Article Figures & Data
Figures
- Figure 1.
SOX18 is overexpressed in ccRCC and is correlated with poor prognosis of ccRCC patients. A, Western blot analysis of SOX18 expression in a panel of cell lines. B, Western blot analysis of SOX18 expression in matched ccRCC tissues (T) and adjacent normal tissues (N). GAPDH was used as a loading control. C, Analysis of SOX18 mRNA levels with qPCR in 28 ccRCC and corresponding normal tissues. D, TCGA data detailing SOX18 mRNA expression in ccRCC tissues compared with adjacent normal tissues. E, Representative IF staining of SOX18 in matched normal kidney and ccRCC sections (magnification = 200 ×). F, χ2 test on SOX18 protein expression in primary tumors, metastatic tumors, and normal tissues stained with IHC. G, Representative IHC staining of SOX18 in matched adjacent normal kidney, primary ccRCC, and bone metastasis tissues (magnification = 400 ×). Differences between SOX18 IHC scores were analyzed using χ2 test. H, The correlation between SOX18 expression and pathological grade and TNM stages was analyzed using a χ2 test. I, The OS rates of patients in high- and low-SOX18 groups were plotted using the Kaplan–Meier method and analyzed using a log-rank test. ***, P < 0.0001.
- Figure 2.
Silencing of SOX18 suppresses ccRCC growth. A–B, Cell proliferation and colony formation were measured using CCK-8 (A) and colony formation assays (B). C, The volume and weight of ACHN-derived xenografts in the SOX18-silenced (shSOX18) and control (shNC) groups. D, Distribution of cell-cycle phases in shSOX18- or shNC-infected cells was investigated using flow cytometry. E, Representative images of EDU incorporation by cells in shSOX18 and shNC groups (magnification = 200 ×). *, P < 0.05; **, P < 0.001; ***, P < 0.0001.
- Figure 3.
The influence of SOX18 on cell apoptosis and gene transcription. A, The expression of MYC, CCND1, MMP-7, VIM, SNAI1, EGF, and HGF genes in ACHN and 769P cells identified by transcriptomics was validated by qPCR analysis. B, Western blot analysis of c-Myc and cyclin D1 expression in SOX18-silenced or control cells. GAPDH was used as a loading control. C, IF staining of c-Myc in xenograft models from shSOX18 or shNC groups (magnification = 200 ×). D, Increased luciferase activity was observed in 293T cells cotransfected with the MYC promoter and SOX18 plasmids compared with that in control cells. E, TUNEL staining of cells infected with shSOX18 or shNC. The ratio of apoptotic cells in the shSOX18 groups was higher than that in the shNC groups. ns, no significant difference; *, P < 0.05; **, P < 0.001; ***, P < 0.0001.
- Figure 4.
SOX18 deletion inhibits cellular migration and invasion by regulating both MMP-7 expression and the EMT process. Transwell invasion assays treated with (A) or without (B) Matrigel showed that SOX18 deletion suppressed migration and invasion in ACHN and 769P cells (magnification = 100 ×). C, Western blotting analysis of MMP-7 and EMT-related proteins (vimentin, snail, N-cadherin, and E-cadherin) expression in cells transfected with shSOX18 or shNC. D–F, IF staining of E-cadherin, N-cadherin, and vimentin in SOX18-silenced or control xenograft models (magnification = 200 ×). **, P < 0.001; ***, P < 0.0001.
- Figure 5.
SOX18 knockdown suppresses cellular migration and invasion through the EGF/EGFR and HGF/c-MET signaling pathways. A, Western blot analysis of EGFR, p-EGFR (Tyr1068), c-MET, p-MET (Tyr1349), p-MET (Tyr1234/5), AKT, p-AKT (Ser473), MEK1/2, p-MEK1/2, ERK1/2, and p-ERK1/2 expression in SOX18-silenced cells. GAPDH was used as a loading control. B, IF staining of EGFR and p-MET (Tyr1234/5) in SOX18-silenced or control xenograft models (magnification = 200 ×). C, The EGF and HGF secretion level in SOX18-silenced or control cell culture was detected by ELISA assays. D, Western blot analysis of EGFR, p-EGFR, AKT, and p-AKT in SOX18-induced or control cells treated with EGFR or c-MET siRNA or negative control. E, Western blot analysis of c-MET, p-MET (Tyr1349), AKT, and p-AKT in SOX18-induced or control cells treated with c-MET siRNA or negative control. F–G, Transwell invasion assays with (F) or without (G) Matrigel indicated that the presence of HGF or EGF could rescue the inhibition of migration and invasion due to an SOX18 deficiency (magnification = 100 ×). *, P < 0.05; **, P < 0.001; ***, P < 0.0001.
- Figure 6.
Ectopic expression of SOX18 overcomes cabozantinib-mediated inhibitory effects. A, Western blot analysis showed that the activation of EGF/EGFR and HGF/c-MET signaling pathways in SOX18-silenced cells was not observed in the presence of HGF and EGF. B, The influence of SOX18 on cell proliferation under increasing concentrations of cabozantinib. C–D, Ectopic expression of SOX18 decreased inhibition of cell migration (C) and invasion (D) that was mediated by cabozantinib at concentrations of 2 or 3 μmol/L. E, Western blot analysis of c-MET, p-MET (Tyr1349), p-MET (Tyr1234/5), AKT, p-AKT (Ser473), MEK1/2, p-MEK1/2, ERK1/2, and p-ERK1/2 in SOX18-induced or control cells treated with cabozantinib at concentrations of 2 or 3 μmol/L. F, Expression of SOX18 in ccRCC and the interaction between SOX18 and related molecular networks. ns, no significant difference; *, P < 0.05; **, P < 0.001; ***, P < 0.0001.
Additional Files
Supplementary Data
- Supplementary Table S1 - Clinicopathological characteristics of patients with ccRCC
- Supplementary Table S2 - Information of antibodies used in the present study
- Supplementary Table S3 - The sequence of gene primer and small interfering RNAs
- Supplementary Table S4 - Univariable and multivariable analysis for ccRCC patients' overall survival using Cox's proportional hazards model
- Supplementary Figures S1-S5 - Figure S1 shows the role of SOX18 on ccRCC cell growth. Figure S2 shows the analysis of RNA-seq and the effect of SOX18 on protein expression and cell apoptosis. Figure S3 shows the effect of SOX18 on cell migration and invasion, and the expression of other proteins. Figure S4 shows the influence of SOX18 on cabozantinib-mediated inhibitory effect on cellular function. Figure S5 shows the influence of SOX18 knockdown on cell migration and invasion under cabozantinib.
Volume 18, Issue 12
