异丹叶大黄素下调膀胱癌细胞中细胞周期蛋白D1表达的分子机制研究  

Molecular mechanism of down-regulating cyclin D1 expression by isorhapontigenin

作　　者：方勇[1] 王章桂[1] 侯琦[2] 卢瑜[3] 

机构地区：[1]浙江大学医学院附属邵逸夫医院肿瘤内科,浙江杭州310016 [2]中国医学科学院协和医科大学药物研究所北京100050 [3]南京军区杭州疗养院,浙江杭州310007

出　　处：《中国病理生理杂志》2014年第1期53-60,共8页Chinese Journal of Pathophysiology

基　　金：浙江省中医药科学研究基金资助项目(No.2013ZB084);浙江省自然科学基金资助项目(No.LY13H160013)

摘　　要：目的:探索异丹叶大黄素(ISO)下调肿瘤细胞中细胞周期蛋白D1表达的分子机制。方法:以ISO作用于人源性膀胱癌UMUC3、RT12和RT4细胞后用Western blotting法检测细胞周期蛋白D1表达水平;以RTPCR法检测细胞周期蛋白D1 mRNA的表达水平。稳定转染细胞周期蛋白D1启动子萤光素酶报告基因至UMUC3细胞并筛选后,检测ISO预处理后萤光素酶活性。ISO预处理UMUC3细胞后,提取核蛋白检测核转录因子表达水平的变化。分别稳定转染GFP和GFP-细胞周期蛋白D1表达构建载体,筛选克隆;ISO预处理后,Western blotting法检测细胞周期蛋白D1的蛋白表达水平,贴壁依赖性细胞生长实验法检测细胞生长能力的变化,流式细胞术检测细胞周期的变化。分别稳定转染人源性细胞周期蛋白D1野生型和-163位点突变型萤光素酶报告基因质粒载体至UMUC3细胞并筛选,ISO预处理后检测细胞的萤光素酶活性。最后利用染色质免疫沉淀的方法,以抗Sp1抗体检测ISO对Sp1与细胞周期蛋白D1启动子结合力的影响。结果:ISO可从mRNA和蛋白水平显著抑制肿瘤细胞的细胞周期蛋白D1水平,并且是从内源性转录水平来抑制细胞周期蛋白D1的转录。ISO可抑制、下调核转录因子Sp1的表达,并抑制Sp1与细胞周期蛋白D1启动子区域的结合力,该结合位点主要位于启动子-92到+27bp的5’-非翻译区。结论:ISO可通过下调Sp1核转录因子表达及其与细胞周期蛋白D1启动子的结合,从内源性转录水平来抑制细胞周期蛋白D1的表达,从而诱导G0/G1细胞周期阻滞并抑制肿瘤细胞增殖。我们的研究将为临床中药单体ISO综合治疗肿瘤提供新的策略。AIM ： To explore the molecular mechanism of down-regulating cyclin D1 expression by isorhaponti- genin （ISO）. METHODS： The protein expression of cyclin D1 in UMUC3, RTI12 and RT4 bladder cancer cells was de- termined by Western blotting after the ceils were treated with ISO. The total RNA isolated from UMUC3 ceils treated with ISO was subjected to RT-PCR for determining the cyclin D1 mRNA level. After stably transfected with cyclin D1 promoter- driven luciferase reporter, UMUC3 cells were treated with ISO to observe the inhibitory effect of ISO on cyclin D1 promoter transcriptional activity. The cytoplasmic and nuclear extracts were isolated from the UMUC3 cells treated with ISO and then subjected to Western blotting with the anti-Spl antibody. After the UMUC3 stable transfectants with either GFP or GFP-cyc- lin DI were treated with ISO, the protein expression of cyclin D1 was detected by Western blotting, and the anchorage-in- dependent growth assay in soft agar and flow cytometry were conducted for cell cycle analysis. Wild-type cyclin D1 lucifer- ase reporter or its mutant at - 163 site were cotransfected with pSUPER plasmid into UMUC3 cells for determining cyelin D1 promoter activity after pretreatment with ISO. Chromatin immunoprecipitation （CHIP） assay was used to analyze the effectof ISO on Spl binding activity to the cyclin D1 promoter region using anti-Spl antibody. RESULTS ： ISO showed a signifi- cant inhibitory effect on cyclin DI mRNA and protein expression at the transcription level. Further studies identified that ISO down-regulated cyclin DI gene transcription via inhibition of Spl transactivation. Moreover, exogenous expression of GFP-cyclin D1 rendered UMUC3 cells resistant to induction of G0/GL cell cycle arrest and inhibition of cancer cell anchor- age-independent growth by ISO treatment. ChIP assay dernonstrated that ISO induced G0/Gl cell cycle arrest and inhibited cancer cell anchorage-independent growth through down-regulating Spl/cyclin D1 axis in bladder cancer cells at the

关 键 词：异丹叶大黄素 膀胱癌 细胞周期蛋白D1 Sp1转录因子 细胞周期阻滞 

分 类 号：R737.1[医药卫生—肿瘤]