化疗脱发中p53对Sonic Hedgehog信号通路调控机制研究

Chemotherapy-induced alopecia (CIA), one of the major and frequent side effects of cancer chemotherapy, represents one of the major unmet challenges in clinical oncology since the molecular mechanism of which remains largely unknown. In previous studies，we had proofed that Shh signaling pathway is a major target of chemotherapeutic agents in chemotherapy-induced feather defect, as well as in CIA mouse. However, how do chemotherapeutic agents interfere in the expression of Shh remains unknown. Previous studies have reported that p53-deficient mice don’t undergo hair loss under Cyclophosphamide（CYP）treatment, though this conclusion was challenged and revised in some following studies. We have found out previously that in p53-deficient mice, the expressions of Shh as well as its downstream genes are not repressed after CYP treatment. This founding rises up two attractive questions:(1) Is that p53 mediate the repression of chemotherapeutic agents on the Shh signaling pathway？(2) If so, how does p53 interfere in the expression of Shh？To solve these two questions, here in this present study, experiments will be carried out to investigate the direct regulations of p53 on Shh signaling pathway, using ChIP-Seq, luciferase reporter assay, as well as EMSA, in wild type and/or p53-deficient mice. Also the indirect regulations of p53 on Shh signaling pathway will be investigated by using RNA-Seq combined with ChIP-Seq and luciferase reporter assay. Our findings in this study will reveal the molecular mechanism of the regulation of p53 on the expression of Shh in CIA, and elucidate a new mechanism on chemotherapeutic agents induced tissue injury.

化疗脱发（CIA）是最常见的化疗副作用之一，但其发生的分子机制尚不清楚。本实验室前期研究表明，Shh信号是化疗药物损伤羽毛和小鼠毛发关键靶点，但是化疗药物如何干扰Shh的表达尚不清楚。前人的研究表明，p53基因缺失小鼠不发生CIA，虽然这个结论在后续的研究中受到挑战和修改。而我们的初步结果表明，p53基因缺失小鼠在环磷酰胺处理后Shh及其下游基因的表达并没有下降。那么，p53是否介导了化疗药物对Shh的抑制？p53又是如何调控Shh的表达？为了探究这些问题，本研究将应用小鼠CIA模型、p53基因缺失小鼠等，利用ChIP-Seq、报告基因、EMSA等技术研究CIA发生过程中p53对Shh的直接调控的机制；利用RNA-Seq结合ChIP- Seq、报告基因等技术研究p53对Shh的可能的间接调控方式，从而揭示p53介导化疗药物调控Shh的分子机制，阐明化疗药物损伤正常组织的新机理。

化疗脱发是目前癌症治疗中仍无法解决的一个重要的问题。文献报道，p53基因缺失的小鼠不发生化疗脱发。我们前期发现，野生型小鼠注射环磷酰胺后皮肤中Shh基因的表达水平显著下降，而p53基因缺失的小鼠中相关基因则没有变化。为了探究化疗脱发中p53是否对Shh信号通路存在调控，我们利用免疫染色、qPCR、RNA-seq、iTRAQ、ChIP-seq、ChIP-PCR以及报告基因等手段展开研究。实验结果表明，注射CYP后，毛囊中Shh基因表达水平显著下降，而p53、pERK、g H2AX、Casp3等基因显著活化；Shh的激动剂SAG能够缓解CYP造成的损伤，而其抑制剂Vismodegib则能引起毛发损伤；RNA-seq的结果显示野生型小鼠中p53的靶基因显著活化，Shh信号通路相关基因则显著下调，而这些基因的变化在p53缺失小鼠中都不存在；磷酸化蛋白组分析结果显示p53以及MAPK信号通路显著活化；p53的激动剂能够损伤毛发，而其抑制剂则能缓解化疗脱发；MAPK的抑制剂或者ERK2基因显性失活都能缓解CIA的发生，但是在p53基因缺失小鼠中，CYP同样能够活化的ERK，说明ERK对Shh的调控依赖p53；ChIP-seq和ChIP-PCR的结果显示p53能够结合到Shh基因的远端增强子、启动子和内含子上，并且过表达p53能够抑制Shh启动子的活性；免疫共沉淀以及报告基因的结果显示p53与STAT1存在相互结合，共同抑制Shh启动子的活性；p53的靶基因EDA2R的表达量在CYP处理后显著上升，而EDAR、EDARADD、TRAF3则显著下降，提示EDA2R能够干扰EDA-EDAR信号传导从而抑制Shh基因的表达；人毛发体外培养系统以及临床化疗病人毛发样品的研究结果表明，在化疗药物引起毛发损伤的同时，Shh基因的表达也显著下降。这些结果表明，CIA中p53一方面能够结合到Shh基因上，并与其他转录因子共同调控Shh的表达；另一方面能够通过其靶基因影响Shh的表达。