Figure 3.

SAMD9 stimulates angiogenesis, EMT, and the Wnt/β‐catenin pathway. a) Differentially expressed genes between SAMD9‐overexpressed KYSE270 cells (SAMD9‐OV) and their control cells (vector). KYSE270 cells were transfected with a SAMD9‐expressing construct or empty vector for 72 h, followed by mRNA sequencing. b) The gene set enrichment analysis (GSEA) shows that SAMD9 is positively associated with EMT, angiogenesis, and the Wnt/β‐catenin pathway in ESCC cells. The GSEA was performed using mRNA sequencing data from SAMD9‐OV and their vector control cells. c) Encyclopedia of Genes and Genomes analysis of primary tumors mRNA sequencing dataset showing that angiogenesis, EMT, and Wnt/β‐catenin pathways are involved in metastatic recurrence of ESCC. The mRNA sequencing data from the primary tumor samples of ESCC patients with or without postoperative metastatic recurrence (Figure 1a). d) Immunohistochemistry analysis shows that expression of CD31 and VEGF is positively regulated by SAMD9 in tumors of animal models. Tumors from subcutaneous xenograft models (Figure 2e) and lung metastatic models (Figure 2g). e) Visualized tumor vasculature images were obtained from xenograft tumors perfused with FITC‐lectin by confocal fluorescence microscopy (n = 5/group). f) Immunofluorescence analysis of tumor tissues from subcutaneous xenograft models (Figure 2e) shows that SAMD9 positively regulates Vimentin and β‐catenin expression, while negatively regulating E‐cadherin expression. Blue: DAPI; Green: indicated proteins. g) TOP/FOP luciferase analysis shows that SAMD9 positively regulates β‐catenin signaling in ESCC cells. Each bar represents the mean of three independent experiments. Significance between the control and treatment groups was determined using an unpaired two‐tailed Student t‐test, and a p‐value less than 0.05 was considered statistically significant. Error bar, SD.