TIMELESS在宫颈癌中的异常表达机制及靶向治疗研究

Chemo- and radiotherapy are important therapeutic strategies for advanced cervical cancer. The sensitivity of chemo- and radiotherapy is largely dependent on DNA repair capacity in cancer. The role of DNA repair in cervical cancer is still elusive. We performed meta-analysis of gene profile database in cervical cancer, which showed upregulation of multiple genes involved in DNA repair pathway including TIMELESS. TIMELESS regulates DNA replicate fork progression and DSB repair. We demonstrated that TIMELSS was overexpressed in 70% of cervical squamous cancer by immunohistochemistry, and that TIMELESS knockdown not only resulted in cell proliferation retardation, cell apoptosis and cell senescence, but also sensitized cancer cells to cisplatin. The upregulation of TIMELESS is possibly contributed by E2F1/4 activation. Through clinical investigation, in vitro assay and animal study, we expect to determine the deregulation mechanism and functional significance of TIMELESS, illuminate the effect of targeting TIMELESS on chemo- and radiotherapy response in cervical cancer, as well as the mechanism of TIMELESS in DNA repair pathway. The expected results will provide TIMELESS as a novel molecular therapy candidate regulating DNA repair pathway in cervical cancer.

放化疗是晚期宫颈癌的重要治疗方案，肿瘤的DNA修复能力影响放化疗敏感性。宫颈癌中DNA修复机制的研究尚有争议。我们的生物信息学分析显示宫颈癌中存在TIMELESS等多个DNA修复基因的表达上调。文献提示TIMELESS参与DNA复制叉进展和DNA修复。前期实验证实TIMELESS在70%宫颈癌中高表达；TIMELESS基因敲减导致细胞增殖阻滞、凋亡和衰老，并增强细胞对顺铂的敏感性。TIMELESS高表达的可能机制为E2F1/4功能激活。课题拟从分子细胞水平研究TIMELESS在宫颈癌中的表达及调控机制，靶向TIMELESS对宫颈癌DNA修复、细胞增殖、衰老、放化疗敏感性的影响以及TIMELESS调控DNA修复的机制，并从临床标本和动物实验多层次验证TIMELESS在宫颈癌中的作用及靶向治疗价值，为靶向TIMELESS及DNA修复通路治疗宫颈癌提供思路。

果蝇中TIMELESS主要参与生物钟调节，在哺乳动物中参与维持 DNA 复制叉稳定性。本研究探索TIMELESS在宫颈癌细胞增殖及顺铂敏感性中的作用。通过生物信息学分析、免疫组化及定量聚合酶链反应研究TIMELESS表达情况。通过免疫组化及荧光素酶报告基因检测研究导致TIMELESS表达上调的转录因子。通过体内及体外研究探索TIMELESS敲低对细胞增殖及顺铂敏感性的影响。通过流式细胞术及β-半乳糖苷酶染色实验研究细胞凋亡及衰老。通过彗星实验、免疫荧光染色及免疫组化研究DNA损伤修复通路。TIMELESS在 52.5%宫颈癌组织中呈高表达状态。E2F1和E2F4促使TIMELESS的转录激活。在体内及体外实验中，TIMELESS敲低抑制细胞增殖，提高顺铂敏感性。TIMELESS敲低诱导细胞凋亡及衰老。TIMELESS敲低导致DNA损伤及顺铂诱导的ATR/CHK1通路激活受阻。在宫颈癌中TIMELESS呈高表达状态并调节细胞增殖及顺铂敏感性，为宫颈癌顺铂增敏提供新思路。