膀胱相关性环状RNA-1（BCRC-1）结合溴结构域蛋白4（BRD4）在膀胱癌中的作用及机制研究

Our previous studies have demonstrated that the bromodomain-containing protein 4((BRD4) /C-MYC/EZH2 is a key pathway to regulate the growth of bladder cancer. 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. Recent studies have revealed the new mode of circRNA to exert its function: it locates in the nucleus and interacts with transcriptional regulatory proteins, promoting the transcription of target genes. In our previous work, we successfully amplified and identified the low expression of bladder cancer related circRNA-1（BCRC-1）. Moreover, we found that BCRC-1 is also enriched in the nucleus. Further studies showed that, BCRC-1 could interact with BRD4. Overexpression of BCRC-1 efficiently blocked the effects of BRD4 on C-MYC transcriptional activation. In the project, we intend to confirm the interaction of BCRC-1 with BRD4 protein, and detect its effects on BRD4 binding to C-MYC promoter. Furthermore, we will establish BCRC-1 stable overexpression/restraint system, and explore the role of BCRC-1 on the biological function of BRD4/C-MYC/EZH2 pathway in bladder cancer. This work will elucidate a new role model of the endogenous circRNA as a blocker to bind transcriptional regulators and inhibit gene transcription, as well as provide a novel strategy for the treatment of bladder cancer.

我们前期研究表明：溴结构域蛋白4（BRD4）/C-MYC/EZH2是调控膀胱癌生长的关键通路。环状RNA（circRNA）是一类结构稳定、具有组织相关性的非编码RNA，近来研究揭示了circRNA新型作用方式：在细胞核内与转录调节蛋白结合，促进靶基因转录。我们前期研究成功扩增、鉴定了膀胱癌低表达的膀胱相关性环状RNA-1（BCRC-1），其在细胞核内富集；初步检测显示：BCRC-1可以与BRD4结合，过表达BCRC-1能够阻断BRD4对C-MYC的转录激活。本研究拟进一步观测膀胱癌BCRC-1与BRD4蛋白的相互作用及其对BRD4结合至C-MYC启动子的影响；通过建立BCRC-1稳定过表达/抑制体系，探讨BCRC-1对BRD4/C-MYC/EZH2通路活性及膀胱癌生物学功能的作用，旨在阐明内源性circRNA作为阻断剂结合转录调节因子、抑制基因转录的全新作用模式，为膀胱癌治疗提供新途径。

近来研究表明环状RNA（circRNAs）可以通过与RNA结合蛋白直接结合进而调控下游基因的表达。然而，具有结合蛋白能力的环状RNA在膀胱肿瘤中的作用仍然不明确。在本研究中，我们首先证实了溴结构域蛋白4（BRD4）在膀胱癌组织及细胞中显著高表达并与膀胱癌病人的不良预后密切相关，BRD4与BCRC-1在膀胱癌组织中的表达呈显著负相关；RIP及RNA pull-down实验进一步证实BRD4与BCRC-1可以在膀胱癌细胞核中直接结合。关于BCRC-1的作用机制，我们证明BCRC-1可以通过与BRD4结合进而降低BRD4对C-MYC启动子区域的转录激活作用。细胞功能学实验及裸鼠成瘤实验证实，过表达BCRC-1可以在体内及体外水平显著抑制膀胱癌细胞的增殖能力，而过表达C-MYC、EZH2能够部分逆转BCRC-1的抑癌作用。此外，我们筛选前期测序数据并验证发现circ0001361在膀胱癌组织及细胞中显著高表达，且其表达水平与膀胱癌分级、侵袭以及淋巴结转移等临床指标呈正相关。细胞及动物实验证实干扰circ0001361的表达能够抑制膀胱癌细胞的侵袭迁移能力。circ0001361可以在细胞浆中与miR-491-5p直接相互作用进而上调下游靶基因MMP-9的表达。最后我们的研究充分证实了环状RNA可以作为miRNA及RNA结合蛋白的“分子海绵”的假说，同时重点揭示了circRNA作为内源性阻断剂结合转录调节因子，抑制基因转录的新型作用模式，为膀胱癌的精准治疗提供了新途径。