Abstract:

Objective  To study the effects of gastrin on regenerating gene I(Reg I) gene transcription factors (TFs) in gastric cancer cell SGC7901. Methods  Reg I gene promoter 1414bp fragment was amplified from gastric cancer cell SGC7901 genomic DNA by nest-PCR. The fragment was inserted into PMD19-T vector and identified by sequencing. The 1414bp fragment and its HindⅢ digestion 800bp and 614bp fragments were digoxin-labeled by random primer assay. After sensitivity detection, the three fragments were used as probes. Transcription factor binding sites (TFBS) in Reg I gene promoter 1414bp fragment were analyzed by online software Genomatix MatInspector. Gastric cancer cell SGC7901 was incubated with gastrin-G17 for 48 hours at final concentrations of 10 ^-7 mol/L and 10 ^-8 mol/L, and the nuclear proteins were extracted, respectively. Effects of gastrin on Reg I gene TFs were detected by Southwestern blotting using digoxinlabeled 1414bp, 800bp and 614bp as probes, respectively. Results Twenty major bands of proteins with different molecular weight were detected with 1414bp probe. After treatment with gastrin, the pattern of bands showed no change. However, the densities of some bands were changed, the densities of band 9, 12, 13, 14, 15 and 16 decreased significantly ( P＜0.05). The densities of 6 bands above mentioned between those in the two groups treated with gastrin at different final concentration had no significant difference ( P >0.05). Of the 6 changed bands detected with 1414bp probe, three bands including band 9, 12 and 13 were detected with proximal 614bp probe. The densities of the three bands also decreased after gastrin incubation ( P＜0.05). Three bands including band 9, 12 and 14 detected with distal 800bp probe. However, only the densities of band 14 decreased after gastrin incubation ( P＜0.05). Band 15 and band 16 were not detected with proximal 614bp or distal 800bp probe. Conclusion The results suggest that Reg I gene may be transcriptionally regulated by cooperation of many TFs in gastric cancer cell SGC7901. Decreasing binding activities of some TFs may be one of pathways of gastrin up-regulating Reg I gene expression in gastric cancer cell SGC7901.

Key words: Reg I gene , Gastrin , Transcription factor , Gastric cancer cell , Southwestern blotting