胃泌素对胃癌细胞SGC7901 Reg Ⅰ基因转录因子的效应

Effects of gastrin on Reg Ⅰ gene transcription factors in SGC7901 cell

目的探讨胃泌素对胃癌细胞SGC7901 Reg Ⅰ(Reg Ⅰ)基因转录因子的效应。方法应用巢式PCR技术从胃癌细胞SGC7901基因组DNA扩增Reg Ⅰ基因启动子1414bp片段,将该片段插入pMD19-T载体,序列分析鉴定。应用随机引物法以地高辛分别标记1414bp及其HindⅢ酶切800bp和614bp片段,经灵敏度检测后,作为探针。应用Genomatix MatInspector在线分析软件分析Reg Ⅰ基因启动子1414bp片段的转录因子结合位点。分别以10-7mol/L和10-8mol/L胃泌素G-17处理胃癌细胞SGC7901 48h,提取核蛋白。应用DNA-蛋白质印迹法(Southernblotting),分别以地高辛标记的1414bp、800bp和614bp片段为探针检测胃泌素对胃癌细胞SGC7901 Reg Ⅰ基因转录因子的效应。结果 1414bp探针可检测到20条蛋白主带。胃泌素孵育后,带型没有变化,但是一些条带的灰度值改变,带9、12、13、14、15和16的灰度值明显降低(P<0.05);不同浓度胃泌素处理组之间上述6个条带的灰度值差异不明显(P>0.05)。614bp探针可检测到灰度值变化的6条主带中的带9、12和13,胃泌素处理后,此3条主带的灰度值明显降低(P<0.05)。800bp探针可检测到灰度值变化的6条主带中的带9、12和14,胃泌素处理后,仅带14的灰度值明显降低(P<0.05)。614bp和800bp探针均未检出带15和带16。结论胃癌细胞SGC7901 Reg Ⅰ基因表达由多个转录因子协同调控。降低几个转录因子的结合活性可能是胃泌素上调胃癌细胞SGC7901Reg Ⅰ基因表达的途径之一。

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 MatInspeetor. 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 bandsincluding 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.