Zeb1通过诱导干性促进前列腺癌的雄激素非依赖

发布时间：2018-05-13 15:17

本文选题：雄激素非依赖前列腺癌 + 上皮间质转化　；参考：《上海交通大学》2014年博士论文

【摘要】：目的:抗雄性激素疗法在前列腺癌临床治疗的初期效果显著,但不能够杀灭所有的肿瘤细胞,最终导致雄激素非依赖前列腺癌(AIPC)的发生,目前仍然是前列腺癌治疗中的焦点和难点。残存的、雄激素非依赖的肿瘤细胞可能具有与其他雄激素依赖的肿瘤细胞不同的生物学特性,这些细胞的形态学及其他特性目前尚不清楚。相关研究表明,上皮间质转化(EMT)在肿瘤的发生、发展、转移及耐药等多方面起着很重要的作用。本课题旨在探索和分析前列腺癌从雄激素依赖转变成雄激素非依赖过程中EMT转录因子Zeb1是否及如何参与前列腺癌的雄激素非依赖的形成。方法:从细胞、组织和动物水平利用实时定量PCR、免疫组化、免疫荧光、免疫印迹等实验技术分别从mRNA水平和蛋白水平来检测Zeb1的表达情况。通过慢病毒干扰和过表达的方法通过一系列体内体外实验来探讨下调和上调Zeb1之后细胞的形态及功能学改变。计数数据采用χ2检验方法,单因素的比较采用t检验。统计分析和作图采用Prism GraphPad5。P0.05被认为具有统计学意义。结果:与雄激素依赖的前列腺癌细胞系(LNCa P,PC3 AR(+)和22RV1)相比,雄激素非依赖前列腺癌细胞系中(C4-2B,PC3和DU145),Zeb1的表达明显增加,伴随着Vimentin表达的增加和E-cadherin表达的减少。在去势后的PTEN条件性敲除小鼠前列腺肿瘤组织中,Zeb1的表达明显升高伴随着上皮标志E-cadherin和间质标志Vimentin的共表达。另外,与良性前列腺增生组织相比,前列腺癌组织标本中Zeb1的表达明显升高,而且与前列腺癌的转移密切相关。进一步过表达Zeb1之后,细胞对抗雄激素药物的耐受性增加,迁移能力增强。体内移植瘤实验发现,去势之后肿瘤仍然能够继续生长。而干扰Zeb1的表达后,细胞对抗雄激素药物的敏感性增加,迁移能力下降。有趣的是,Zeb1的表达伴随着一些多能干细胞标志物如Sox2的变化,以及细胞克隆形成能力和成球能力的改变。同时,对小鼠进行去势处理之后,小鼠前列腺组织中Zeb1与Sox2的表达均出现升高。进一步免疫沉淀发现Zeb1能够特异性的与Sox2蛋白相结合。结论:EMT转录调控因子Zeb1可能通过诱导干细胞样特性促进前列腺癌雄激素非依赖的形成和转移。调控Zeb1抑制上皮间质转化过程,或许可以为前列腺癌的临床治疗提供潜在的可能分子靶标。
[Abstract]:Objective: antiandrogen therapy is effective in the early stage of clinical treatment of prostate cancer, but it can not kill all tumor cells, leading to the development of androgen independent prostate cancer (AICC). At present, prostate cancer treatment is still the focus and difficulty. The remaining androgen independent tumor cells may have different biological characteristics from those of other androgen dependent tumor cells, the morphological and other characteristics of which are unclear. Related studies have shown that EMTs play an important role in carcinogenesis, development, metastasis and drug resistance. The purpose of this study was to explore whether and how EMT transcription factor Zeb1 is involved in the formation of androgen independence in prostate cancer from androgen dependent to androgen independent. Methods: the expression of Zeb1 was detected by real-time quantitative PCR, immunohistochemistry, immunofluorescence and Western blotting at cell, tissue and animal levels, respectively. By means of lentivirus interference and overexpression, a series of experiments in vitro and in vivo were carried out to investigate the morphological and functional changes of Zeb1 after down-regulation and up-regulation. 蠂 2 test was used for counting data and t test was used for single factor comparison. The use of Prism GraphPad5.P0.05 in statistical analysis and mapping is considered statistically significant. Results: compared with androgen-dependent prostate cancer cell lines (LNCA PnPC3AR3 () and 22RV1), androgen independent prostate cancer cell lines increased the expression of C4-2BmPC3 and DU145ZEb1, accompanied by the increase of Vimentin expression and the decrease of E-cadherin expression. In castrated PTEN conditioned knockout mice, the expression of Zeb1 was significantly increased with the coexpression of epithelial marker E-cadherin and interstitial marker Vimentin. In addition, the expression of Zeb1 in prostate cancer tissues was significantly higher than that in benign prostatic hyperplasia tissues, and was closely related to the metastasis of prostate cancer. After further overexpression of Zeb1, cell resistance to androgen drugs increased and migration ability increased. In vivo transplanted tumor experiments found that the tumor can continue to grow after castration. After interfering with the expression of Zeb1, the sensitivity of the cells to androgen drugs was increased, and the migration ability was decreased. Interestingly, the expression of Zeb1 was accompanied by changes in some pluripotent stem cell markers such as Sox2, as well as the ability of cell clone formation and spherulation. At the same time, the expression of Zeb1 and Sox2 in the prostate tissues of mice increased after castration. Further immunoprecipitation revealed that Zeb1 could specifically bind to Sox2 protein. Conclusion Zeb1 may promote androgen independent development and metastasis of prostate cancer by inducing stem cell-like characteristics. Regulating the inhibition of epithelial mesenchymal transformation by Zeb1 may provide a potential molecular target for the clinical treatment of prostate cancer.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

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