Blockage of ETS homologous factor inhibits the proliferation and invasion of gastric cancer cells through the c-Met pathway

Figure 1 ETS homologous factor and c-Met expression in gastric cancer tissues and cell lines. A: Quantitative PCR analysis of mRNAs revealed the upregulation of ETS homologous factor (EHF) mRNA in gastric cancer (GC) tissues compared with their matched adjacent tissues; B: Western blotting results showed that the expression of EHF and c-Met were both upregulated in GC tissues; C: Immunohistochemical staining of EHF indicated that increased expression of EHF was higher in GC tissues than that in matched adjacent tissues (400 ×); D: The mRNA expression level of EHF was enhanced in GC cell lines compared with that in normal gastric epithelial cells; E: EHF protein expression was increased in GC cell lines in which c-Met was also upregulated. ^aP < 0.05 vs matched adjacent tissues; ^eP < 0.001 vs normal gastric epithelial cells. T: GC tissues; N: Matched adjacent tissues.

Figure 2 Transfection of ETS homologous factor-targeted small interfering RNA reduces c-Met expression in gastric cancer cell lines. A: Quantitative PCR was performed to evaluate the mRNA levels of ETS homologous factor (EHF) in gastric cancer (GC) cells following transfection for 72 h; B: Western blotting was used to detect the protein expression of EHF and c-Met after small interfering RNA transfection. ^bP < 0.01 vs negative control (NC) group; ^eP < 0.001 vs NC group.

Figure 3 The effects of ETS homologous factor silencing on gastric cancer cell proliferation. A: Cell viability was evaluated by the Cell Counting Kit-8 after transfection for 1, 3, 5, and 7 d; B and C: Cell colony formation was measured to verify the changes in gastric cancer (GC) cell proliferation following ETS homologous factor downregulation. ^eP < 0.001 vs negative control (NC) group.

Figure 4 Downregulation of ETS homologous factor induces apoptosis and cell cycle arrest in gastric cancer cells. A and B: The analysis of apoptosis was performed by flow cytometry to detect the induction of apoptosis caused by ETS homologous factor (EHF) silencing following V-FITC/propidium iodide (PI) staining; C and D: The cell cycle arrest induced by EHF downregulation was assessed by PI staining and further analyzed by flow cytometry. ^aP < 0.05 vs negative control (NC) group; ^bP < 0.01 vs NC group; ^eP < 0.001 vs NC group. GC: Gastric cancer.

Figure 5 ETS homologous factor regulates the cell migration and invasion abilities of gastric cancer cells. A and B: The transwell assay was performed to assess the cell migration ability of gastric cancer (GC) cells following ETS homologous factor (EHF) downregulation; C and D: Invasion assays were performed by adding Matrigel into the Transwell chamber to determine the effects of EHF silencing on the invasion ability of GC cells. ^eP < 0.001 vs negative control (NC) group.

Figure 6 ETS homologous factor promotes the malignant biological behaviors of gastric cancer cells through the c-Met pathway. Western blotting was conducted to investigate the effects of ETS homologous factor (EHF) silencing on the expression and activities of signaling molecules in the c-Met pathway. A: Alterations in the Ras- extracellular signal-related kinase 1/2 (Erk1/2) cascade after EHF inhibition; B: The molecular changes in phosphatase and tensin homolog (PTEN) and glycogen synthase kinase-3β (GSK3β) and their potential targets following EHF silencing; C: The altered activities of signal transducer and activator of transcription 3 (STAT3) and the changed expression of its downstream effectors after EHF downregulation. GC: Gastric cancer; NC: Negative control group.