环状RNA hsa_circ_0000396作为膀胱癌分期预测标志物的实验及临床研究

Circular RNA (circRNA), a novel type of endogenous non-coding RNA, is closely related to the invasion and metastasis of bladder cancer. Recently, circRNA has been found to be secreted from bladder cancer cells into urine by exosomes, which provides a new way for noninvasive prediction of muscle-invasive bladder cancer. In our previous studies, high-throughput circRNA sequencing and experimental verification demonstrated that the novel circular RNA hsa_circ_0000396 was significantly increased in muscle-invasive bladder cancer and affected its malignant biological behavior. In further studies, cell experiments in vitro and metastatic tumor models in vivo will be conducted to clarify the function of hsa_circ_0000396. Moreover, experiments including bioinformatics prediction, RNA pull-down, RNA immunoprecipitation and etc. will be applied to reveal the molecular regulatory mechanism of hsa_circ_0000396 in the invasion and metastasis of bladder cancer. Finally, digital PCR assays of circRNA in urine exosomes will be established and the predictive value of hsa_circ_0000396 for muscle-invasive bladder cancer will be evaluated by a multi-level systematic clinical study. Taken together, this study will not only help to identify the complicated molecular mechanism of circRNA in bladder cancer progression, but also provide novel insights into noninvasive detection of bladder muscle-invasive cancer.

环状RNA（circular RNA, circRNA）作为一类新兴内源性非编码RNA，与膀胱癌侵袭转移密切相关，并可通过外泌体由膀胱癌细胞分泌至尿液中，为肌层浸润性膀胱癌的术前无创性预测提供了一条新途径。本项目前期通过高通量测序筛选及验证，发现新环状RNA hsa_circ_0000396在肌层浸润性膀胱癌组织中显著升高并影响其恶性生物学行为。现拟应用体外细胞和体内转移瘤模型明确其功能；通过生物信息学预测、RNA pull-down及RNA结合蛋白免疫沉淀等手段揭示其调控膀胱癌侵袭转移的分子机制；建立尿液上清外泌体中circRNA的微滴式数字PCR检测方法，通过临床大样本、多层次系统研究，评价hsa_circ_0000396对肌层浸润性膀胱癌的预测价值。本研究不仅为初步阐明circRNA在膀胱癌侵袭转移中的作用机制奠定基础，而且还为膀胱癌术前分期预测提供了新的依据。

膀胱癌（Bladder cancer，BC）是泌尿系统中最常见恶性肿瘤之一，其发病率与死亡率一直居高不下。由于缺乏灵敏的诊断方法，近四分之一的BC患者初次就诊时已经处于疾病中晚期阶段，易发生远处转移，预后差。尽管近年来，包括外科手术，放、化治疗以及免疫疗法在内的临床治疗手段有所提升，但是BC患者的整体治疗效果不佳，5年生存率依旧处于较低水平。因此，寻找高效灵敏的诊断和分期预测分子生物标志物，探究其促进肌层侵袭转移等分子机制，将为改善BC患者预后提供重要参考。环状RNAs（circRNAs）是一类特殊的非编码RNAs，能稳定存在于组织，细胞及多种体液中，研究表明，circRNAs可作为重要调节因子，广泛参与癌症发生发展的全过程。本课题前期应用高通量测序技术，初步建立了膀胱癌相关circRNAs差异表达谱，为寻找高敏感度、强特异性的膀胱癌标志物提供了基础。我们进一步通过功能富集分析，临床组织样本验证及Sanger测序等方法确定hsa_circ_0000396（circSCL38A1）为研究靶点。通过体内外功能试验分析该分子可发挥促癌基因功能，促进BC侵袭性；采用RNA pull down 及RNA 免疫共沉淀实验（RIP）证实circSCL38A1可与ILF3相互作用，二者共定位于细胞核中；通过泛素化IP实验证实circSCL38A1可通过调节泛素化修饰抑制ILF3降解；通过CUT&Tag-seq与RNA-seq数据联合分析，最终确定TGF-β2为circSCL38A1-ILF3复合体的下游靶点；此外，通过临床样本证实，显示circSCL38A1可装载至血清外泌体，并具有成为BC诊断和分期预测标志物潜能。