NamiRNA正向调控G蛋白偶联雌激素受体表达在乳腺癌发病中作用研究

MicroRNAs (miRNAs) are small non-coding RNAs that canonically inhibit the translation or promote degradation messenger RNAs (mRNAs) through an RNA-RNA interaction with the 3-untranslated region (UTR) of target genes in the cytoplasm. However, the function of abundant miRNAs in the nucleus remained unclear. Notably, it was found that miRNAs tend to have a lower expression in cancer, which is acknowledged as an important molecular characteristic of cancer cells. Yet many challenges remain to illustrate the mechanisms how biologic behavior of tumors is properly controlled by such low expression miRNAs. In our previous research, we found that the function of nuclear miRNAs is completely different from cytoplasmic miRNAs. In contrast to the negative regulation, the nuclear miRNAs could activate gene transcription by binding to enhancer regions, termed as NamiRNA (Nuclear Activating miRNA). Here, our current proposed project try to explore positive regulation of low expression miRNAs in breast cancer and focus on investigating the function of miR-339 in the regulation of enhancer activities and expression of GPER1. We will illuminate the mechanisms through which GPER1 gene expression is up regulated by miR-339, and further explore the function of miR-339 and GPER1 throughout each stage of breast cancer. In the end, we hope to uncover a new perspective towards understanding the function of tumor associated miRNAs in breast cancer development, and provide a new and effective strategy to inhibit breast cancer development through NamiRNA intervention.

miRNA传统上在细胞浆中通过结合靶基因3’UTR抑制翻译或降解mRNA进而发挥负向调控作用，这很难解释许多定位于细胞核内miRNA作用机制。肿瘤相关miRNA低表达是肿瘤细胞重要分子特征，而低表达miRNA如何精准调控肿瘤生物学行为仍面临诸多挑战。我们前期研究工作发现定位于细胞核内miRNA与公认的miRNA在胞浆中负向调控机制不同，可通过结合增强子序列进而发挥基因转录激活作用，命名为NamiRNA （Nuclear Activating miRNA）。本项目以乳腺癌低表达miRNA与潜在靶基因为研究对象，确定乳腺癌中低表达miR-339改变增强子活性与GPER1正向调控关系，力图阐明miR-339对GPER1正向调控分子机制，探讨miR-339对GPER1正向调控对乳腺癌细胞生物学行为影响，从全新角度诠释肿瘤相关miRNA在乳腺癌发生发展中的作用，寻找miRNA干预对乳腺癌治疗新策略。

摘要.抑癌基因在肿瘤中表达下调或失活与肿瘤的发生发展有重要关系。抑癌基因的低表达除了基因突变的原因外，还和表观遗传学调控元件紧密相关。主要的表观遗传调控方式包括启动子DNA甲基化、组蛋白修饰和非编码RNA调控。目前认为抑癌基因低表达大多由于启动子DNA高甲基化和组蛋白异常修饰，及少数非编码RNA的调控。我们的研究工作以乳腺癌为例，发现乳腺癌中低表达的抑癌基因启动子DNA甲基化状态和正常对照组比较，约70%没有明显变化。而这些抑癌基因增强子上的组蛋白修饰H3K27ac却表现出明显的差异表达，也就是H3K27ac在乳腺癌组织的富集要明显低于正常对照组，提示增强子的H3K27ac修饰异常可能与乳腺癌中抑癌基因低表达相关。.乳腺癌作为全球最主要的恶性肿瘤之一，位列我国女性癌症发病首位，恶性乳腺癌患者5年生存率低，尚无有效治疗手段，因此寻找新的靶点和有效治疗药物迫在眉睫。本研究中我们收集了157对不同分子分型的乳腺癌组织的总RNA进行了QPCR验证，结果表明miR-339和抑癌基因GPER1在Luminal A/B及三阴性乳腺癌三种分子亚型的乳腺癌中表达下调，而在HER2富集型和癌旁对照组织的表达没有明显差异。这提示我们miR-339可以作为非HER2+富集型乳腺癌尤其是三阴性乳腺癌的潜在靶点。后续我们在非HER2+富集性乳腺癌细胞系T47D和4175中过表达外源性miR-339，结果表明这两个非HER2+富集性乳腺癌细胞中抑癌基因GPER1的表达上调，它们的增殖和克隆形成都受到明显抑制；相同结果在动物试验中更进一步得到验证。此外，我们利用NamiRNAs-增强子-靶基因激活的调控模型详细阐述了乳腺癌中抑癌基因低表达的分子机制。.总之，我们的研究对miR-339是如何通过增强子开关调控抑癌基因GPER1从而发挥抑癌作用进行了详细阐述，为后续进一步将miR-339开发为非HER2富集型乳腺癌治疗的新型核酸药物提供较为全面的理论基础和方法。同时NamiRNAs-增强子-靶基因激活的调控模型的提出将拓宽我们对miRNAs调控功能的认知，为现今的miRNAs负向调控理论提供一个很好的补充。