摘要目的 观察Ki-67核心启动子在人胃癌细胞中的转录活性.方法 使用缺失分析法分别从5'端和3'端对Ki-67基因启动子逐段缺失,得到不同长度的8个和3个截短DNA片段.分别插入pGL3-Basic载体后转染人胃癌SCG-991细胞,使用双荧光素酶检测系统鉴定转录活性,确定Ki-67基因核心启动子.比较Ki-67核心启动子与另2种肿瘤启动子hTERT和Survivin的转录活性.结果 自5'端缺失得到的-223～+771截短片段在SCG-991细胞内转录活性达到病毒SV40启动子活性的56.5%;自3'端缺失的-223～+30截短片段转录活性更强,为-223～+771片段活性的2.1倍,是hTERT启动子的1.7及Survivin启动子和15.3倍.结论 Ki-67核心启动子区域为-223～+30,在胃癌SCG-991细胞中的转录活性超过SV40启动子,以及hTERT启动子及Survivin启动子.

abstractsObjective To identify the proximal core promoter of Ki-67 gene and its transcriptional activities in human gastric cancer SCG-991 cells. Methods Various lengths of DNA fragments,8 of which were 5' truncations including the initiating ATG codon and 3 of which were 3' truncations encompassing the transcription initiation,were amplified by PCR from the 5' flanking sequence of Ki-67 gene and inserted into luciferase reporter vector pGL3-Basie,and tested by dual-luciferase reporter assay system to identify the core promoter essential for transcriptional activation. Then transcriptional activity of Ki-67 core promoter was compared with that of hTERT and Survivin promoter,respectively. Results The transcriptional activity of the proximal -223 ~ + 771 fragment in gastric cancer SCG-991 cells was equivalent to 56.5% of SV40 promoter/enhancer. The transcriptional activity of 3' truncations of -223~+30 fragments in SCG-991 cells was approximately 2.1-fold activity of -223～+ 771 fragments. In contrast,in normal umbilical vein epithelial cells,no significant transcriptional activity was observed in either 5' -truncated -320～+771 fragments or 3' truncations of -223～+30 fragments. The -223～+30 region demonstrated higher transcriptional activity than hTERT and Survivin promoter in SCG-991 cell lines. Conclusion These findings suggest that the proximal - 223～+30 region upstream of the transcription start site functions as the core promoter essential for transcriptional activation of Ki-67.