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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 p16 INK4A in human prostate cancer cell line PC-3 and its possible molecular mechanism. Methods: PC-3 cells were treated with DHA at different concentrations (25, 50 and 100 μmol / L) for 48 h, and the untreated DHA group was also set up. Then, the apoptosis rate and level of reactive oxygen species (ROS) in each group were detected by FCM. The methylation level of p16 INK4A gene was detected by bisulfite sequencing and the real-time fluorescent quantitative PCR Western blotting was used to detect the expression of m RNA and protein of DNA methyltransferase 1 (DNMT1) and p INK4A respectively. The localization and expression of p16 INK4A protein were detected by immunofluorescence . Results: Compared with the blank control group, the apoptotic rate and the ROS level of PC-3 cells in each concentration of DHA group were significantly increased (P <0.05). After DHA treatment, the methylation level of p16 INK4A gene in PC-3 cells was significantly decreased (P <0.05), while the expression of DNMT1 mRNA and protein was also significantly decreased (P <0.05) INK4A mRNA and protein expression levels were significantly increased (P all <0.05). The expression of p16 INK4A protein in the nucleus and cytoplasm was significantly higher than that in the blank control group. The fluorescence intensity of p16 INK4A protein in DHA group was significantly increased (P <0.05). CONCLUSION: DHA may restore the expression of p16 INK4A protein by down-regulating the expression of DNMT1 gene and eventually induce the apoptosis of prostate cancer PC-3 cells, accompanied with ROS produce.