CHD1L基因在膀胱尿路上皮癌发生发展中作用及机制研究

The amplification and dysfunction of an oncogene is one of the key genomic changes during bladder cancer initiation and development. Therefore, identification and characterization of new oncogenic genes is still important for bladder cancer research. Further more, understanding the molecular mechanisms of oncogenic genes and developing potential intervening targets are urgent for bladder cancer prevention and effective treatment. We have found first time that CHD1L gene overexpression is positively associated with the degree of bladder tumor malignancy and clinical TNM type, and is correlated to short survival time. Our further in vitro study demonstrated that siRNA-mediated knockdown of CHD1L expression in BT5637 cells inhibits cellular growth and invasion. However, the functions and mechanisms of CHD1L in the course of bladder cancer development are unclear. We therefore hypothesize that CHD1L could be a driver gene, particular, its unique macro domain of CHD1L protein, which plays pivotal roles in the oncogenesis and/or metastasis of bladder cancer. In order to prove our hypothesis, we set up following aims: (1) in vivo mice exnograft study to observe the tumor formation capacity or metastasis. (2) various CHD1L protein domains (wild type, mutation of helicase domain and micro domain, truncated N, C terminal domain) will be constructed and transfected into normal urothelial cells (HU609) cells for in vitro and in vivo study. (3) To understand the molecular mechanism of CHD1L-mediated bladder cancer development, RNA-seq and DNase-1-ChIP approaches will be used for transcriptome and CHD1L-mediated regulatory region study. The oncogenic pathways will be identified using experimental and computational approaches. (4) To investigate the protein-protein interaction of CHD1L and identify its partners using protein mass spectrometry (LS/MS/MS), signaling pathway will be inferred. In summary, this research project will help us to understand the role and mechanisms of CHD1L in the development of urothelial carcinoma of bladder and provide a novel biomarker for bladder cancer diagnosis and prognosis.

前期首次发现CHD1L在膀胱尿路上皮癌中高表达且与膀胱尿路上皮癌不良预后密切相关。体外细胞实验显示抑制CHD1L 高表达能致肿瘤细胞生长减慢、侵袭力减弱。CHD1L 在膀胱尿路上皮癌癌变过程的功能和分子机制尚不清楚。根据前期实验结果和相关文献，推测CHD1L 高表达是膀胱尿路上皮癌癌变过程的一个关键因素，CHD1L信号传导是促进膀胱尿路上皮癌高度侵袭性转移的前提。为验证假设，首先通过动物实验验证CHD1L 在体內成瘤及转移中的作用;构建一系列缺失突变体检测CHD1L抑制膀胱尿路上皮癌细胞生长的关键结构域;利用RNA-seq和DNase-1-ChIP技术方法从CHD1L过度表达或siRNA沉默膀胱尿路上皮癌细胞中鉴定 CHD1L 关键作用蛋白及其结合的结构域;验证并研究CHD1L 与节点蛋白之间的相互作用及分子机理。为寻找新的膀胱尿路上皮癌治疗靶点和肿瘤诊断标志物奠定基础。

1、CHD1L对膀胱癌BT5637细胞增殖的影响.摘要：目的：探索CHD1L参与膀胱癌BT5637细胞增殖、侵袭、转移相关功能,，为进一步研究其致瘤分子机制奠定基础。方法：通过qPCR和western blot实验检测膀胱癌BT5637细胞株中CHD1L的表达情况，应用RNAi技术，导入合成的特异性针对CHD1L基因的siRNA敲减BT5637细胞珠中CHD1L的表达后，通过MTT比色法显示证实显著抑制了膀胱肿瘤细胞的增殖能力，细胞划痕实验来检测转染后细胞的迁移能力变化，细胞迁移及体外侵袭实验观察转染空细胞组、阴性对照组（NC-inhibitor）细胞与CHD1L-inhibitor组BT5637细胞迁移及侵袭能力。裸鼠移植瘤模型观察RNA干扰沉默CHD1L对移植瘤生长的影响。结果：qPCR 验证结果显示膀胱癌BT5637细胞中CHD1L呈高表达，通过RNA干扰降低CHD1L的表达能引起膀胱癌细胞BT5637的增殖抑制。MTT实验结果显示： CHD1L-inhibitor组BT5637细胞与-NC-inhibitor对照组相比，活细胞数量明显降低(P<0.05)，增殖能力受到显著抑制。划痕实验结果显示：第一天与第二天的迁移距离对比，转染CHD1L-inhibitor组BT5637细胞明显小于NC-inhibitor对照组(P<0.05)，迁移能力显著减弱。体外细胞迁移实验结果显示：转染CHD1L-inhibitor组BT5637细胞与-NC-inhibitor对照组相比，迁移及侵袭细胞数量均明显降低(P<0.05)，迁移及侵袭能力均受到显著抑制。BT5637-siRNA组裸鼠移植瘤生长速度减慢，30d后瘤重(0．38±0．24)g，明显低于BT5637-neu（0.6±0.23）g、BT5637细胞(0.69±0.40)g；统计学分析差异均有显著性。结论：CHD1L可能在膀胱癌中起致癌作用。.2、敲低GSG2（Haspin）通过靶向KIF15（Kinase-12）抑制膀胱癌的发展和进程.摘要：膀胱癌是全世界泌尿科系统中最常见的恶性肿瘤。 GSG2与膀胱癌之间的关系尚未得到证实，目前仍不清楚。在这项研究中，证明了与正常组织相比，膀胱癌组织中的GSG2上调，其高表达与更高级的恶性等级和更低的存活率相关。进一步的研究表明，GSG2的过表达/敲低可以促进/抑制膀胱癌细胞