基于高通量测序技术的膀胱癌转录组分子标记的初步筛选

Bladder cancer is one of the most common malignant tumors, and there is close connection between its development and gene mutation, but many genetic variations associated with bladder cancer have not been revealed. We performed high-throughput transcriptome sequencing on muscle-invasive UCB tissue and found: there were more than 1800 cancer-specific differentially expressed genes between the tumor and none-tumor tissue. Our data also revealed that more than 300 genes showed differential splicing patterns between them, and most of the gene splicing changes had not been reported before, and also three drug-resistance genes were identified. One of them had never been reported in bladder cancer. This project intends to adopt high-throughput sequencing technology to perform transcriptome sequencing and bioinformatics analysis on normal and cancerous tissue samples in another 12 patients with bladder cancer, combining the analysis results of the 13 patients, screens and identifies the new differentially expressed genes , alternative splicing, gene fusion and somatic mutation specific to bladder cancer. At last we would verify our hypothesis and identify the candidate targets by performing reverse transcription-polymerase chain reaction and Sanger sequencing on more than 200 cases of bladder cancer. And furtherly we will design in vivo and in vitro functional experiments to verify them. Our job will surely improve the molecular diagnostics, monitoring and personalized therapy for bladder cancer.

膀胱癌是常见的恶性肿瘤之一，其发生发展与基因的变异存在着密切的关系，但还有很多与膀胱癌相关的基因变异未被揭示。 申请人前期对单个膀胱癌患者正常和癌组织的转录组进行测序发现：有 1800 多个基因表达差异显著；300 多个基因的剪接异构体的表达比例发生显著变化，大部分剪接改变的基因之前未见报道，并发现3个耐药基因，其中一个未见报道。 本项目拟采用高通量测序技术，对另外12 例膀胱癌患者的正常和癌组织样品进行转录组测序和生物信息学分析，然后结合 13 例病人的分析结果，筛选出肿瘤特异的新的差异表达基因、肿瘤特异的差异可变剪接基因，基因融合和体突变，并通过大量膀胱癌样品（n>200），采用Real-time PCR方法和Sanger测序技术验证生物信息学分析的结果筛选出候选靶点，并设计细胞及动物水平进行体内外功能实验进行验证，推动膀胱癌分子诊断、肿瘤监测和个性化治疗的发展。

膀胱癌是泌尿系统常见恶性肿瘤，但目前其分子机制仍不十分清楚。本项目对12例膀胱癌患者的癌和癌旁组织样品进行高通量全转录组测序，并对测序结果进行质量检测，选择8例膀胱癌患者的测序结果进行生物信息学分析，筛选差异表达基因、可变剪切、融合基因，并对差异表达显著的基因进行KEGG通路富集和GO功能分析。结果显示：肿瘤差异表达基因2388个，其中表达上调的基因1641个，表达下调的基因747个；肿瘤差异可变剪接基因多种形式合计4782种，其中包含A3SS、A5SS、MX、ES 和 RI等形式；肿瘤特异的基因融合共18种；差异表达显著的基因主要富集在细胞黏附相关分子通路、粘着斑通路、cGMP-PKG 信号通路等。.结合前期测序结果进行综合分析，应用RT-PCR和IHC的方法验证筛选出的差异表达基因（CDH1、VEGFA、PTPRF、CLDN7、miRNA-24、DDX46、CDCA8、Cofilin 1）在组织中的表达情况，同时结合患者临床资料，分析这些基因的mRNA和蛋白的表达情况，进一步验证了CDH1、VEGFA、PTPRF、CLDN7、DDX46、miRNA-24、CDCA8和Cofilin1基因与膀胱癌的发生发展密切关系。.此外，我们初步探讨了部分基因（miRNA-24、CARMA3、DDX46、CDCA8、Cofilin 1）在膀胱癌中的作用机制。通过细胞及动物实验对miR24、CARMA3基因及Cofilin1进行体内外功能验证。结果发现miRNA-24和Cofilin 1均能影响膀胱癌细胞的增殖、周期及凋亡等；结合双荧光素酶报告试验明确了miR-24 基因可以通过调控CARMA3 基因的表达而影响膀胱癌细胞功能；ChIP和荧光素酶证明了TCF7L2参与了Cofilin 1诱导的膀胱癌的发生发展。.本课题探讨了膀胱癌分子作用机制，寻找膀胱癌特异性分子标志物，将为膀胱癌早期诊断、治疗、复发监测及个性化治疗提供理论依据。