MEG3基因敲除对小鼠前列腺正常功能的影响和与前列腺癌转移的机制研究

The incidence of prostate cancer (PCa) in China is increasing significantly in recent years due to an aging male population. It is important to understand the molecular mechanisms underlying PCa etiology and progression. Long non-coding RNAs (lncRNAs) are important regulators of various physiological processes and have also been involved in cancer development. The lncRNA MEG3 has been shown to be downregulated in different tumor types; however, the biological significance of this downregulation in regulating cancer biology and tumor cell behavior remains, in general, unclear. Work from our lab has shown that 1) MEG3 is primarily expressed in prostate basal/stem cells and its expression is downregulated in PCa as well as in metastases; 2) Underexpression of MEG3 in PCa is associated with advanced clinical stage and poor patient overall survival; 3) In experimental settings, MEG3 overexpression suppressed PCa cell proliferation, colony and sphere formation, and Matrigel invasion in vitro; 4) In vivo, MEG3 overexpression suppressed tumor growth and the suppressive function was turned around by inhibiting MEG3 expression; 5) Mechanistically, MEG3 directly binds to EZH2, resulting in impaired expression of EZH2 downstream targets such as DAB2IP. However, whether and how MEG3 downregulation causally and directly contributes to PCa development and/or progression are unclear. My goal in this proposal is to address these KEY questions. First of all, I’ll create the prostate-specific MEG3 knockout mouse model, deleting MEG3 gene in prostate basal or luminal cells, respectively. Then I’ll carefully monitor mouse behavior and organ development in different stages, including heart, brain, spleen, lung, kidney and as well as in urogenital system, including prostate, bladder, testis via HE staining and IHC staining; 2) I’ll analyze the network of MEG3 binding proteins, identify 3-5 prominent candidates by employing MEG3 RNA pull-down and proteomics technique, such as MS-SPEC and focus on one candidate X by using functional screening assay; 3) I’ll investigate RNA network targeted by MEG3-X and identify the most important genes by employing RNA-seq; 4) the therapeutic effects of MEG3 will be evaluated via systemic delivery. Accomplishment of the goals proposed herein will provide new perspectives in understanding the loss of MEG3 among other cancer types, and also lay a solid groundwork for developing RNA based novel therapeutics.

以往研究显示非编码RNA-MEG3在肿瘤中广泛缺失 。我们的早期研究发现：1)MEG3主要在前列腺基底细胞表达，在前列腺癌中表达明显下调且与恶性程度有关；2) MEG3对前列腺癌细胞和肿瘤生长有抑制作用；3)MEG3与EZH2直接结合调节其下游基因的表达。但是，MEG3表达下调是否与前列腺癌发生直接相关尚不明确。为了回答这一问题，本项目拟从以下三方面进行研究：1)建立前列腺MEG3特异敲除小鼠模型，观察小鼠在不同发育时期前列腺发育的异常以及是否有肿瘤形成或转移；2)利用蛋白质组学技术分析MEG3结合蛋白调控网络，鉴定新的MEG3特异结合蛋白；3) 利用转录组学技术分析MEG3与特异结合蛋白的RNA调控网络和重要靶基因；4)采用原位注射方法评价MEG3对前列腺癌的治疗效果。本课题将为阐明MEG3在其它肿瘤中缺失的原因提供新的视角，也为RNA治疗药物开发提供重要的研究基础。

我们的早期研究发现：1)MEG3主要在前列腺基底细胞表达，在前列腺癌中表达明显下调且与恶性程度有关；2) MEG3对前列腺癌细胞和肿瘤生长有抑制作用；3)MEG3与EZH2直接结合调节其下游基因的表达。但是，MEG3表达下调是否与前列腺癌发生直接相关尚不明确。为了回答这一问题，本项目成功建立三种不同的MEG3特异敲除小鼠模型，发现基底细胞中缺乏MEG3的成年小鼠容易发生不明原因死亡。当在前列腺管腔细胞中敲除MEG3后，小鼠前列腺结构和形态的变化在早期不明显。由于前列腺癌一般发生于老年人，结合很多体外研究数据，我们预测老年小鼠将有一些异常。体外研究发现：1）通过蛋白质组学技术分析MEG3结合蛋白调控网络，成功鉴定出数百个MEG3特异结合蛋白，发现Meg3对细胞周期、染色质结构重塑、DNA损伤修复中不可或缺。2）转录组学技术分析也发现MEG3对相似信号通道中发挥关键作用。3）当恢复MEG3在前列腺癌细胞中的表达水平后，细胞生长明显减慢，肿瘤发生率也降低。提示了MEG3对前列腺癌的潜在治疗效果。本课题第一次探索了MEG3对成年小鼠生长和发育的影响，并在小鼠身上探索了MEG3治疗前列腺癌的有效性。这些成果为彻底阐明MEG3本身的生理功能和病理功能奠定了重要基石。