竞争性内源RNA做为EMT调控网络核心元件的系统性研究

Epithelial-to-mesenchymal transition (EMT) is a fundamental developmental process and has been implicated in metastasis, the development of cancer stem cells and resistance to chemotherapies. Recently, numerous lncRNAs that were aberrantly overexpressed in metastatic cancers have been shown to promote metastasis by regulating ZEB1 functionality via a competing endogenous RNA (ceRNA) based mechanism. While ceRNA has been shown to regulate EMT in various cancers, whether ceRNA represents an intrinsic component of the EMT regulatory circuits has not been explored. ..Recently, we developed a systematic approach that combined computational algorithms with experimental methods to reconstruct EMT regulatory networks utilizing time-course transcriptome data of cells undergoing TGF-beta-induced EMT. Critically, we showed that canonical EMT marker genes TGFBI, which have been consistently associated with EMT across a wide-range of tissues and cell types, are highly upregulated and function as ceRNA during TGF-beta-induced EMT. Critically, miRNAs other than miR-200s were key EMT regulators in our EMT cellular model, which strongly suggested that ceRNA could represent a conserved element of the EMT regulatory network...Here, we hypothesized that ceRNA represent an intrinsic component of EMT regulatory network and its function is essential for EMT. To address this question, we will first reconstruct EMT regulatory networks consisted of ceRNA, microRNA and master transcription factors by integrative mining time-course transcriptome data collected from three distinct cellular models of EMT with different miRNA as key EMT regulators. We will then analyze the role of ceRNA in EMT via a combination of computational simulation, single-cell analysis of cellular states upon TGF-beta treatment and classical molecular biology techniques such as siRNA-mediated silencing of target ceRNAs. Finally, we will evaluate the utility of conserved EMT-associated ceRNAs via construction of ceRNA-based models for cancer classification. ..Both ceRNA and EMT have been the focus of our laboratory and we have established competent tool-sets to computationally analyze ceRNA, reconstruct EMT regulatory networks and perform experimental validations. The technical expertise together with our established results on roles of ceRNA in EMT will enable an effective execution of the proposed project. The discoveries from proposed research will shed crucial insights into EMT regulatory circuits and establish EMT-associated ceRNAs as broadly applicable cancer markers that could be valuable for precision medicine for cancer.

多个在肿瘤中高表达的lncRNA已被确认为miR-200的竞争性内源RNA（ceRNA），进而调控上皮细胞向间质细胞转变（EMT）。但是，目前尚不清楚ceRNA能影响EMT是肿瘤突变的结果，还是ceRNA本身就是EMT调控必需的核心元件。在前期工作中，我们发现在依赖不同miRNA的EMT模型中，进行EMT的细胞均显著上调EMT相关miRNA的ceRNA。因此，本课题提出ceRNA是EMT核心调控元件的假说。我们将采用计算与实验相结合的方法，在多种EMT细胞模型中构建、验证含有ceRNA的EMT调控网络，阐明ceRNA动态变化调控EMT的机理，并建立基于EMT相关ceRNA的肿瘤精准分型方法。本课题组在计算分析ceRNA动态变化，构建、验证EMT调控网络等方面有丰富的经验。本课题的成果将能全方位扩展EMT核心调控网络，确认EMT相关ceRNA作为肿瘤标记物的价值，并对肿瘤的精准医疗做出贡献。

上皮细胞向间质细胞转变(EMT)是发育和肿瘤转移的一个核心过程。尽管大量研究揭示了基于转录因子SNAIL和miRNA-34,ZEB和miR-200c形成的双向负反馈网路可以调控EMT，但是并不清楚是否存在其他机制。ceRNA是目前比较有争议的一个假说，主流的研究指出在生理条件下ceRNA不太可能具有生物学功能。在前期工作中，我们发现在依赖不同miRNA的EMT 模型中，进行EMT的细胞均显著上调EMT相关miRNA的ceRNA。在本课题执行过程中，我们采用计算与实验相结合的方法，在A549和MCF10A EMT细胞模型中构建、验证了含有ceRNA的EMT调控网络，确认了在生理状态下，单个mRNA 就可以具有ceRNA功能，同时ceRNA的动态变化在时间轴上与双向负反馈网路耦合，进而精准的调控EMT过程中由E向M态转化。本课题的成果首次揭示了双稳态与ceRNA的内在联系，在机制上深入扩展EMT核心调控网络，同时纠正了 ceRNA领域的误解，确认了ceRNA 在生理状态下，特别是肿瘤转移过程中具有重要的功能。本课题的成果揭示了EMT动态变化的新机制，确认了EMT相关ceRNA作为肿瘤标记物的价值，对开发基于肿瘤转移的动态变化的精准医疗新方法提供了新思路。