GEN1 regulates cell proliferation, migration, apoptosis and ferroptosis in gastric cancer

Figure 1 GEN1 was over-expressed in gastric cancer. A: Gene expression profiling interactive analysis were used to investigate the expression of GEN1 in gastric cancer (GC); B: Real-time quantitative polymerase chain reaction detected GEN1 mRNA levels in GC cells (n = 3); C: Knockdown of GEN1 was successfully done in AGS cells by transfecting with short hairpin (sh) GEN1-1, shGEN1-2, or shGEN1-3 (n = 3). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. STAD: Stomach adenocarcinomas; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; CON: Control; shCtrl: Negative control with scrambled shRNA; sh: Short hairpin.

Figure 2 The interference efficiency of shGEN1 on GEN1 in AGS cells. A: Observation of the state of negative control with scrambled shRNA and shGEN1 infected cells after 72 hours under a fluorescence microscope; B: Real-time quantitative polymerase chain reaction detected the expression levels of GEN1 mRNA after lentiviral infection (middle) (n = 3); C: Western blotting detected GEN1 protein levels after lentiviral infection (right) (n = 3). ^bP < 0.01. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GFP: Green fluorescent protein; shCtrl: Negative control with scrambled shRNA; sh: Short hairpin.

Figure 3 Silencing of GEN1 repressed AGS cell proliferation and cell cycle progression. A: Cell counting kit-8 assays assessed the proliferation of AGS cells transfected with negative control with scrambled shRNA cells (shCtrl) or short hairpin GEN1 (n = 3); B and C: Flow cytometry assays determined the percentage of AGS cells transfected with shCtrl or shGEN1 at different stages (n = 3). ^bP < 0.01. ^cP < 0.001. OD: Optical density; shCtrl: Negative control with scrambled shRNA; sh: Short hairpin.

Figure 4 Inhibition of GEN1 intensified cell apoptosis and decreased cell migration in AGS cells. A-C: Flow cytometry (A), wound-healing (B) and transwell assays (C) analyzed the apoptosis and migration of AGS cells transfected with negative control with scrambled shRNA or shGEN1 (n = 3). ^bP < 0.01. ^cP < 0.001. shCtrl: Negative control with scrambled shRNA; sh: Short hairpin.

Figure 5 Enrichment analysis of GEN1-related genes. A: The Kyoto Encyclopedia of Genes and Genomes enrichment analysis was applied with 264 genes associated with GEN1 (r > 0.4, P < 0.001); B: Visualization of the genes of the cell cycle pathway associated with GEN1 through cytoscape. Red represented a positive correlation, and the darker the color, the stronger the correlation; C: Western blotting detected CCNA2, CCNB1, CCNB2, cyclin-dependent kinase (CDK) 1, CDK2 and polo-like kinase 1 protein levels in AGS cells transfected with negative control with scrambled shRNA cells or shGEN1 (n = 3). sh: Short hairpin; CDK: Cyclin-dependent kinase; PLK1: Polo-like kinase 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; ATP: Adenosine triphosphate.

Figure 6 GEN1 downregulation impelled cell ferroptosis in AGS cells. A: The adenosine triphosphate content in AGS cells transfected with negative control with scrambled shRNA cells or shGEN1; B: Dihydroethidium staining analysis of reactive oxygen species production in AGS cells; C: Western blotting detected ACSL4, GPX4, and FTH1 protein levels in AGS cells. ^cP < 0.001. ATP: Adenosine triphosphate; shCtrl: Negative control with scrambled shRNA; sh: Short hairpin; DAPI: 4’,6-diamidino-2-phenylindole; DHE: Dihydroethidium; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 7 GEN1 knockdown sensitizes gastric cancer cells to ferroptosis and cisplatin. A: Ferrostatin-1 rescues cell viability in GEN1-knockdown AGS cells. Data are mean ± SD (n = 3); B: Dose-response curves of cisplatin in negative control with scrambled shRNA and shGEN1 AGS cells. Half-maximal inhibitory concentration values: ShCtrl = 5.9 μM, shGEN1 = 2.8 μM (P < 0.001, unpaired t-test). ^bP < 0.01. ^cP < 0.001. ¹P vs negative control with scrambled shRNA. ²P vs shGEN1 (two-way analysis of variance). shCtrl: Negative control with scrambled shRNA; sh: Short hairpin; IC₅₀: Half-maximal inhibitory concentration.