DDX46调节MDM2-p53反馈环促进膀胱癌增殖转移的机制研究

Bladder cancer is one of the most common malignant tumors, but its molecular mechanism is still unclear. .The applicant has finished the RNA-seq sequencing of bladder cancer tissues and found DDX46 was abnormally highly expressed in bladder cancer tissue compared with corresponding adjacent normal tissue, and our IHC date based on 20 pairs of bladder cancer tissues and adjacent normal tissues paraffin section has preliminarily verified DDX46’s high expression, cellular level studies also confirmed that it promote the proliferation and metastasis. Moreover genome-wide expression profile chip showed that  MDM2 gene expression significantly lower which is the important regulatory proteins of p53 and the inhibition of p53 signaling pathways. Therefore we speculated that DDX46 involved in the development of bladder cancer by regulation of MDM2-p53 feedback loop..We plan to study from the following three aspects: first of all, analysis the correlation of clinical and expression among DDX46、MDM2 and p53 by detecting gene and protein expression in bladder cancer tissues; then design in vivo and vitro functional experiments after overexpression and interference DDX46 and detect specific genes and their translated proteins changes on the p53 pathway at the same time; finally research DDX46 role in regulating the expression of MDM2 and p53, and correlation between it and MDM2 in function. This project will lay the foundation for the further study of bladder cancer molecular mechanism.

膀胱癌是泌尿系常见的恶性肿瘤，其分子机制仍不十分清楚。.本课题组前期通过转录组测序发现DDX46基因在膀胱癌组织中特异性高表达，通过IHC在20对组织切片中验证其高表达；细胞水平研究也证实DDX46敲减后抑制膀胱癌细胞的增殖和转移；对DDX46干扰的细胞样本进行全基因组表达谱芯片分析后发现：p53的重要调节蛋白MDM2基因表达显著下调，且下游p53信号通路显著受到抑制。由此我们推测DDX46可能通过调节MDM2-p53反馈环参与了膀胱癌的发展过程。.本课题拟通过：(1)检测和分析DDX46、MDM2、p53基因及其蛋白在膀胱癌组织中的表达及临床相关性；(2)在细胞和动物水平进一步研究DDX46对膀胱癌增殖和转移功能的影响，分析验证p53通路上关键基因及蛋白变化；(3)研究DDX46对MDM2与p53表达的调节作用以及其与MDM2之间的功能相关性。本课题将为膀胱癌分子机制的深入研究奠定基础。

膀胱癌是泌尿系统常见恶性肿瘤，由于发生发展分子机制仍不清楚，使得其临床治疗进展甚微。.在前期测序的基础上发现DDX46基因在膀胱癌中高表达。本课题组首先通过IHC的方法验证了DDX46基因在膀胱癌中的高表达，且其表达水平与患者的TNM分期密切相关。其次，在细胞和动物水平，通过celigo细胞计数、MTT实验、凋亡及克隆形成、划痕实验、Transwell侵袭及体内裸鼠成瘤实验分析发现敲低表达DDX46基因抑制膀胱癌细胞的增殖、周期、侵袭和迁移及在体内的裸鼠成瘤能力。然后，通过人类基因组表达谱芯片技术分析敲低表达DDX46基因后基因的差异表达，对差异表达基因进行IPA分析，发现差异表达基因在p53通路富集，并用WB检测p53通路关键蛋白，发现Bcl2表达减少，Capase3、Bax和P14ARF表达增加，MDM2表达减少，p53表达增加。最后进行DDX46与MDM2-p53反馈环的关系分析：1）进行MDM2回复实验，结果表明MDM2过表达能回复DDX46干扰引起的增殖抑制和凋亡。2）用CHX处理膀胱癌细胞系，WB检测敲低表达DDX46基因后MDM2、p53表达变化，结果表明MDM2和p53的半衰期明显缩短。3）MG132处理细胞系，发现MG132处理后DDX46蛋白表达水平下降，说明存在除蛋白酶以外的降解途径。4）提取总蛋白，对MDM2或p53抗体进行免疫共沉淀，用Ub抗体检测发现p53和MDM2之间存在互作关系且p53上存在泛素化。.本项目深入分析了DDX46基因通过MDM2-p53反馈环影响膀胱癌的机制，进一步推动了膀胱癌分子机制的研究。