Figure 6.

Downregulation of SDPR Induces GC EMT and Stemness through the ERK-NF-κB Signaling Pathway

(A) Tumorsphere-formation assays were performed to detect the effect of SDPR on sphere formation ability. The number of spheres were counted under a microscope in five randomly selected fields. **p < 0.01; ***p < 0.001. (B) The ability of cell migration and invasiveness tested using Transwell chamber migration assays. The cells were counted under a microscope in five randomly selected fields. **p < 0.01; ***p < 0.001. (C) Fluorescence images of mice after tail-vein injections with SDPR-silencing cells and control cells. (D) Luciferase signals of lung metastasis nudes in SDPR-silencing group mice and control group mice; the data were expressed as mean ± SD. ***p < 0.001. (E) Kaplan-Meier survival curves and univariate analyses (log rank) for the mice after injections with SDPR-silencing cells and control cells. *p < 0.05; **p < 0.01. (F) Western blot of EMT markers (E-cadherin, vimentin, and N-cadherin), stemness markers (CD44 and SOX2), and SDPR protein expression levels in MGC803 cells transfected with small interfering RNA targeting SDPR (si-SDPR) (#1–2) and MKN45 cells transfected with SDPR plasmid. (G) Immunofluorescence microscopy colocalization analysis of SDPR and ERK in MGC803 and MKN45 cells. Magnification, 180×. (H) P-ERK, ERK, P-IKKα-β, IKKβ, P-IKBα, IKBα, p-p65, and p65 were quantitated by western blot after transfection with NC and si-SDPR (#1–2), pcDNA, and SDPR, as indicated. GAPDH served as a loading control. (I) Lysates from MGC803 and MKN45 cells were immunoprecipitated using anti-SDPR or anti-ERK antibodies and immunoblotted with the indicated antibodies. Twenty percent of the lysate used for immunoprecipitation was loaded as the input control. (J) qRT-PCR analysis was performed for miR-577 expression in MGC803 cells transfected with pcDNA or SDPR plasmid and MKN45 cells transfected with si-SDPR (#1–2). Endogenous U6 RNA was used as a control. *p < 0.05; **p < 0.01; ***p < 0.001.