阻断Sp1蛋白对Ki-67基因转录的影响  

Sp1-siRNA and mithramycin effectively inhibit the Ki-67 gene transcription

作　　者：徐为[1] 李望 钱国伟 田卉 李连涛 刘俊杰 陈菲菲 李慧忠 章龙珍 郑骏年 

机构地区：[1]徐州医学院附属医院肿瘤中心,江苏221002 [2]江苏省肿瘤生物治疗重点实验室

出　　处：《中华实验外科杂志》2011年第4期605-607,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目，江苏省自然科学基金资助项目

摘　　要：目的观察阻断转录因子Sp1蛋白及其和顺式作用元件的结合，对Ki-67基因转录的抑制作用。方法Sp1小干扰RNA（Sp1-siRNA）、Sp1蛋白竞争剂光神霉素分别作用于宫颈癌Hela细胞、人肾癌OS—RC-2和肺癌A549细胞。Westernblot检测Hela细胞Sp1蛋白表达。细胞免疫组织化学检测3种肿瘤细胞Ki．67蛋白表达。Sp1—siRNA或光神霉素与Ki-67基因核心启动子pGLBK235共转染上述3种肿瘤细胞，双荧光素酶报告基因系统检测启动子活性。结果Sp1—siRNA处理的Hela细胞Sp1蛋白表达显著减少。Sp1-siRNA、光神霉素处理的3种肿瘤细胞Ki-67蛋白均显著减少。Sp1-siRNA共转染的质粒pGLBK235启动子活性在Hela、OS-RC-2、A549细胞中分别降至单独转染pGLBK235的45．9％、64．5％、46．0％；光神霉素共转染的pGLBK235启动子活性随着药物浓度的升高而降低，其中药物浓度为500nmol／L时其活性最低，在Hela、OS—RC一2、A549细胞分别降至单独转染pGLBK235的34．7％、44．2％、29．8％。结论抑制Spl蛋白或是阻断其与Sp1顺式作用元件的结合，可以有效抑制Ki-67基因转录。Objective To investigate the regulation of Ki-67 gene transcription by the inhibition of Spl protein with Spl small interfering RNAs （Spl-siRNA） and Spl binding sites competitor mithramycin. Methods Spl -siRNA and mithramycin （ a drug known to block activity of Spl family members by binding to GC-rich regions） were used to down-regulate Ki-67 gene transcription in Hela ceils, OS-RC-2 cells and A549 cells. Immunoblotting assay was performed to detect the change of Spl protein in He]a cells. Immu- nohistoehemistry was used to detect the change of Ki-67 protein in Hela ceils, OS-RC-2 ceils and A549 cells. The dual-lueiferase reporter assay system was used to identify the difference of transcriptional activity between Ki-67 promoter plasmid pGLBK23 terated by Spl-siRNA or mithramycin and plasmid pGLBK23. Results After treated by Spl-siRNA, Spl protein in Hela cells was increased significantly. Spl-siRNA and mithramycin significantly decreased the Ki-67 protein in Hela cells, OS-RC-2 cells and A549 ceils. The transcription activities of Ki-67 promoter were reduced to 45.9% , 64. 5% and 46. 0% by Spl-siRNA in Hela cells, OS-RC-2 cells and A549 cells, respectively. The transcriptional activities of Ki-67 promoter were affected by mithramyein in a dose-dependent manner. Mithramycin（ 500 nmol/L） inhibited the Ki-67 gene transcription to 34. 7%, 44. 2% and 29.8% in Hela cells, OS-RC-2 cells and A549 cells, respec- tively. Conclusion Inhibition of Spl protein by Spl-siRNA and mithramyein obviously decrease Ki-67 gene transcription.

关 键 词：Ki-67启动子 转录因子SP1 小干扰RNA 光神霉素 

分 类 号：R730.5[医药卫生—肿瘤]