Figure 8.

miR-3174 Decreases Autophagic Cell Death and Apoptosis in GC Cells by Inhibiting the Expression of ARHGAP10

(A) Mitochondrial membrane potentials were determined and analyzed by confocal microscopy after transfection of cells with a JC-1 fluorescence probe. (B and C) The apoptosis of cells (B, MKN45; C, BGC823) were detected by flow cytometry. (D and E) After transfecting cells (D, MKN45; E, BGC823) with GFP-mRFP-LC3, cells were plated into a 35-mm confocal culture dish, and autophagic-associated puncta were measured in a manner similar to that described in Figure 4A (63× objective magnification; scale bar, 20 μm) after 48 hr. (F and G) LC3-II protein levels were detected in MKN45 (F) and BGC823 (G) cells by western blotting with or without chloroquine (CQ, 10 μM for 2 hr) stimulation. (H and I) The protein levels of p-Akt, mTORC1, p70s6k, p-p70s6k, BECN1, p62, p53, Bax, pan-caspase-3 (inactivated form of caspase-3), and cleaved caspase-3 (c-caspase-3) in GC cells (H, MKN45; I, BGC823) were assessed with western blotting. (J) Correlations between ARHGAP10 expression and the IC₅₀ values for anti-cancer drugs in 28 GC cell lines obtained from the GDSC database were measured. Anti-cancer drugs (IC₅₀ values) that were significantly negatively correlated with ARHGAP10 expression are marked in green (p < 0.01), and those whose IC₅₀ values were significantly positively correlated with ARHGAP10 expression are marked in red (p < 0.01). (K and L) Left panels, cell viability was measured using CCK-8 assays in MKN45 (K) or BGC823 (L) cells with the treatments shown in the figure. Right panels, IC₅₀ values for CDDP were also calculated in MKN45 or BGC823 CDDP-sensitive cells (K) and their CDDP-resistant cells (L) after exposure to CDDP for 48 hr. β-actin was used as an internal control. Graph represents mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001.