长链非编码RNAs作为膀胱癌分期预测标志物的实验及临床研究

Bladder cancer is one of the most common malignancy in urinary tract system. Staging of the primary lesion, especially the differentiation of non-muscle invasive bladder cancer (pTis, pTa and pT1) and muscle-invasive bladder cancer (pT2-pT4) is pivotal to guide therapeutic approach. Searching for accurate methods to predict muscle-invasive bladder cancer has become a key clinical challenge. Long non-coding RNAs (lncRNAs) were reported to be embedded in exosomes that could be secreted by tumor cells into surrounding body fluids, and several studies had linked lncRNAs to the pathogenesis of human cancers. These characteristics of lncRNAs suggested that the lncRNAs could be served as novel noninvasive biomarkers for predicting tumor development and progression. In our previous studies, high-throughput lncRNAs chip analysis and RT-qPCR were performed to analyze the expression difference of lncRNAs in tissue samples of non-muscle invasive bladder cancer, muscle-invasive bladder cancer and normal mucosa. Seventeen lncRNAs were found differently expressed in tissues of muscle-invasive bladder cancer. In the following studies, RT-qPCR will be employed to detecte the expression of the above-mentioned lncRNAs in exosomes of urine supernatant in differential stage of bladder cancer, and the predicting model of muscle-invasive bladder cancer will be establish, which may show us medically values on predicting muscle-invasive bladder cancer. Furthermore, the vitro assays including cell invasion, cell metastasis and the vivo nude mice experiments combined with lncRNA-mRNA coexpression analysis will be applied to demonstrate the roles of lncRNAs in muscle-invasive bladder cancer. The potential experimental results may provide novel insights into noninvasive detection of bladder muscle-invasive cancer and help clarify the molecular mechanisms involved in the pathogenesis of bladder cancer.

膀胱癌是泌尿系统最常见的恶性肿瘤，术前对肌层浸润性膀胱癌（pT2-pT4）进行有效预测是治疗方案选择的关键，也是临床亟待解决的难题。长链非编码RNAs（lncRNAs）与肿瘤侵袭转移密切相关，并可通过exosomes由肿瘤细胞分泌到体液中，为肌层浸润性膀胱癌无创性预测提供了一条新途径。本项目前期通过lncRNAs芯片和RT-qPCR技术，初步获得由17个lncRNAs组成的肌层浸润性膀胱癌组织lncRNAs表达谱。现拟以尿液上清exosomes作为载体，采用RT-qPCR对不同分期膀胱癌及健康对照尿液上清中上述lncRNAs进行检测，构建膀胱癌分期预测模型，评价其对肌层浸润性膀胱癌的预测价值。最后通过细胞实验、动物模型及生物信息学研究，分析上述lncRNAs对膀胱癌侵袭转移的影响。本研究将为膀胱癌的无创性分期预测提供依据，并为初步揭示lncRNAs在膀胱癌侵袭转移中的作用机制奠定基础。

膀胱癌是世界范围内泌尿系统最常见的恶性肿瘤，其发病率和死亡率在我国均居男性泌尿生殖系肿瘤的首位，严重威胁人类健康。临床上膀胱癌患者初诊时约70%为非肌层浸润性膀胱癌（Ta、Tis、T1），经尿道膀胱肿瘤切除术（TURBT）微创治疗，预后相对较好，但仍有30%的患者会进展成恶性程度高、预后较差的肌层浸润性膀胱癌。非编码RNAs与肿瘤发生、侵袭转移密切相关，可通过外泌体由肿瘤细胞分泌到体液中，为膀胱癌的早期诊断和分期预测开辟了一条新途径。本课题前期应用高通量lncRNAs芯片，初步建立起膀胱癌组织lncRNAs差异表达谱，为寻找膀胱癌分期诊断中高敏感度、强特异性的生物标志物提供了高通量数据基础。本研究中，我们采用RT-qPCR验证lncRNAs差异表达谱内各分子在独立样本集中的表达情况，发现RP11-396O4.6（ENST00000622374）在膀胱癌组织中表达下调，并可抑制膀胱癌细胞增殖，为初步揭示lncRNA在膀胱癌发生、发展中的作用机制奠定了基础。随后，我们针对游离lncRNAs和外泌体lncRNAs，分别构建uc004cox.4、GAS5膀胱癌早期诊断模型以及外泌体lncRNA MALAT1、PCAT-1和SPRY4IT1早期诊断模型，两模型的诊断效能均明显高于传统尿液脱落细胞学。基于血液游离lncRNAs，我们建立了针对膀胱癌早期诊断的高性能3-lncRNAs（MEG3、SNHG16和MALAT1）模型；随后，以血清外泌体为检测载体，我们发现了lncRNA PCAT-1、UBC1和SNHG16在外泌体内高度富集且稳定存在，三者构成的模型可早期发现膀胱癌，其中UBC1对非肌层浸润性膀胱癌的复发监测具有一定的价值；我们还分别构建了基于7个尿液游离miRNAs分子的膀胱癌诊断模型和基于4个血清游离miRNAs分子的肌层浸润膀胱癌预测模型，显著提高了膀胱癌早期诊断和分期预测水平。本课题的研究成果将为膀胱癌的无创性诊断、分期预测和预后监测提供依据，研究过程中，我们还发现体液中circRNA、piRNAs具有成为早期诊断和预后监测的新型生物标志物的可能，若对以上体液非编码 RNA 进行系统研究，探讨其在膀胱癌发生发展中的作用机制，将为膀胱癌的诊断、分期预测及预后监测提供重要线索。