长链非编码RNA MEG3结合蛋白调控乳腺癌发生发展的分子机制及临床意义

The presence of unexpected huge number of long non-coding RNA opens up a new research direction in the micro-regulation of gene expression in cancer, and it is crucial to identify lncRNA binding proteins in order to gain deep insight into the role of lncRNA and how it works. In previous work, we have developed an affinity purification method that can efficiently identify RNA binding proteins, and revealed key proteins for mRNA export (PNAS, 2011; NAR, 2013). Our preliminary data show that the expression of MEG3 is significantly decreased in breast cancer samples and re-expression of MEG3 in breast cancer cell line inhibits its growth, however，proteins bound to MEG3 that mediate its role in growth inhibition have not been identified yet, nor has the molecular mechanism on its regulation of occurrence and progression of breast cancer been elucidated. Thus we will first define proteins bound to MEG3 at the molecular level in this project, then screen for key factors directly underlying the role of MEG3 as growth suppressor. Secondly, we will explore the mechanism of MEG3 binding protein in the progression of breast cancer at the cellular level，by checking the expression of genes in P53 and PI3K pathway as well as in angiogenesis. Thirdly, we will examine breast cancer tumorigenesis in nude mice using stable cell line expressing MEG3 binding proteins. Finally, we will determine the expression of MEG3 and its binding proteins in clinical breast cancer samples and perform correlation analysis according to clinical indexes. The project aims to elucidate the mechanism on the regulation of occurrence and progression of breast cancer by MEG3 and its binding proteins, which may eventually lead to potential target in targeted cancer therapy.

长链非编码RNA（lncRNA）的发现指出了肿瘤基因表达微观调控的新方向，而鉴定其结合蛋白是阐明其作用机制的关键。申请人曾建立分离RNA结合蛋白的亲和纯化技术并鉴定出介导mRNA出核功能的主要蛋白（PNAS, 2011; NAR, 2013）。预实验发现lncRNA MEG3在乳腺癌中低表达并抑制乳腺癌细胞系增殖，但介导MEG3 抑制肿瘤细胞增殖功能的结合蛋白及其调控乳腺癌发生发展的分子机制均不清楚。故将从分子水平鉴定MEG3结合蛋白并确定直接介导MEG3抑制肿瘤细胞增殖的关键蛋白；在细胞水平上检测MEG3结合蛋白对P53通路、PI3K通路以及血管生成基因表达的影响探讨其调控乳腺癌进程机制；然后验证MEG3结合蛋白稳转细胞株在裸鼠中诱发乳腺癌的能力，并在临床样本中检测MEG3及其结合蛋白的表达，分析与临床指标的相关性。本研究将阐明MEG3及其结合蛋白作用机制，为乳腺癌靶向治疗提供潜在靶点。

长链非编码RNA（lncRNA）MEG3与乳腺癌密切相关，但MEG3在乳腺癌发生发展中的分子机制尚不清楚，而鉴定其结合蛋白是阐明其作用机制的关键。在基金委的支持下，本项目旨在研究MEG3在乳腺肿瘤发生发展中的作用及其潜在机制。首先将传统MS2-MBP亲和纯化技术与Flp-In T-REx定点稳转技术相结合，构建可诱导型MEG3稳转细胞株，通过亲和纯化和质谱技术鉴定结合于MEG3上的蛋白，并进一步优化了交联方法以及洗脱盐浓度等条件，最终得到了稳定的体内MEG3结合蛋白纯化的MS2-MBP亲和纯化方法，该方法的建立将为lncRNA的研究奠定方法学基础。其次，研究证实MEG3在乳腺正常细胞中高表达，在乳腺癌细胞中低表达。在MCF10A细胞中，利用CRISPR-dCAS9系统成功敲低MEG3，功能实验结果表明敲低MEG3能够明显促进细胞迁移，在MDA-MB-231以及MCF7细胞中，利用Lenti-X Tet-On 3G病毒系统，构建了可诱导型MEG3过表达稳转细胞系，结果表明MEG3过表达能够明显抑制乳腺癌细胞的增殖与迁移。再次，为了明确介导MEG3抑制细胞迁移的下游靶基因，我们将MEG3过表达与敲低细胞皆送样RNA-seq，对差异基因进行功能交叉分析，最后通过划痕实验进一步明确MEG3通过抑制IGFBP3的表达抑制乳腺癌细胞迁移。最后，MEG3抑制IGFBP3表达的分子机制方面，我们推测核定位的MEG3可能通过结合转录因子进而调控IGFBP3表达，故我们将预测的可能调控IGFBP3转录的转录因子与MEG3结合蛋白纯化后质谱结果交叉比对，得到了7个可能的侯选蛋白，目前后续研究正在进行中。综上，本项目为lncRNA在乳腺癌的发病机制和靶向治疗方面提供了新的线索。