基于单细胞转录组的膀胱癌干细胞新标志物鉴定及“干性”分子机制研究

Bladder cancer treatment is challenged as the high rate of recurrence and metastasis. Cancer stem cell (CSC) targeted treatment is the key to reduce these rates. Traditional CSC research is limited with simple cell surface molecular markers to precisely isolate this kind of special cells, so that unable to further study the genetic mechanisms of "stemness" in CSCs. Single cell sequencing technology brings new opportunity to study CSCs on a higher accuracy. .In this study, we carry on genetic analysis of bladder cancer stem cell (BCSC) based on single cell RNA-seq. We recently completed the sequencing of 953 single cells, which were sorted from 4 samples of human bladder transitional cell carcinoma based on traditional BCSC surface marker CD44. We will cluster the single cell expression profile, to speculate the exact CSC subgroup and analyze its gene expression characteristics compared with other types of cells. We will speculate the potential surface markers of BCSC subgroup and perform the validation through tumorsphere formation assay and Xenotransplantation. On the other hand, combining transcriptome sequencing data as well as clinical information of bladder cancer from multiple databases, we will determine several key genes which relate to tumor progression, and the function of key genes will be investigated by lentiviral overexpression screening. This work has the opportunity to reveal the genetic basis of “stemness” of bladder cancer and explore potential new biomarkers as well as potential clinical targets of BCSCs, thus providing theoretical basis for BCSC targeting therapy in the future.

膀胱癌是常见的泌尿系统恶性肿瘤，极易复发和转移，有效针对膀胱癌干细胞（BCSC）的治疗是减少其复发和转移的关键。现有的BCSC标志物单一有限，无法精确分离BCSC，其“干性”产生机制不详。申请人在前期单细胞基因组的研究基础（共同一作European Urology和Cell Research各一篇）之上，进一步从单细胞转录组水平对BCSC展开研究，完成了四例样本953个细胞的测序。本项目拟基于单细胞表达谱精确定位BCSC亚群并提取其表达特征：（1）推测潜在细胞表面分子标记，通过成球培养和异种移植最终确定BCSC新标志物。（2）通过基因注释及数据库临床关联，筛选肿瘤“干性”关键基因，通过基因过表达实验进行功能验证，并进一步推测肿瘤“干性”产生的分子机制。本研究有望鉴定一批BCSC的新标志物，揭示调控其“干性”的分子机制，为BCSC的靶向治疗提供潜在靶点和科学依据。

膀胱癌是常见的泌尿系恶性肿瘤，极易复发和转移。膀胱癌的异质性是膀胱癌治疗过程中复发和转移的重要原因，深入地研究膀胱癌内部异质性以及膀胱癌内部不同细胞亚型对膀胱癌的影响则成为进一步认识和改善膀胱癌治疗方法的重要途径。本项研究通过三个方面围绕这一主题展开了研究：.1）基于单细胞RNA-seq的膀胱癌表达异质性与细胞演化研究：通过对膀胱癌单细胞数据的生物信息学分析，揭示膀胱癌表达层面的异质性，对不同细胞类型进行了聚类分析，探索其在肿瘤生物学表型中所起的作用，构建膀胱癌演化模型。基于上述研究，我们发现高级别膀胱癌与低级别膀胱癌在细胞组成上存在差异，具有“干性”的“basal”类别细胞亚型在高级别膀胱癌中明显较多。同时，这一类细胞在膀胱癌演化过程中处于早期阶段，表明其对于膀胱癌发生于发展发挥着重要作用。.2）膀胱癌干细胞表达特征与“干性”分子基础研究：基于已知细胞标志物对膀胱癌进行分选，采集膀胱癌干细胞表达谱，通过与其他类型细胞的差异分析与共表达网络构建，探索潜在的“干性”相关的关键基因，并进行功能验证，为基于膀胱癌干细胞的治疗提供了潜在靶点。基于上述研究，我们首次构建了原代膀胱癌的表达谱，并且通过生物信息学手段筛选出了维持膀胱癌“干性”的重要基因COL5A1，并通过细胞学实验证实了其维持“干性”的功能，为膀胱癌针对肿瘤干细胞的治疗提供了潜在靶点。.3）膀胱癌预后模型研究：分别基于单一组学和整合多组学数据构建膀胱癌预后模型，并在多个验证集中进行验证和评价。通过上述研究，我们分别构建了基于lncRNA和多组学数据的膀胱癌预后模型，且两个模型均具有较强的鲁棒性，为膀胱癌的临床预后提供新的选择。