卵巢上皮性癌组织中p16INK4A基因表达缺陷的意义及其与甲基化的关系  

Disfigurement of p16INK4A gene expression in development of ovarian cancer and the mechanism

作　　者：李敏[1] 黄在菊[1] 董卫红[1] 李晓艳[1] 王晓翊[1] 贺晓红[1] 王瀚[1] 王泽华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院妇产科,武汉430022

出　　处：《中华妇产科杂志》2006年第6期408-412,共5页Chinese Journal of Obstetrics and Gynecology

基　　金：国家自然科学基金资助项目(30070786)

摘　　要：目的研究抑癌基因p16INK4A在卵巢上皮性癌(卵巢癌)组织及细胞系中的表达变化,分析其表达变化与甲基化的关系。方法选取7种卵巢癌细胞系、18份卵巢癌组织和10份正常卵巢组织为研究对象。采用甲基化特异性PCR方法检测p16INK4A基因甲基化状态;RT-PCR技术检测p16INK4A基因的mRNA表达;蛋白印迹(western blot)法检测P16INK4A蛋白的表达。5-杂氮-2′-脱氧胞苷对p16INK4A基因甲基化的卵巢癌细胞进行去甲基化处理,再次进行p16INK4A基因的mRNA和蛋白表达的检测,以及p16INK4A基因甲基化的分析。检测5-杂氮-2′-脱氧胞苷处理前后卵巢癌细胞的生长情况;并将处理前后的细胞接种于裸鼠,观测肿瘤的体积、重量。结果3种卵巢癌细胞系(Anglne、SW626和OVCAR3细胞)、6份卵巢癌组织中存在p16INK4A基因甲基化,卵巢癌细胞和卵巢癌组织中的甲基化率分别为3/7和33%(6/18)。卵巢癌细胞系、卵巢癌组织和正常卵巢组织中,p16INK4A基因的mRNA相对含量的平均值分别为0·34±0·11、0·81±0·13、1·52±0·12,蛋白相对含量的平均值分别为0·56±0·14、1·32±0·12、2·09±0·11,卵巢癌细胞系、卵巢癌组织分别与正常卵巢组织相比,差异均有统计学意义(P<0·05)。有甲基化表现的卵巢癌细胞和组织中p16INK4A基因的mRNA和蛋白表达均下降。5-杂氮-2′-脱氧胞苷处理能使p16INK4A基因甲基化的卵巢癌细胞中的p16INK4A基因的mRNA和蛋白重新表达或表达增高。与去甲基化处理前比较,去甲基化处理后Anglne、SW626和OVCAR3细胞的生长速度均减慢;接种去甲基化处理的OVCAR3细胞的裸鼠中,肿瘤体积和重量明显减小,分别为(0·243±0·022)cm3、(0·035±0·004)g。结论p16INK4A基因的表达下降或缺失在卵巢癌的发生中起重要作用,DNA甲基化是其表达缺陷的原因,去甲基化处理可以恢复p16INK4A基因的表达并抑制卵巢癌细胞的增殖。Objective To evaluate the expression of p16INK4A gene in ovarian cancer and analyze the relation between this alteration and the promoter methylation of p16INK4A DNA. Methods The expression of p16INK4A mRNA was detected with RT-PCR in ovarian cancer cell lines and tissues, and the protein was detected by western blot. Methylation specific PCR （MSP） was used to check whether it was methylated in the promoter of p16INK4A, and normal ovarian tissues were used as control. The demethylating agent, 5-aza-2＇-deoxycytidine, was used to treat methylated ovarian cancer cells and then, ovarian cancer cell growth was measured in vivo and in vitro. Results Three ovarian cancer cell lines （Anglne, SW626, OVCAR3）and six ovarian cancer specimens were found methylated in p16INK4A DNA; the methylation rate was 3/7 and 33% （6/18） in ovarian cancer cell lines and specimens respectively. All the methylated cell lines and ovarian cancer tissues expressed decreasing p16INK4A mRNA and protein. Compared with the control, both the expression of p16INK4A mRNA and protein were decreased significantly or absolutely deleted in ovarian cancer cells（ P 〈0. 05 ）. The decrease was partly due to the methylation of p16INK4A. 5-aza-2＇-deoxycytidine could reactivate the expression of p16INK4A in methylated cells and decrease the cell growth in vivo and in vitro. Conclusion The disfigurement of p16INK4A gene plays an important role in the development of ovarian cancer, and this alteration is partially caused by methylation of p16INK4A DNA.

关 键 词：卵巢肿瘤 蛋白质P16 基因 P16 DNA甲基化 脱氧胞苷 

分 类 号：R737.31[医药卫生—肿瘤]