双氢青蒿素对前列腺癌PC-3细胞UHRF1的影响及调控机制研究

发布时间：2018-04-28 12:56

本文选题：前列腺癌 + 双氢青蒿素　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:探讨双氢青蒿素(Dihydroartemisinin,DHA)作用人前列腺癌PC-3细胞株后,泛素样含PHD和环指域1(Ubiquitin-like with PHD and ring finger domains 1,UHRF1)的表达改变及可能诱导的甲基化调控机制。方法:不同浓度(0、25、50和100μmol/L)DHA处理PC-3细胞株48 h,DMSO处理的未加药组为空白对照组。MTT法检测细胞增殖活性。FCM法检测细胞凋亡率、周期分布及活性氧水平(Reactive oxygen species,ROS)。亚硫酸氢盐基因组测序法(BGS)检测p16INK4A基因启动子区域的甲基化水平。实时荧光定量PCR检测UHRF1、甲基化转移酶1(DNA methyltransferase 1,DNMT1)及p16INK4A mRNA表达。蛋白质印迹法检测UHRF1、DNMT1及p16INK4A蛋白的表达水平。细胞免疫荧光法检测p16INK4A蛋白的定位及表达。结果:1.MTT和FCM结果表明DHA能通过诱导PC-3细胞凋亡和G1/S期阻滞而抑制细胞增殖,并伴有ROS水平的升高。2.BGS结果显示DHA作用PC-3细胞后,p16INK4A基因启动子区域的甲基化水平明显降低。3.RT-PCR和Western blotting结果显示DHA诱导UHRF1和DNMT1 mRNA及蛋白表达水平明显降低,而p16INK4A表达水平升高。4.细胞免疫荧光结果显示p16INK4A蛋白在细胞核及细胞质中均有表达。DHA作用PC-3细胞后,p16INK4A蛋白定位未发生改变,但p16INK4A蛋白的荧光强度显著增加。结论:DHA可能通过抑制UHRF1和DNMT1的表达,下调p16INK4A基因启动子区域的甲基化水平,从而解除p16INK4A蛋白的表达抑制。最终DHA抑制PC-3细胞增殖,诱导细胞凋亡及G1/S期阻滞,并伴有ROS的生成。DHA作用PC-3细胞的机制可能与UHRF1对p16INK4A的调控有关。
[Abstract]:Aim: to investigate the effect of dihydroartemisinine dihydroartemisinin (DHA) on the expression of PHD and 1(Ubiquitin-like with PHD and ring finger domains 1 UHRF1 in human prostate cancer cell line PC-3 and the possible mechanism of methylation regulation induced by dihydroartemisinine dihydroartemisin (DHA). Methods: the PC-3 cell lines treated with different concentrations of 渭 mol/L)DHA and 25 渭 mol/L)DHA for 48 h were treated with DMSO for 48 h. The proliferative activity of PC-3 cells was detected by MTT assay. The apoptosis rate, cell cycle distribution and reactive oxygen speciesrose were detected by FCM method. The methylation level of promoter region of p16INK4A gene was detected by bisulfite genome sequencing. The expression of UHRF1, 1(DNA methyltransferase 1 DNMT1 and p16INK4A mRNA were detected by real-time fluorescence quantitative PCR. The expression of UHRF1 DNMT1 and p16INK4A protein was detected by Western blot. The localization and expression of p16INK4A protein were detected by immunofluorescence assay. Results 1. The results of MTT and FCM showed that DHA could inhibit the proliferation of PC-3 cells by inducing apoptosis and G 1 / S arrest. The results showed that the methylation level of the promoter region of p16INK4A gene in PC-3 cells was significantly decreased after DHA treatment. 3. The results of RT-PCR and Western blotting showed that DHA induced a significant decrease in UHRF1, DNMT1 mRNA and protein expression, while the expression level of p16INK4A was increased by .4. The results of cellular immunofluorescence showed that the localization of p16INK4A protein in PC-3 cells did not change after the expression of p16INK4A protein in nucleus and cytoplasm, but the fluorescence intensity of p16INK4A protein increased significantly. ConclusionDHA may inhibit the expression of p16INK4A protein by inhibiting the expression of UHRF1 and DNMT1 and down-regulating the methylation level in the promoter region of p16INK4A gene. Finally, DHA inhibited the proliferation of PC-3 cells, induced apoptosis and arrest of G 1 / S phase, accompanied by the production of ROS. DHA may play a role in the regulation of p16INK4A by UHRF1.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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