“HSF1-miR-449b-DNA损伤应答基因XPC”通路促进膀胱癌发生、发展的作用与机制的研究

The XPC protein play important roles in the DNA damage respone, including the process of cell cycle arrest, apoptosis, and DNA repair, which is critical for the maintenance of gemomic stability. We have firstly reported that the attenuated expression of XPC protein is associated with the carcinogenesis of bladder cancer. In the following unpublished data, the overexpression of the protein of heat shock factor (HSF1) is observed in bladder cancer and the “HSF1-miR449b-XPC” regulation pathway is potentially exsited in both HEK293 and bladder cancer cells. We aim to investigate the regulatory mechanism of XPC gene silencing in bladder cancer and the role of “HSF1-miR449b-XPC” pathway in this project. Firstly, we will make an effort to prove that miR449b being a direct target of transcriptional factor HSF1 and XPC being a direct target of miR449b. Secondly , we will explore the existance and importance of the “HSF1-miR-449b-XPC” pathway in bladder cancer. Combined the associated studies of clinical ,bladder cancer cells and animal models, with the strategies of gene interference (shRNA-HSF1, mir449 mimmics), we will try to prove the overexpression of HSF1 inhibit the XPC-mediated DNA damage response and is contributed to the carcinogenesis of bladder cancer , via promoting the expression of mir-449b. With our studies in the future, it is not only hopeful to expose the novel role of HSF, Mir449b in the DNA damage response and carcinogenesis, and also provide the new cancer diagonosis marker based on XPC defection.

DNA修复基因XPC促进细胞凋亡、细胞周期阻滞或DNA修复等过程，有助于维持基因组稳定性。我们前期工作已首次证实膀胱癌发生、发展与XPC表达缺陷密切相关。基于后续研究初步发现热休克转录因子1（HSF1）在膀胱癌中高表达以及潜在“HSF1-miR449b-XPC”转录调控通路的重要线索，本研究将深入探讨膀胱癌XPC基因沉默机制。从分别证实“HSF1— miR449b” 、“mir449b—XPC”的关键转录调控作用入手，确立“HSF1-miR449b-XPC”级联调控通路；结合临床关联性研究，联合细胞学与动物学研究，采用外源基因干预策略，证实膀胱癌HSF1异常高表达通过上调miR-449b抑制XPC介导的DNA损伤应答，促进肿瘤发生、发展的作用与机制。本研究将揭示HSF1、Mir-449b在DNA损伤应答与肿瘤中的崭新角色，有助于阐明肿瘤高遗传不稳定性机制，有望为膀胱癌早期诊断提供新策略

DNA修复基因XPC促进细胞凋亡、细胞周期阻滞或DNA修复等过程，有助于维持基因组稳定性。我们前期工作已首次证实膀胱癌发生、发展与XPC表达缺陷密切相关。为进一步探讨膀胱癌中XPC表达缺陷的机制，在生物信息学预测的基础上，本研究首先利用Luciferace报告基因检测等技术证实了XPC是miR-449b的新型靶基因，然后利用启动子报告系统和ChIP等技术进一步证实了HSF1对miR-449b具有关键转录激活作用，从而证实了 “HSF1-miR-449b-XPC ”通路存在。进一步的循证医学研究表明，HSF1、miR-449b的高表达以及XPC表达缺失与膀胱癌临床病理学分级相关。为深入研究“HSF1-miR449b-XPC”通路促进膀胱癌发生、发展的机制，我们分别建立干扰HSF1以及过表达miR-449b的膀胱癌细胞模型，并进行了DNA修复能力、细胞侵袭转移、基因组稳定性等细胞学与动物学研究。结果显示过表达miR-449b降低细胞的DNA 损伤修复能力，增加DNA损伤诱导突变频率以及提高细胞的侵袭转移能力和致瘤力。干扰HSF1后则得到了相反的结果，但同时过表达miR-449b则能完全回补干扰HSF1的效应。进一步机制研究发现HSF1通过调控miR-449b表达促进膀胱癌细胞EMT过程，而抑制HSF1后可逆转EMT过程。由此可见miR-449b可促进膀胱癌细胞EMT过程。综上所述，本研究揭示了HSF1、miR-449b在DNA损伤应答与肿瘤发生中的崭新角色，为进一步以XPC基因表观遗传沉默作为膀胱癌早期诊断或预警、预后指标的研究提供了重要的实验基础。