摘要
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.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第10期1261-1263,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(30400442)