Fig 2: Knockdown of APOC2 inhibits the biological behavior, apoptosis resistance, and EMT in GC. (A–D) PCR and western blot were used to evaluate the knockout efficiency of the APOC2-specific small interfering RNAs (siRNAs) (siAPOC2#1, siAPOC2#2, and siAPOC2#3) in AGS and BGC-823 cells. (E, F) Cell mobility analyzed using a wound healing assay. (G, H) Migration and invasion of siAPOC2#1 and siControl cells were detected using Transwell migration and invasion assays. (I, J) Colony formation assay of AGS and BGC-823 cells transfected with siAPOC2#1 and siControl. (K, L) Propidium iodide (PI) and Annexin V staining levels were determined by flow cytometry to assess cell apoptosis in AGS and BGC-823 cells transfected with siAPOC2#1 and siControl. (M, N) Western blot analysis of the protein levels of E-cadherin, N-cadherin, vimentin, Snail, Slug, Twist1, MMP-2, and MMP-9 in AGS and BGC-823 cells transfected with siAPOC2#1 and siControl. Data are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, based on Student's t-test

Fig 3: Knockdown of APOC2 suppresses metabolic activity and inhibits PI3K/AKT/mTOR signaling in GC. (A, B) Representative images of lipid droplets (LDs) in AGS and BGC-823 cells transfected with Lenti-shAPOC2#1 and Lenti-shControl as evaluated using BODIPY 493/503 staining (green) and Oil Red O staining; cell nuclei were stained with 4',6-Diamidino-2-Phenylindole (blue). (C, D) Concentration of total cholesterols (TCs) in AGS and BGC-823 cells transfected with Lenti-shAPOC2#1 and Lenti-shControl. (E, F) Concentration of triglyceride (TGs) in AGS and BGC-823 cells transfected with Lenti-shAPOC2#1 and Lenti-shControl. (G) Cell energy phenotype of Lenti-shAPOC2#1 and Lenti-shControl AGS and BGC-823 cells. Baseline phenotype is indicated by an open marker. Stressed phenotype is indicated by a filled marker. (H, I) The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of AGS and BGC-823 cells transfected with Lenti-shAPOC2#1 and Lenti-shControl tested by Seahorse XF cell analysis. (J–O) Glycolytic capacity of AGS and BGC-823 cells transfected with Lenti-shAPOC2#1 and Lenti-shControl was measured by glycolytic stress test, the key parameters of glycolytic flux, including glycolysis and glycolytic capacity, were shown. (P, Q) GO (P) and KEGG pathway (Q) analysis of these differentially expressed genes revealed that the PI3K/AKT signaling was inhibited in APOC2 knockdown cells using GSEA. (R) KEGG annotations of the identified proteins from GC and PM tissues. (S, T) After the cells were transfected with Lenti-shAPOC2#1 and Lenti-shControl, the proteins in the PI3K/AKT/mTOR pathway of AGS and BGC-823 cells were evaluated using western blot. Data are shown as mean ± SD; ns, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, based on Student's t-test

Fig 4: APOC2 promotes tumor progression and PM of GC cells in vivo via the PI3K/AKT/mTOR pathway. (A–C) Photograph of subcutaneous tumors excised from nude mice treated with 740Y-P or intraperitoneal injection of AGS cells stably infected with Lenti-shControl or Lenti-shAPOC2 lentiviral particles (N = 6 per group). Tumors were observed and recorded by tumor volume (B) and tumor weight (C). (D–F) Photograph of subcutaneous tumors excised from nude mice treated with LY294002 or intraperitoneal injection of AGS cells stably infected with Lenti-oeControl or Lenti-oeAPOC2 lentiviral particles (N = 6 per group). Tumors were observed and recorded by tumor volume (E) and tumor weight (F). (G) Representative images of the macroscopic appearance and hematoxylin and eosin (H&E) staining of PM nodules in nude mice treated with the PI3K pathway activator by 740Y-P or intraperitoneal injection of AGS cells stably infected with Lenti-shControl or Lenti-shAPOC2 lentiviral particles (N = 6 per group). (I, J) The total number (I) and weight (J) of PM nodules in the respective groups. (H) Representative images of the macroscopic appearance and H&E staining of PM nodules in nude mice treated with PI3K pathway inhibitor by LY294002 or intraperitoneal injection of AGS cells stably infected with Lenti-oeControl or Lenti-oeAPOC2 lentiviral particles (N = 6 per group). (K, L) The total number (K) and weight (L) of PM nodules in the respective group. (M) Western blot was used to detect proteins expression of p-PI3K, p-AKT, p-mTOR, E-cadherin, N-cadherin, vimentin, Snail, Slug, Twist1, MMP-2, and MMP-9 from five patients with GC in tumor tissues, ANTs, and PM tissues. Data are shown as mean ± SD; ns, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, based on Student's t-test

Fig 5: APOC2 mediates EMT via PI3K/AKT/mTOR signaling in GC. (A, B) Western blot analysis of the protein levels of p‐PI3K, p‐AKT, p‐mTOR, E cadherin, N‐cadherin, vimentin, Snail, Slug, Twist1, MMP‐2, and MMP‐9 in GC cells transfected with Lenti‐shAPOC2#1 and Lenti‐shControl, as well as cells treated with or without the PI3K pathway activator 740Y‐P. (C, D). Western blot analysis of the protein levels of p‐PI3K, p‐AKT, p‐mTOR, E‐cadherin, N‐cadherin, vimentin, Snail, Slug, Twist1, MMP‐2, and MMP‐9 in AGS and BGC‐823 cells transfected with Lenti‐oeAPOC2 and Lenti‐oeControl, as well as cells treated with or without PI3K pathway inhibitor by LY294002. Data represent mean ± SD of three independent experiments. Data are shown as mean ± SD; ns, no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, based on Student's t‐test