Fig 1: Effects of trefoil factor family 2 (TFF2) expression on gastric cancer cell proliferation and apoptosis and the effect of stable Sp3 knockdown in BGC-823 gastric cancer cells. (A) Western blot analysis of TFF2 and Sp3 expression in gastric cancer cells. BGC-823 cells were grown and stably transfected with HA-TFF2, pU6-siSp3, or control vectors, and cell lysates were subjected to western blot analysis for TFF2 and siSp3 expression. GAPDH was used as a loading control. (B) Bromodeoxyuridine (BrdU) incorporation assay. The 4 types of BGC-823 cell sublines were grown and incubated with BrdU for up to 22 h and then subjected to spectrophotometer analysis of optical absorbance levels at 450 nm. Data are expressed as the means (nm) ± SD of 3 separate experiments and were analyzed by Student's t-tests. The BrdU incorporation assay showed that Sp3 promoted the proliferation of BGC-823 cells (pcDNA6.0 + pU6 vs. pcDNA6.0 + siSp3, P<0.01), whereas TFF2 reduced cell proliferation (pcDNA6.0 + siSp3 vs. TFF2 + siSp3, P<0.05). (C) Flow cytometric analysis of BCG-823 cells following incubation with cisplatin for 36 h and subsequent staining with Annexin V and propidium iodide (PI). Dots in Annexin V−/PI−, Annexin V+/PI−, and Annexin V+/PI+ indicate intact cells, cells undergoing early apoptosis and dead cells, respectively. Data are shown as the means (%) ± SD of 3 separate experiments and were analyzed by Student's t-tests. We compared the frequency of apoptotic cells for pcDNA + pU6 (45.85%), pU6 + TFF2 (51.03%), siSp3 + pcDNA (38.12%), and siSp3 + TFF2 (41.17%) cells. In cells with a high expression of Sp3 and overexpression of TFF2, gastric cancer cell apoptosis was significantly higher than that in the control cells (P<0.05). In Sp3 knockdown gastric cancer cells, apoptosis decreased significantly (P<0.05). In cells with Sp3 knockdown and overexpression of TFF2, the apoptosis level did not decrease obviously. These results indicated that TFF2 and Sp3 alone significantly promoted the apoptosis of gastric cancer cells. TFF2 and Sp3 in combination further enhanced the levels of gastric cancer cell apoptosis. Accordingly, TFF2 and Sp3 had a synergistic effect on gastric cancer cell apoptosis. (D) Tumor cell invasion assay for the four BGC-823 cell sublines. Data are shown as the means ± SD of 3 separate experiments and were analyzed by Student's t-tests. Compared with the empty vector group, gastric cancer cells with a high expression of Sp3 and overexpression of TFF2 did not differ significantly with respect to invasive ability (P>0.05). In cells with Sp3 knockdown, the invasive ability decreased significantly (P<0.05). In gastric cancer cells with Sp3 knockdown and TFF2 overexpression, the invasive ability markedly increased. These results indicated that TFF2 and Sp3 alone significantly promoted the invasive ability of gastric cancer cells. The combination of TFF2 and Sp3 did not further enhance the invasive ability of gastric cancer cells. This suggested that TFF2 and Sp3 had an antagonistic effect on gastric cancer cell invasion. ns, not significant; ***P<0.01, **P<0.05, and *P<0.1 compared to the control. Groups in the bar charts are as follows: pU6 cont (control), pcDNA6.0 + pU6; pU6 TFF2, TFF2 + pU6; siSp3 cont, pcDNA6.0 + siSp3; and siSp3 TFF2, TFF2 + siSp3.