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pG13-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Kj-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-225～＋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。
Objective 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-Basic, and tested by dual-lnciferase 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 ceils 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＇ -trun- cated -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.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第1期34-36,共3页
Chinese Journal of Experimental Surgery
基金
基金项目：国家自然科学基金资助项目（30873021）
作者简介
通信作者：郑骏年
