化学小分子选择性识别miRNA-1587 G-四链体、调控生物功能及其机理研究

MicroRNAs (miRNAs) are endogenous short RNAs playing important regulatory roles in human genome. The functions of miRNAs are accomplished by interacting with mRNAs or RNA binding proteins directing RNA posttranscriptional regulation, which are highly related to their structures. . This project will focus on the recognition of G-quadruplexes in mature miRNAs and the regulation of their biological functions using chemical molecules. Recently, we found a G-rich miRNA, miRNA-1587, was overexpressed in Hela cell lines. In this research, we will probe the formation of miRNA-1587 G-quadruplex in different conditions and identify its targeting mRNAs or RNA binding proteins in cancer cells. Then, we will investigate the roles of G-quadruplex in miRNA-1587 biological function, and explore the correlation between the structure of the miRNA and human disease. In addition, we will screen out the selectively binding ligands for miRNA-1587 G-quadruplex in our natural products library. According to the screening and molecular modeling results, we will design and synthesize superior ligands recognizing the miRNA-1587 G-quadruplex. Finally, we will test the chemical molecules to confirm their capacity of inducing G-quadruplex formation of miRNA-1587 and their biological activity in living cells. . Our research will develop novel strategy to regulate the biological functions of miRNAs by their structural transition induced by chemical molecules, providing pivotal molecular mechanism for the action of miRNA and the considerable drug-precursors for human cancers.

本项目主要研究富鸟嘌呤miRNA G-四链体结构的识别、生物功能和化学小分子调控。以在Hela癌细胞中特异性高表达的miRNA-1587为研究对象，探测其形成的G-四链体结构和性质；从天然分子库中筛选对miRNA-1587 G-四链体具有识别性能的化学分子，进一步设计、合成对miRNA-1587 G-四链体具有高选择性识别功能的新型化学小分子，在细胞中研究化学小分子识别miRNA-1587 G-四链体，调控miRNA-1587的生物功能，并探讨其调控功能的分子机理。为miRNA功能调控的研究提供新策略、研究方法和理论依据。. 本研究将极大加深对miRNA G-四链体、性质与生物功能的认识，丰富和发展选择性识别miRNA G-四链体的化学小分子，探讨外源性化学分子选择性调控miRNA生物功能的新机理。此研究对于深入理解生命体系中核酸结构及化学分子的调控具有重要的科学意义。

miR-1587与人类一些疾病相关，并且富含鸟嘌呤，可形成特殊结构，因而对miR-1587结构、性能及靶向调节的研究具有重要的科学意义。.本项目使用质谱，CD谱及NMR谱等方法揭示了miR-1587在K离子或铵离子条件下可形成稳定的特殊二级结构G-四链体；系统地探讨了白屈菜红碱、两面针碱、药根碱、假巴马汀和血根碱等天然生物碱小分子识别miR-1587G-四链体的性能，发现两面针碱、药根碱和假巴马汀等小分子具有高亲合性识别和结合miR-1587G-四链体的性能；并且发现合成的药根碱衍生物不仅对miR-1587G-四链体具有识别性能，还能诱导单分子miR-1587G-四链体形成二聚体。.利用基因芯片技术与计算机预测软件筛选出十四个miR-1587的候选靶基因，使用Q-RT-PCR与蛋白印迹实验探讨了miR-1587过表达后候选靶基因在mRNA和蛋白表达水平的改变，构建了野生型与突变型萤光素酶报告基因质粒，发现miR-1587对TAGLN基因的性能具有显著的影响，因此选择TAGLN为miR-1587的靶点，进一步探讨miR-1587的生物功能。本研究结果表明：诱导miR-1587G-四链体结构的形成，将导致miR-1587与TAGLN的结合受到影响，表现为萤光素酶强度的上升，miR-1587的功能受到了抑制。加入识别和结合化学小分子(pseudopalmatine)，稳定miR-1587G-四链体结构，可以进一步抑制miR-1587的功能。反之，G-四链体去稳定剂TMPyP4的加入破坏了其G-四链体结构，使得 miR-1587能够与TAGLN更有效的结合，从而促进miR-1587的功能。.本项目揭示了miR-1587G-四链体与其功能之间的关系，为miR-1587功能的调控提供了新策略、研究方法和实验依据；此研究对于理解生命体系中核酸特殊结构及使用小分子探针调控其生物功能具有重要的学术意义。