草酸钙尿石症患者肾组织γ-谷氨酰基羧化酶基因启动子序列分析及其活性研究

Sequence and functional analysis of GGCX promoter region in renal tissues of patients with calcium oxalate urolithiasis

目的分析草酸钙尿石症患者肾组织γ-谷氨酰基羧化酶（GGCX）基因启动子序列多态性，并探讨该序列变化对启动子活性的影响。方法分别以42例草酸钙尿石症患者肾组织（尿石组）与31例非结石肾组织（肾肿瘤癌旁正常组织，对照组）基因组DNA为模板，PCR扩增GGCX基因5’-侧翼启动子区1329bp片段（-1270-＋58bp），电泳鉴定后，分别对两组PCR产物测序。筛选差异启动子序列克隆至萤光素酶报告基因载体pGL3-basic，转染293细胞，双萤光素酶检测启动子活性。结果与Ensembl正常序列比较，尿石组启动子片段测序共发现4处变异位点，-804bp处TT缺失（refsnp ID：rs11433794），-704bp处A→G、-511bp处A→G、-115bp处T→C。其中尿石组rsll43794SNP发生明显高于对照组（P〈0．05）。构建含rs11433794SNP位点的萤光素酶重组子pGL3-GGCX Promoter（804TT），与正常序列的重组子pGL3-GGCX Promoter比较，pGL3-GGCX Prorooter（804TT）转录活性明显下降（P〈0．01）。结论GGCX启动子区rs11433794SNP位点可能与草酸钙尿石症发生存在一定关系，该SNP位点可导致启动子转录活性下调，导致GGCX基因表达下降。

Objective To investigate the polymorphisms of human GGCX promoter region and analyze its transcriptional activity. Methods The sequence of GGCX promoter region （ - 1270- ＋ 58 bp） in 48 patients with calcium oxalate urolithiasis ,and 16 patients without urolithiasis was screened ,and then a functional difference in transcriptional activities of diversity sequence and normal sequence was investigated by dual luciferase reporter assay. Results Five variable sites were found （ - 804 bp ： TT deletion, refsnp ID：rs11433794; -704 bp：A→G： - 511 bp： A→G： - 115 bp： T→C; - 20 bp： C→T）, and patients with calcium oxalate urolithiasis exhibited a higher frequency of-804 bp TT deletion than controls （P 〈 0.05 ）. Dual luciferase reporter assay revealed that - 804 bp TT deletion resulted in significantly lower promoter activity （ P 〈 0.01 ）. Conclusion GGCX promoter rs11433794 SNP may confer host genetic susceptibility to calcium oxalate urolithiasis. This SNP possible decreases the transcriptional activity of GGCX promoter and induces a low GGCX-producing capability.