一个影响前列腺癌中L-plastin启动子转录活性的多态性位点的成功鉴定  

Identification of A Single Base Polymorphism in L-plastin Promoter which Affects its Transcription Activity in Prostate Cancer

作　　者：林天歆[1,2] 黄健[1] 蔡清清[3] 谢文练[1] 许可慰[1] 叶枫[4] 尹心宝[1] 胡明[1] 

机构地区：[1]中山大学附属第二医院泌尿外科,广州510120 [2]中山大学附属第二医院医学研究中心 [3]中山大学肿瘤医院内科 [4]中山大学附属第二医院体检中心,广州510120

出　　处：《中华泌尿外科杂志》2006年第S2期29-33,共5页Chinese Journal of Urology

基　　金：国家自然科学基金资助项目(30500507);广东省自然科学基金资助项目(021842;05001758;06104605)

摘　　要：目的研究已发现的前列腺癌中L-plastin启动子一个多态性位点对其转录活性的影响和意义。方法扩增前列腺癌细胞株LNCaP中L-plastin启动子序列,发现在距离转录起始点-1751上存在多态性位点T后,利用定点突变技术构建文献报道的启动子序列质粒(-1751C),测定含-1751T质粒和含-1751C质粒的荧光素酶活性。用巢式PCR扩增前列腺癌细胞株和癌组织中该位点序列,并进行单链构象多态性分析。结果成功构建L-plastin启动子,并发现一个位于-1751的多态性位点(C/T);成功构建启动子序列含-1751C质粒;荧光素酶活性测定表明(-1751C)质粒启动子转录活性为(-1751T)质粒的4~5倍,2者受到雄激素刺激后活性均升高;单链构象多态性分析表明该位点多态性普遍存在于前列腺癌患者和细胞株。结论鉴定了一个普遍存在于前列腺癌的L-plastin基因启动子的多态性位点,含有不同碱基位点的启动子的转录活性明显不同。Objective To investigate the effect of the single base polymorphism in L-plastin promoter on promoter transcriptional activity after a single base polymorphism at-1751 has been identified and study the characters of this polymorphism and its significance. Methods L-plastin promoter was amplified from prostate cancer cell line LNCaP and cloned into plasmid PGL3-basic which containing the luciferase gene downstream the promoter, sequencing result indicated a single base polymorphism at-1751 with nucleotide T, and literature-reported promoter(-1751 C) was constructed with the site-mutagenesis method. In vitro luciferase activity was assessed for these 2 different base variant promoters. Nested PCR, single strand conformation polymorphism(SSCP) analysis and silver staining methods were performed to identified the sequence at point-1751 on L-plastin promoter in 4 prostate cancer cell lines, 40 prostate cancer samples and 9 pairs of prostate tumor-normal tissues. Results Sequence of L-plastin promoter was amplified and cloned into vector pGL3-basic and polymorphism at site-1751 (C→T)in L-plastin promoter was identified. Clone containing reported promoter(-1751 C)sequence was constructed with site-mutagenesis method. The luciferase activity of the plasmid-1751 C transfected LNCaP cells was 4 to 5 times higher than that of plasmid-1751 T transfected cells with or without 24 hour DHT stimulation. The results of nested PCR,SSCP and silver staining indicated site-1751 polymorphism was a common phenomenon which prevalently occured in prostate cancer patients and cell lines. Conclusions A prevalently existing single base polymorphism was identified at site-1751 of L-plastin promoter in the prostate cancer cell lines and tissues, the transcriptional activity of L-plastin promoter was different with variant base sequence.

关 键 词：前列腺肿瘤 癌 L-PLASTIN 多态性位点 

分 类 号：R737.25[医药卫生—肿瘤]