PC-1基因在肿瘤组织中的表达及其启动子区的克隆和活性分析

背景与目的：PC－1是在骨转移和雄激素非依赖的前列腺癌细胞株C4－2中高表达的新基因。本文拟研究PC－1基因在多种人肿瘤组织和正常组织中的表达水平,并对该基因的启动子区进行克隆及活性分析。方法：以PC－1基因特异性DNA序列为探针与10对肿瘤和正常组织的总RNA进行杂交;以C4－2细胞基因组DNA为模板,PCR扩增PC－1基因翻译起始位点上游DNA序列。PCR产物定向克隆到含荧光素酶报告基因的载体pGL3-Basic上,将重组质粒瞬时转染C4－2细胞,荧光素酶定量分析检测启动子活性。结果：多肿瘤组织中PC－1基因的表达水平明显高于相应的正常组织中的表达;翻译起始位点上游340bp的片段没有启动子活性,而ATG前1099bp、1337bp,1579bp,1831bp和4939bp长度的片段都表现出启动子的功能活性。结论：PC－1基因在人前列腺癌以及多种肿瘤组织中被特异激活,表明该基因的功能可能和肿瘤的发生有关;研究的初步结果表明4939bp长度的启动子活性最强,以1831bp和4939bp之间可能含有转录增强子元件。

Expression of PC-1 gene in tumor tissues,cloning and analysis of its putative promoter region

Background & Objective: PC 1 is a novel gene which is overexpressed in bone metastasis and androgen independent prostate cancer cell line C4 2. The objective of this study was to evaluate the expression level of PC 1 gene in multiple human tumor and normal tissues, and clone a series of putative PC 1 gene promoter regions and analyze their promoter activities. Methods:PC 1 gene specific DNA sequence was used as probe to hybridize with total RNA from 10 pairs of tumor and normal tissues; The C4 2 cell genomic DNA was used as a template in the polymerase chain reaction to amplify PC 1 upstream regions from the translation initiation codon. The PCR product was directly cloned into the luciferase reporter vector pGL3 basic. C4 2 cells were transiently transfected with above recombinant plasmids and the putative promoter activities were analyzed by luciferase assay. Results:PC 1 gene expression level is remarkable higher in multiple tumor tissues than in their matched normal tissues;there is no promoter activity in the 340 bp fragment from the upstream of initiation codon, while 1099 bp, 1337 bp, 1579 bp, 1831 bp, and 4939 bp fragments had the promoter activities. Conclusion: The PC 1 gene expression is specifically activated in multiple human tumor tissues, suggesting that PC 1 gene expression might be involved in cancer development. Our primary data shows that the highest promoter activity of PC 1 gene is within the 4939 bp DNA fragment and maybe there exists an enhancer element between the 1831 bp and 4939 bp area.