MiceMice were treated with lv-oe-NC + sh-NC, lv-oe-NC + sh-CK2, lv-oe-SOX2 + sh-NC, or lv-oe-SOX2 + sh-CK2. (A) Quantitation of tumor volume of mice (n = 12). (B) Quantitation of tumor weight of mice (n = 12). (C) Expression of SOX2, miR-30e, USP4, SMAD4, and CK2 in mouse tumor tissues determined by qRT-PCR (n = 12). (D) Quantitative western blot analysis of SOX2, USP4, SMAD4, and CK2 proteins in mouse tumor tissues (n = 12). (E) Quantitation of Ki67 expression in mouse tumor tissues detected by immunohistochemistry. (F) Quantitation of lung metastatic nodules after H&E staining in mice (n = 12). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All measurement data were shown as mean ± standard deviation. Unpaired data between the two groups were compared by unpaired t test. Comparisons among multiple groups were performed using one-way ANOVA. Comparisons of tumor volume data between groups at different time points were conducted using repeated-measures ANOVA.
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