Ki-67启动子转录调控结构与功能研究  

Structural and functional analysis of human Ki-67 gene promoter

作　　者：李中林[1] 钱国伟[2] 徐为[2] 李望[2] 张宝福[2] 刘俊杰[2] 陈菲菲[2] 田卉[2] 章龙珍[2] 郑骏年[2] 

机构地区：[1]徐州医学院附属医院神经外科,江苏221002 [2]徐州医学院肿瘤生物治疗实验室

出　　处：《中华实验外科杂志》2010年第9期1296-1298,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30972976）

摘　　要：目的 观察Ki-67核心启动子调控元件对转录调控的影响.方法 根据Ki-67启动子结构软件分析结果,对Ki-67核心启动子（-223～＋30）内的5个调控区域,包括（＋13～＋30）及4个潜在Sp1结合位点（-198～-172）、（-170～-144）、（-63～-37）和（-14～＋12）进行内部缺失,目的片段缺失的Ki-67核心启动子插入pGL3-Basic载体,构建报告基因重组体质粒.将重组体分别转染肿瘤Hela、SW1990、SGC-7901、A549细胞株,使用双荧光素酶报告基因检测系统测定其转录活性,并与Ki-67核心启动子质粒pGLBK2＋转录活性比较.结果 缺失（＋13～＋30）的启动子重组体质粒在4种肿瘤细胞的转录功能均有显著提高,分别达到缺失前的1.2、2.0、1.7、1.4倍.缺失潜在Sp1结合位点的4个启动子重组体在4种肿瘤细胞的转录功能均有显著降低,其中缺失（-170～-144）后转录活性最小,在4种肿瘤细胞中分别降至未缺失前的16.2%、10.4%、6.7%、12.2%.结论 Ki-67启动子（＋13～＋30）区包含负性调控元件,对其缺失后形成的新的Ki-67核心启动子（-223～＋12）是更好的调控肿瘤基因治疗的启动子.（-198～-172）、（-170～-144）、（-63～-37）和（-14～＋12）可能存在潜在的Sp1顺式作用元件,其中-170～-144区域的顺式作用元件最为重要.Objective To investigate the transcriptional regulatory elements of the core promoter of Ki-67 gene. Methods The truncated fragment of Ki-67 core promoter BK235 （ -223 to ＋ 12） was directly cloned into pGL3-Basic vector, then transfected into Hela cells, SW1990 cells, SGC-7901 cells and A549 cells. The transcriptional activity of BK235 in each carcinoma cell line was identified using the dualluciferase reporter assay system. Four internally deleted vectors of Ki-67 promoter BK235 （-223 to ＋ 12）were prepared, whose Sp1 binding site including （- 198 to - 172）, （- 170 to - 144）, （-63 to -37）and （-14 to ＋ 12） was deleted respectively, and their transcriptional activity was tested by dual-luciferase reporter assay system to locate the core regulatory element. Results The transcriptional activity of Ki-67 core promoter BK235 （-223 to ＋ 12） in Hela cells, SW1990 cells, SGC-7901 cells and A549cells was respectively 1.2-fold, 2.0-fold, 1.7-fold and 1.4-fold of Ki-67 core promoter BK2 ＋ （-223 to ＋ 30）. Deletion of the putative Spl binding site within BK235 resulted in the decreases of promoter activities, especially deletion of （- 170 to - 144） yielded lowest transcriptional activity, equivalent to 16. 2%,10.4%, 6.7% and 12. 2% of BK2 ＋ （-223 to ＋30） promoter in Hela cells, SW1990 cells, SGC-7901cells and A549 cells, respectively. Conclusion A new core promoter BK235 （ -223 to ＋ 12） of Ki-67gene was cloned and It had a better transcriptional performance than BK2 ＋ （-223 to ＋ 12）. All of four Spl binding sites of Ki-67 promoter had a positive effect on transcriptional regulation function and - 170 to -144 was the core regulatory sequence of Ki-67 promoter.

关 键 词：KI-67基因 启动子 转录调控 

分 类 号：Q78[生物学—分子生物学]