雄激素受体-miR-124环式互调控抑制前列腺癌的增殖

发布时间：2018-06-02 05:42

本文选题：前列腺癌 + 雄激素受体　；参考：《上海交通大学》2015年博士论文

【摘要】：目的:前列腺癌是男性泌尿生殖系统中最常见的恶性肿瘤之一。在前列腺癌的发生、发展过程中雄激素受体(androgen receptor,AR)信号通路发挥重要的作用。AR通过介导下游多种编码及非编码基因(例如microRNA)的表达,调控前列腺癌的增殖。已有研究表明,microRNA-124(miR-124)在前列腺癌中发挥抑癌基因的功能,但是其上游调控基因及相关分子机制尚不明确。本论文以AR及miR-124为研究对象,基于已有的研究成果通过解析两者之间的调控模式,探索AR及mi R-124参与调控前列腺癌增殖的可能机制。方法:在AR阴性的前列腺癌细胞系PC3中构建AR过表达亚克隆细胞系PC3/AR和对照PC3/neo;在AR阳性的前列腺癌细胞系LNCaP中使用包含AR靶向shRNA(或对照)的慢病毒感染获得AR持续敲降细胞系LNCaP-sh-AR和对照LNCaP-sh-control;在该细胞系中使用包含成熟miR-124(或对照)的慢病毒感染获得稳定过表达miR-124细胞系LNCaP-miR-124和对照LNCaP-control。采用实时定量反转录PCR方法检测AR表达量改变对miR-124表达产生的影响及miR-124表达变化对AR表达产生的影响。通过雄激素应答实验,检测雄激素能否引起miR-124的表达应答。通过细胞增殖实验、细胞侵润实验、裸鼠皮下植瘤实验,在体外及体内验证过表达miR-124对前列腺癌细胞增殖的抑制作用。通过重亚硫酸盐测序法检测pri-miR-124-3启动子区域的甲基化程度。使用Prism GraphPad5软件进行统计分析和作图。采用学生式t检验比较不同组间差异,当p0.05时认为两者具有统计学意义上的显著差异。结果:本研究中,我们发现AR与miR-124之间存在环式互调控,即AR激活后可以促进miR-124的转录表达,同时miR-124则在转录后水平上抑制AR的表达,在正常生理状态下实现稳态。在前列腺癌细胞中,由于pri-miR-124-3启动子区域高度甲基化阻碍了AR与启动子的互作,导致miR-124的表达降低及互调控稳态的破坏。通过外源恢复miR-124的表达,在体外及体内均能抑制前列腺癌细胞的增殖及成瘤能力。结论:AR与miR-124通过环式互调控方式实现的稳态,在前列腺癌细胞中由于启动子区域甲基化遭到破坏。通过外源恢复miR-124表达可以有效抑制前列腺癌的增殖。
[Abstract]:Objective: prostate cancer is one of the most common malignant tumors in male genitourinary system. Androgen receptor AR-signaling pathway plays an important role in the pathogenesis and development of prostate cancer. AR regulates the proliferation of prostate cancer by mediating the expression of multiple coding and non-coding genes (such as microRNAs) downstream. It has been shown that microRNA-124miR-124) plays a role as a tumor suppressor gene in prostate cancer, but its upstream regulatory gene and its related molecular mechanisms are not clear. In this thesis, AR and miR-124 were taken as the research objects. Based on the existing research results, the possible mechanism of AR and miR-124 involved in the regulation of prostate cancer proliferation was explored by analyzing the regulatory models between them. Methods: construction of AR overexpression subclone cell line PC3/AR and control PC3 / neoc in AR negative prostate cancer cell line PC3 and AR positive prostate cancer cell line LNCaP using lentivirus containing AR targeted shRNAs (or controls) to obtain AR persistence LNCaP-sh-AR and control LNCaP-sh-control cell lines were used to obtain stable expression of miR-124 LNCaP-miR-124 and control LNCaP-control cells by using lentivirus infection containing mature miR-124 (or control). Real time quantitative reverse transcription PCR was used to detect the effect of AR expression on miR-124 expression and miR-124 expression on AR expression. Androgen response test was used to determine whether androgen can induce miR-124 expression response. The inhibitory effect of overexpression of miR-124 on the proliferation of prostate cancer cells in vitro and in vivo was verified by cell proliferation assay, cell infiltration test and subcutaneous tumor implantation in nude mice. The methylation of pri-miR-124-3 promoter region was detected by bisulfite sequencing. Use Prism GraphPad5 software for statistical analysis and mapping. Student t test was used to compare the difference between different groups. When p0.05, the difference between the two groups was statistically significant. Results: in this study, we found that there is cyclic regulation between AR and miR-124, that is, AR activation can promote the transcription expression of miR-124, while miR-124 inhibits AR expression at posttranscriptional level and achieves homeostasis in normal physiological state. In prostate cancer cells, the hypermethylation of pri-miR-124-3 promoter region hinders the interaction between AR and promoter, which leads to the decrease of miR-124 expression and the destruction of the stable state of mutual regulation. By restoring the expression of miR-124, the proliferation and tumorigenesis of prostate cancer cells were inhibited in vitro and in vivo. Conclusion the homeostasis of miR-124 and AR is destroyed by promoter region methylation in prostate cancer cells. Restoring miR-124 expression by exogenous can effectively inhibit the proliferation 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】：2015
【分类号】：R737.25

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