人PCAN1基因启动子的克隆及其上游调控区域的鉴定

Molecular cloning of the human PCAN1 promoter and identification of its 5′ regulatory regions

目的克隆人前列腺癌基因1(PCAN1)5′上游2.6 kb启动子片段,构建pGL3-p2.6 kb载体,测定其启动子活性,初步鉴定该片段内的DNA调控区域。方法采用PCR法从人基因组DNA中扩增PCAN1基因5′上游2.6 kb启动子片段,并构建到荧光素酶报道基因载体pGL3-basic中,构成pGL3-p2.6 kb;瞬时转染前列腺癌细胞LNCaP,通过双荧光素酶活性检验测定PCAN1启动子活性。使用Erase-a-system对pGL3-p2.6kb载体中2.6 kb片段进行一系列5′侧翼缺失,产生12个缺失体,分别瞬时转染LNCaP细胞,通过检测荧光素酶的活性观察缺失突变对PCAN1启动子活性的影响。结果克隆的PCAN1 2.6 kb启动子片段经测序鉴定正确无误;pGL3-p2.6 kb转染LNCaP细胞后,双荧光素酶活性测定结果显示2.6 kb片段具有明显的启动子活性;5′缺失突变分析显示,PCAN1基因上游-1599 bp至-1541 bp、-347 bp至-84 bp的缺失,与2.6 kb片段相比,启动子活性分别升高2.24倍和2.53倍,-1541 bp至-1226 bp的缺失,与2.6 kb片段相比,启动子活性降低2.11倍。结论克隆的人PCAN1基因5′上游2.6 kb片段具有较强的启动子活性,PCAN1基因上游2.6 kb区域内分别存在2个负调控区和1个正调控区。

Objective To analyze the regulatory regions upstream of the human PCAN1 gene, a reporter plasmid containing of a 2.6 kb PCAN1 promoter was constructed by the PCR method, and DNA cis-acting elements within this promoter were further identified. Methods A 2.6 kb fragment upstream of the PCAN1 gene was amplified by PCR using human genomic DNA as a template, and it was then inserted into the upstream of the luciferase reporter gene in pGL3-basic vector to generate pGL3-p2.6kb. Its promoter activity was determined with dual-luciferase reporter assay after it has been transfected into prostate cancer LNCaP cells. A series of 5＇ deletion mutant-luciferase reporter plasmids derived from pGL3-p2.6kb were obtained via Erase-a-base System. The mutant plasmids were transfected into the LNCaP cells and the promoter activities were determined by dual-luciferase reporter assay. Results The sequence of the 2.6 kb promoter fragment proved to be correct by DNA sequecing. Dual-luciferase reporter assay showed that the 2.6 kb fragment presented a significant promoter activity after pGL3-p2.6 kb was co-transfected with pRL-TK into LNCaP cells. Analysis of the mutants showed that PCAN1 promoter activities increased 2.24-fold and 2.53-fold after the - 1 599 bp to - 1 541 bp and - 347 bp to - 84 bp within the promoter were deleted, while the promoter activity decreased 2.11-fold after the - 1 541 bp to - 1 226 bp was deleted. Conclusion A cloned 2.6 kb fragment upstream of the PCAN1 gene presents strong promoter activity. With deletion analysis two negative and one positive regulatory region were identified within the promoter.