miR-101与其靶向circRNA相互调控机制及在膀胱癌中的作用研究

It is now being realized that non-coding RNAs represent most of the human transcriptome, and functionalizing the non-coding space will undoubtedly lead to important insight about basic physiology and disease progression. Our previous study has confirmed that, the down-regulation of miR-101 is closely associated with the development of bladder cancer, and could be used as a target for exploring novel treatment strategies. The exact mechanism involved in the down-regulation of miR-101 in bladder cancer has still to be fully discovered. Evidence from computational analyses of expression data in Archaea and Mammalia suggests that the highly stable expressed endogenous circular RNAs (circRNAs) are more prevalent than previously thought. Recently, the tissue-specific circRNAs have been reported to act as an effective miRNA sponge and affect miRNA target gene activity. Bioinformatic analysis shows that two bladder cancer-specific circRNAs could be transcribed from the coding region of RNA-binding protein 9, and both of them have miR-101 binding sites. We also found that the two bladder cancer-specific cicrRNAs were obviously up-regulated in bladder cancer tissues, and over-expression of this circRNA could further suppress the expression of miR-101. In the project, we will systematically identify the regulation between miR-101 and its targeted cicrRNAs, and create the bladder cancer cell-targeted fusion protein of single-chain antibody against Uroplakin Ib and low molecular weight protamine to transport siRNA and shRNA. This work will build a solid theoretical foundation for functionalizing circRNAs, and develop a more selective strategy for targeted therapy in bladder cancer.

基因组绝大多数转录产物为非编码RNA，阐明其作用已成为重要研究方向。本课题组前期研究证实：miR-101作用下调与膀胱癌进展密切相关，可作为研究膀胱癌发展机制及治疗策略的重要靶点，其内在调控机理有待进一步阐明。最新研究揭示了非编码RNA研究的全新领域：环状RNA（circRNA）作为一类细胞内稳定高表达、具有组织特异性的新型非编码RNA，可以以内源性竞争性RNA的形式抑制miRNA。生物信息学分析提示，RAN结合蛋白9编码区转录过程中能够产生两种circRNA，均具有miR-101的结合位点，且表达具有膀胱癌特异性。我们初步研究亦表明，膀胱癌中这两种circRNA表达均显著升高，过表达靶向circRNA能够进一步下调miR-101表达。本课题拟在此基础上，系统解析膀胱癌中miR-101与其靶向circRNA相互调控机制，并构建膀胱癌细胞特异性核酸转染体系，有望为膀胱癌的靶向治疗提供新策略。

越来越多的证据表明环状RNA（circRNAs）在调控基因表达中发挥着至关重要的作用。然而，circRNAs在膀胱肿瘤中的作用仍然不明确。在本研究中，我们发现miR-101相关的环状RNA（BCRC4）在膀胱癌组织中的表达低于癌旁组织。过表达BCRC4可以促进膀胱癌细胞凋亡并抑制T24T和UMUC3细胞的增殖，并且上调BCRC4可以提高miR-101的表达水平，并能在RNA和蛋白质水平抑制组蛋白-赖氨酸N-甲基转移酶（EZH2）的表达。藤黄酸（GA）作为膀胱癌治疗的天然抗癌化合物，可以增加T24T和UMUC3细胞中的BCRC4的水平，并呈现浓度依赖性。此外，我们对膀胱癌组织及癌旁组织进行RNA高通量测序，鉴定了6,154种具有差异性表达的circRNA。总体上，我们发现共有524个circRNA在膀胱癌组织中明显低表达，47个circRNA表达量升高。我们进一步发现，circHIPK3，又称为膀胱癌相关环状RNA-2（BCRC-2），在膀胱癌组织和细胞系中明显低表达。它的表达与膀胱癌分级，侵袭以及淋巴结转移呈负相关。过表达circHIPK3可在体外实验中显著抑制膀胱癌细胞的迁移侵袭和血管生成能力，并能在体内裸鼠实验中抑制膀胱癌的生长和转移。关于环状RNA的作用机制，我们证明circHIPK3具有miR-558的两个关键结合位点，并且可以大量吸附miR-558，导致乙酰肝素酶（HPSE）的表达明显下调。最后，我们的研究结果证实了circRNA作为“microRNA分子海绵”的假设，提出了治疗膀胱癌的新靶点。