DNA甲基化调控的miR-3189-3p在膀胱癌多药耐受机制中的系统研究

Chemoresistance hinders the clinical therapy of bladder cancer. For a better mechanistic understanding of the chemoresistance of cancer, we have previously performed the 2nd generation sequencing based tri-omic studies of eight bladder cancer cell lines with distinct chemoresistance behavior, which identified a panel of DNA methylation regulated microRNA genes that expressed differentially between chemosensitive and chemoresistant cancer cell lines. The miR-3189 was found to be hypermethylated/silenced in 5637(sensitive) and hypomethylated/expressed in H-bc (resistance) cells. In this proposed study, we plan to 1) establish the stable BCa cell lines integrated with tet-on regulated miR-3189 expression system and conduct a comprehensive functional analysis of the miR-3189-3p and its target genes in the context of BCa cells' multi-chemoresistance, 2) study the transcription regulation of the miR-3189 and its host gene GDF15 with respects of the cis- and trans- elements and DNA methylation , 3) profile the true target of miR-3189-3p via the biotin-miR capture/seq approach, 4) evaluate the correlation between miR-3189 and the carcinogenesis as well as chemoresistance of bladder cancer by clinical sample analyze.

化疗耐受是导致膀胱癌临床治疗困难的重要原因。我们前期通过对8种化疗药物耐受性不同的膀胱癌细胞系（共8株）进行了多重组学整合分析，发现了一批受DNA甲基化调控，且与化疗敏感性相关的microRNA。其中miR-3189的DNA甲基化水平与化疗耐受负相关，而表达水平与化疗耐受正相关。本项目中我们将以miR-3189和其宿主基因GDF15为对象，系统地开展以下研究：1) 在膀胱癌细胞中构建四环素诱导表达系统，研究miR-3189及靶基因在膀胱癌化疗耐受中的作用和机制，2)从顺反式元件等多个层面，系统地研究miR-3189及GDF15的协同转录调控，3) 采用biotin-miR捕获/RNA-seq的手段确立其下游靶基因谱式，4) 通过对膀胱癌患者的样本分析，确立miR-3189的甲基化水平与膀胱癌发生和化疗耐受的临床相关性。此项研究将有助于阐明膀胱癌临床化疗耐受发生机制并为精准化疗提供可能靶点。

项目执行过程中，由于实验数据与预期结果产生偏差，我们围绕着表观遗传调控膀胱癌化疗耐受机制的科学问题，对项目方向及时进行了调整。通过一系列体内和体外实验，如系谱追踪、极限稀释、体外成球等，我们首先明确了膀胱癌中化疗耐受及肿瘤发生、复发的关键干细胞群为SOX2阳性标记。其次我们在临床样本、动物模型和细胞水平明确了低剂量DNA甲基化抑制剂地西他滨可以通过抑制STAT信号途径有效抑制膀胱癌干细胞的干性，当其与化疗药物联用时，可以产生对于肿瘤细胞的有效杀伤。此外，我们还发现RNA甲基化酶METTL3在膀胱癌组织中异常高表达，通过调控NF-kB/MYC/AFF4信号网络调控膀胱癌的进展与干性。上述结果明确了DNA甲基化和RNA甲基化在膀胱癌发生发展及化疗耐受中的重要地位，对于膀胱癌临床治疗的新方法具有重要指导意义。负责人以通讯作者发表高水平SCI论文4篇。