Fig 2: CARD11 is highly expressed in gastric cancer and is associated with poor prognosis. (A) The ENCORI database predicted the expression of CARD11 in tissues of patients with gastric cancer. ***P<0.001 vs. the cancer group. (B) The ENCORI database predicted the association of CARD11 overexpression with overall survival. Cutoff-High, median 50% and Cutoff-Low, median 50%. The expression levels of CARD11 in normal gastric epithelial cells and in gastric cancer cells were detected by (C) reverse transcription-quantitative PCR and (D) western blotting. **P<0.01 and ***P<0.001 vs. the GES-1 group. CARD, caspase recruitment domain-containing protein; ENCORI, the encyclopedia of RNA interactomes.

Fig 3: The transcription factor KLF5 positively regulates the transcription of CARD11 in gastric cancer. (A) HumanTFDB predicted the binding of the transcription factor KLF5 to the CARD11 promoter. (B) The binding sites between KLF5 and CARD11. The expression of KLF5 in gastric cancer cells was detected by (C) RT-qPCR and (D) western blotting. ***P<0.001 vs. GES-1 group. The expression of KLF5 in gastric cancer cells transfected with oe-KLF5 was detected by RT-qPCR (E) and western blot (F). ***P<0.001 vs. Control group. ###P<0.001 vs. oe-NC group. (G) The luciferase activity of gastric cancer cells co-transfected with oe-KLF5 and CARD11-WT or CARD11-MUT was detected by dual luciferase reporter assay. ***P<0.001 vs. oe-NC group. (H) The expression of CARD11 in gastric cancer cells adding anti-KLF5 was detected by ChIP assay. ***P<0.001 vs. Control group. ###P<0.001 vs. IgG group. The expression of CARD11 in gastric cancer cells transfected with oe-KLF5 was detected by (I) RT-qPCR and (J) western blotting. ***P<0.001 vs. Control group. ###P<0.001 vs. oe-NC group. KLF5, Krüppel-like factor 5; CARD, caspase recruitment domain-containing protein; RT-qPCR, reverse transcription-quantitative PCR; oe, overexpression; NC, negative control; WT, wild type; MUT, mutant; ChIP, chromatin immunoprecipitation.

Fig 4: Interference of CARD11 expression inhibits gastric cancer cell metastasis. The (A) migratory and (B) invasive abilities of gastric cancer cells transfected with si-CARD11 were detected by wound healing and Transwell assays. (C) The expression levels of EMT-related proteins in gastric cancer cells transfected with si-CARD11 were detected by western blot analysis. ***P<0.001 vs. the control group. ###P<0.001 vs. the si-NC group. CARD, caspase recruitment domain-containing protein; si, small interfering; EMT, epithelial-to-mesenchymal transition.

Fig 5: KLF5 regulates CARD11 to promote malignant progression of gastric cancer might by activating mTOR pathway. The viability, proliferation and cell cycle of gastric cancer cells transfected with si-CARD11 and oe-KLF5 was analyzed by (A) CCK-8, (B and C) EdU staining and (D) flow cytometry analysis. The (E) migration and (F) invasion of gastric cancer cells transfected with si-CARD11 and oe-KLF5 was detected by wound healing and Transwell assays. (G) The expression of EMT-related proteins in gastric cancer cells transfected with si-CARD11 and oe-KLF5 was detected by western blotting. (H) The expression of mTOR pathway related proteins n gastric cancer cells transfected with si-CARD11 and oe-KLF5 was detected by western blotting. ***P<0.001 vs. Control group. #P<0.05, ##P<0.01 and ###P<0.001 vs. si-CARD11 group. KLF5, Krüppel-like factor 5; CARD, caspase recruitment domain-containing protein; si, small interfering; oe, overexpression; EMT, epithelial-to-mesenchymal transition; p-. phosphorylated.