双氢青蒿素对前列腺癌PC-3细胞中p16^(INK4A)蛋白表达的调控作用及其机制探讨  

Regulation effect of dihydroartemisinin on p16^(INK4A) protein expression in human prostate cancer PC-3 cells and its possible mechanism

作　　者：杜施娟 许舸[1] 邹文琴[1] 罗子国[1] 

机构地区：[1]重庆医科大学生命科学研究院,重庆400016

出　　处：《肿瘤》2017年第1期41-49,共9页Tumor

基　　金：重庆市教委科学技术研究项目(编号:KJ110324)~~

摘　　要：目的 :探讨双氢青蒿素(dihydroartemisinin,DHA)对人前列腺癌PC-3细胞株中p16^(INK4A)表达的影响及其可能的分子机制。方法 :用不同浓度(25、50和100μmol/L)的DHA处理PC-3细胞48 h,同时设置DHA未处理的空白对照组。然后,采用FCM法检测各组细胞的凋亡率和活性氧(reactive oxygen species,ROS)水平;亚硫酸氢盐基因组测序法检测p16^(INK4A)基因的甲基化水平;实时荧光定量PCR法和蛋白质印迹法分别检测DNA甲基化转移酶1(DNA methyltransferase 1,DNMT1)和p^(INK4A)的m RNA及蛋白表达水平;细胞免疫荧光法检测p16^(INK4A)蛋白的定位及表达情况。结果 :与空白对照组相比,各浓度DHA处理组的PC-3细胞凋亡率和ROS水平均明显升高(P值均<0.05)。DHA处理后,PC-3细胞中p16^(INK4A)基因的甲基化水平明显降低(P<0.05),DNMT1 mRNA和蛋白的表达水平也明显降低(P值均<0.05),而p^(INK4A) mRNA和蛋白表达水平明显升高(P值均<0.05)。p16^(INK4A)蛋白在细胞核及细胞质中均有表达;与空白对照组相比,DHA处理组的p16^(INK4A)蛋白荧光强度明显提高(P<0.05)。结论:DHA可能通过下调DNMT1表达,使p16^(INK4A)基因的甲基化水平降低,从而恢复p16^(INK4A)蛋白的表达,最终诱导前列腺癌PC-3细胞凋亡,并伴有ROS的产生。Objective： To investigate the effect of dihydroartemisinin （DHA） on the expression of p16INK4A in human prostate cancer PC-3 cells, and to explore its possible molecular mechanism. Methods： The PC-3 cells were treated with different concentrations of DHA （25, 50, and 100μmol/L） for 48 h, while PC-3 cells without DHA treatment were used as the control. Then the apoptotic rate of PC-3 cells and the level of reactive oxygen species （ROS） in PC-3 cells of different groups were detected by FCM. The methylation level of p1 6INK4A gene was determined by bisulfite genomic sequencing. The expression levels of DNA methyltransferase 1 （DNMT1） and pl 6INK4A were examined by real-time fluorescent quantitative PCR and Western blotting, respectively. The cellular location and expression of p16INK4A protein in PC-3 cells were detected by immunofluorescence staining. Results： Compared with the control group, the apoptotic rate of PC-3 cells and ROS level in different concentrations of DHA-treated groups were significantly increased （all P 〈 0.05）. After treatment with DHA, the methylation level of p16INK4A gene in PC-3 cells was decreased （P 〈 0.05）, the expression levels of DNMT1 mRNA and protein were decreased （both P 〈 0.05）, but the expressions of p16INK4A mRNA and protein were up-regulated （both P 〈 0.05）. P16INK4A protein was expressed in both nucleus and cytoplasm of PC-3 cells. Compared with the control group, the fluorescence intensity of p16INK4A protein in DHA-treated group was significantly increased （P 〈 0.05）. Conclusion： DHA may down-regulate the expression of DNMT1, reduce the methylation level of p16INK4A gene, thereby restore the expression of p16INK4A protein, furthermore induce the apoptosis of prostate cancer PC-3 cells and produce ROS.

关 键 词：前列腺肿瘤 青蒿素类 细胞凋亡 DNA甲基化 P16INK4A基因 双氢青蒿素 

分 类 号：R737.25[医药卫生—肿瘤]