After pcDNA3.1ZEO(-)/CagAand PGL/GP were identified by double restriction
enzyme digestion, PCR and sequencing, the
gastric cancer cell lines AGS and SGC-7901 cells were co-transfected with pcDNA3.1ZEO(-)/CagA and PGL/GP for 48 h. Alternatively, AGS and SGC-7901 cells were transfected by PGL/GP for 36 h later, and infected with Helicobacter pylori for additional 12 h. Meanwhile, the transfected and infected cells were treated using the JAK2 signaling pathway inhibitor
AG490 and the ERK signaling pathway inhibitor
U0126. The untreated cells and empty-vector-transfected cells were used as the control. Finally,
luciferase activity was detected using the
luciferase reporter assay system in transfected and infected cells. The levels of
gastrin mRNA was determined by TaqMan® real-time quantitative PCR.
RESULTS: After co-transfection with pcDNA3.1ZEO(-)/CagA and PGL/GP, the activities of
luciferase were increased by 251.3, 106.1 and 2.4 times in AGS cells and 35.8, 22.7 and 13.4 times in SGC-7901 cells, respectively, as compared with that of the control, pcDNA3.1 ZEO(-)/CagA + PGL3/Basic and pcDNA3.1 ZEO(-) + PGL/GP groups. The activities of
luciferase in PGL/GP transfection and HP
infection group were also increased by 1673.2, 33.5, 1.4 times in AGS cells and 1180.2, 72.2 and 1.5 times in SGC-7901 cells, respectively, as compared with that of the control, PGL3/Basic + HP and PGL/GP groups. There were statistically significant differences between them (P < 0.05), which suggested that the transcription activity of
gastrin promoter increased significantly. But after adding the inhibitor
AG490 and
U0126, respectively, the activities of
luciferase were significantly decreased by 95.7% (
U0126) and 33.0% (
AG490) in co-transfected AGS cells and 94.8% (
U0126) and 86.2% (
AG490) in co-transfected SGC-7901 cells with pcDNA3.1ZEO(-)/CagA and PGL/GP (P < 0.05). In the PGL/GP transfection and HP
infection group, the activities of
luciferase were significantly decreased by 24.6% (
U0126) and 25.8% (
AG490) in AGS cells and 57.3% (
U0126) and 14.1% (
AG490) after adding the inhibitor
AG490 and
U0126, respectively (P < 0.05). The results showed that the
gastrin promoter activities were significantly inhibited. The
gastrin mRNA levels were 3.0 and 4.5 times higher in HP-infected AGS and SGC-7901 cells, respectively, than that in the control groups. In the cells transfected with pcDNA3.1ZEO(-)/CagA, the
gastrin mRNA levels were raised 10.8 and 2.3 times (AGS cells) and 10.9 and 16.2 times (SGC-7901 cells), respectively, as compared with that of control and pcDNA3.1ZEO(-) groups. All of the differences were statistically significant (P < 0.05).
CONCLUSION: These results suggest that CagA may activate the
gastrin promoter and up-regulate the expression of
gastrin gene, and CagA is one of the important
proteins in regulating
gastrin gene expression. The ERK/MAPK and JAK/STAT signaling pathways may be involved in the controlling of
gastrin gene expression by CagA.