NS调节核糖体蛋白合成促进前列腺癌细胞增殖机制的研究

发布时间：2018-05-07 13:33

本文选题：核干因子 + 核糖体生物合成　；参考：《天津医科大学》2015年博士论文

【摘要】：目的探讨NS调节核糖体蛋白合成对前列腺癌细胞增殖的影响,并明确其分子机制。方法首先,利用Oncomine数据库逐个检索RP(ribosomal protein,RP)在良性前列腺增生(Benign prostatic hyperplasia,BPH)组织与PCa(Prostate cancer,PCa)组织中表达的差异;然后,收集国人的PCa及增生组织标本,提取RNA反转录行RT-qPCR检测以上核糖体蛋白mRNA的表达水平;进而,挑选了核糖体大小亚基蛋白中RP差异表达最为明显的两个,通过western blot检测二者在PCa组织中的蛋白表达情况,并分析其与PCa患者临床病理特征以及PSA生化复发的关系。构建NS低表达PC3细胞系,通过蔗糖梯度离心检测NS低表达后对核糖体大小亚基生物合成的影响;通过RT-qPCR检测NS低表达后核糖体蛋白mRNA水平表达情况;筛选变化最明显的核糖体蛋白,通过体内、外实验探讨该核糖体蛋白对PCa发生发展中的作用。构建c-Myc高表达PC3细胞系,再使用c-Myc小分子抑制剂10058-F4处理PC3细胞,通过RT-qPCR及Western blot检测细胞中NS的表达水平,使用DCFH-DA探针检测细胞内ROS的水平;用H2O2上调PC3细胞内的ROS,过氧化氢酶Catalase降低PC3细胞内的ROS,进而行western blot及qPCR检测细胞内的ROS对NS表达的影响;通过细胞计数法及MTT分别检测c-Myc、ROS及NS对PC3细胞增殖能力的影响。结果通过Oncomine网络数据库分析,我们发现在PCa组织内存在核糖体大、小亚基蛋白的高表达,其中RPS蛋白有15种、RPL蛋白有14种。我们通过RT-PCR检测这些核糖体蛋白在国人PCa及前列腺增生组织内的表达时,发现在国人PCa组织中各有11种RPS和RPL存在差异性表达,其中RPS2和RPL36的表达差异性最为显著。我们分别分析了RPS2和RPL36与PCa患者临床病理学资料的关系,发现此二种蛋白与患者的病理及预后密切相关。降低PC3细胞中NS的表达水平后核糖体大亚基合成受抑制;RT-qPCR结果显示NS低表达后大亚基核糖体蛋白mRNA水平均下调;其中RPL19下调最为明显,其在蛋白水平的变化和NS具有一致性;靶向沉默PC3细胞中的RPL19后,发现PC3细胞增殖减慢、凋亡增加且侵袭能力减弱;体内实验显示,沉默RPL19后,PC3在裸鼠体内的成瘤能力下降。上调PC3细胞内的c-Myc可同时上调细胞内ROS和NS的mRNA及蛋白质表达水平;细胞内的ROS水平可增加NS的蛋白表达水平。细胞增殖实验结果显示:c-Myc高表达促进PC3细胞的增殖;提高细胞内的ROS水平可促进PC3细胞的增殖;降低PC3细胞中的NS的水平,可以抑制PC3细胞的增殖。结论在前列腺癌中存在c-Myc-ROS-NS信号通路调节NS的异常表达,NS的异常表达进而通过调节核糖体大亚基蛋白(尤其是RPL19)促进前列腺癌细胞的增殖。
[Abstract]:Objective to investigate the effects of NS on the proliferation of prostate cancer cells and its molecular mechanism. Methods first, the difference of the expression of RP(ribosomal protein Oncomine in benign prostatic hyperplasia (BPH) and PCa(Prostate cancer-PCA (BPH) was searched by Oncomine database, and then the specimens of PCa and proliferative tissue were collected from Chinese people. RNA reverse transcription RT-qPCR was extracted to detect the mRNA expression level of ribosomal protein, and then two of the ribosome size subunit proteins were selected to detect the expression of RP in PCa tissues by western blot. The clinicopathological features and biochemical recurrence of PSA in patients with PCa were analyzed. PC3 cell line with low expression of NS was constructed and the effect of low expression of NS on biosynthesis of ribosomal subunit was detected by sucrose gradient centrifugation, and the expression of ribosomal protein mRNA after low expression of NS was detected by RT-qPCR. The role of ribosomal protein in the development of PCa was studied in vivo and in vitro. C-Myc overexpression PC3 cell line was constructed, then PC3 cells were treated with c-Myc small molecule inhibitor 10058-F4. The expression of NS was detected by RT-qPCR and Western blot, and ROS was detected by DCFH-DA probe. H2O2 up-regulated ros in PC3 cells, catalase Catalase decreased rose in PC3 cells, and then western blot and qPCR were used to detect the effect of ROS on NS expression, and the effects of c-Myc Ros and NS on the proliferation of PC3 cells were detected by cell count and MTT, respectively. Results through the analysis of Oncomine network database, we found that there were high expression of ribosomal protein and small subunit protein in PCa tissue, of which there were 15 kinds of RPS protein and 14 species of RPS protein. When we detected the expression of these ribosomal proteins in Chinese PCa and prostatic hyperplasia by RT-PCR, we found that there were 11 different RPS and RPL expressions in Chinese PCa, and RPS2 and RPL36 were the most significant. We analyzed the relationship between RPS2 and RPL36 and the clinicopathological data of PCa patients, and found that these two proteins were closely related to the pathology and prognosis of PCa patients. The results of RT-qPCR showed that the mRNA level of large subunit ribosomal protein was down-regulated after decreasing the expression level of NS in PC3 cells, in which RPL19 down-regulation was the most obvious, and the changes in protein level were consistent with NS. After targeting RPL19 in PC3 cells, the proliferation of PC3 cells slowed down, the apoptosis increased and the invasiveness decreased. In vivo experiments showed that the tumorigenic ability of pPC3 in nude mice was decreased after RPL19 silencing. Upregulation of c-Myc in PC3 cells could increase the expression of mRNA and protein in both ROS and NS cells, while ROS level in cells could increase the protein expression level of NS. The results of cell proliferation assay showed that the high expression of w _ c-Myc could promote the proliferation of PC3 cells, increase the level of ROS in cells could promote the proliferation of PC3 cells, and decrease the level of NS in PC3 cells, which could inhibit the proliferation of PC3 cells. Conclusion there is an abnormal expression of NS regulated by c-Myc-ROS-NS signaling pathway in prostate cancer and the abnormal expression of NS can promote the proliferation of prostate cancer cells by regulating ribosomal large subunit protein (especially RPL19).
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.25

【参考文献】