启动子区"ncRNA云"在RNA激活中的作用机制及CRISPR-on基因激活靶向治疗膀胱癌的研究

Urinary bladder cancer is the most common urogenital malignant tumor in China. The treatment of bladder cancer, especially muscle invasive or metastatic bladder cancer, remains challenging. Our preliminary results have provided that up-regulation of tumor suppress genes p21、E-cadherin, PAWR and INST6 by small activating RNA inhibit human cancer. Based on these data, we hypothesize that the "cloud" of sense and antisense ncRNA around promoters play a key role in RNA activation. These hypotheses will be tested by pursuing the following two aims: Firstly, we will screen and clone a set of sense and antisense ncRNA and miRNA around RNAa promoters in bladder cancer, and identify them in bladder cancer cell lines and bladder cancer tissues. Furthermore, we will also investigate the mechanism involves epigenetic changes such as histone modifications and interacting with chromatin modifying enzymes. However, it is still inefficient to obtain small activating RNAs by traditional design and screening method, which impedes the further research of RNA activation. Recently studies showed endogenous gene activation using dCas9-based activators. In this study we will also investigate whether CRISPR-on can activate tumor suppress genes which couldn't be activated by traditional RNAa. We will screen a set of single guide RNAs( sgRNAs ) in bladder cancer cell, and the functional sgRNAs are also tested for their abilities of translational application into bladder cancer therapy and explore their action mechanism. Successful completion of these experiments may provide novel strategies for gene function research and the management of bladder cancer.

膀胱癌是我国发病率最高的泌尿系肿瘤。尤其是浸润性或转移性膀胱癌，其有效治疗方法的探索极为重要。在我们前期研究ncRNA激活抑癌基因P21、E-cadherin、PAWR和INST6等表达的基础上，本项目将继续探索膀胱癌细胞中RNA激活关键因素- - 启动子区"ncRNA云"在RNA激活中的作用机制，重点研究靶基因启动子区sense与antisense ncRNA、miRNA功能。我们推测"ncRNA云"起核心作用，通过与染色质修饰酶相互作用影响包括组蛋白密码在内多种表观遗传学的修饰。对传统小RNA激活失败的候选抑癌基因，将采用新型的RNA引导基因激活系统CRISPR-on技术，在膀胱癌细胞中筛选出靶向抑癌基因启动子区的sgRNA；进一步研究基因激活后的生物学效应及其激活机制。本项目将为膀胱癌的基因功能研究和肿瘤靶向治疗提供了新的策略。

膀胱癌是一个重要的公共卫生问题。肌层浸润性膀胱癌极易发生局部及远处的转移，预后较差，是造成膀胱癌患者死亡的重要原因。开发干预膀胱癌进展的新手段，是目前治疗MIBC的关键。近年来，靶向基因启动子区的RNA激活技术(RNAa)和CRISPR-ON（CRISPRa）系统介导的基因靶向激活技术为基因的功能研究和肿瘤靶向治疗提供了一种全新的策略。本研究证实，多个膀胱癌、肾癌中靶向易感抑癌基因启动子区的ncRNA能够激活p16、DIRAS1等基因的表达，并在体内及体外实验中验证其对肿瘤生物学行为的影响。本研究进一步对RNAa激活失败抑癌基因通过CRISPRa系统进行靶向激活，筛选出多条能够激活KLF4、LHPP等抑癌基因的sgRNA，并且在体内及体外实验中验证其对膀胱癌生物学行为的抑制作用。总之，本研究进一步为RNAa激活技术治疗泌尿系肿瘤的提供了新的治疗靶点；为传统RNAa激活失败的抑癌基因提供了一种新的激活思路，为膀胱癌基因功能研究和分子靶向治疗提供新的实验证据。