分割照射下，ATM 与TGFβ1/Smad 信号交联（cross-talk)对宫颈癌放射拮抗的调控作用及机制研究

Radiotherapy is the mainstay of treatment for patients with locally advanced cervical cancer. However, local relapse has continued to be the dominant cause of recurrence and death for them. The cells that survive fractionated radiation therapy show significant changes in some signaling pathways including ATM and TGFβ1, which may play an important role in the regulation of radiation resistance. Previously we obtained an stable radioresistant sub-line from the human cervical cancer cell line Hela by prolonged exposure to X-rays. We found that the radiosensitivity in acquired radioresistant Hela cells could be recovered by TGFβ1 signaling inhibition. Moreover,compared with irradiated cells treated with vehicle, the expression of phosphorylated ATM protein was significant reduced when radioresistant Hela cells were irradiated following a 48 hour exposure to LY2109761, which indicateed the cross-talk between ATM and TGFβ1 signaling might be involved in the regulation of radioresistance. The present project is to explore the mechanisms of cross-talk between ATM and TGFβ1 signaling pathways and to investigate their role in the regulation of radiation resistance in fractionated radiation. Some critical molecules of ATM and TGFβ1 signaling pathways will be estimated when one or other pathway is inhibited or activated in the radioresistant cervical cancer cells. We firstly use RNA intervention, immunoprecipitation, Chip assay and EMSA methods to explore the expressions, the activity, the sub-location of those aforementioned cross-talk and to investigate the relationship between the corss-talk and radiosensitivity. secondly, we will perform gene expression profilling by transcriptome microassay and analyze pathway network by bioinformatics methods to define the relevant molecular mechanisms. Thirdly, we will confirm the results in vitro by xenograft in nude mice and tissue from cervical cancer patients. The radiation resistance regulatory mechanisms of the cross-talk between ATM and TGFβ1signaling pathway in fractionated radiation will be clarified at molecular, cellular and intact living organisms levels, which will provide new molecular targets and new concept for the reverse of radiation resistance in cervical caner.

局部复发是中晚期宫颈癌放射治疗失败的主要原因。分次照射可激活肿瘤细胞ATM和TGFβ1信号通路，促进肿瘤细胞对放射拮抗。我们前期研究提示，ATM和TGFβ1/smad信号通路之间可能存在cross-talk。在此基础上，本项目利用前期在分割照射下获得的耐辐射的宫颈癌细胞株，采用RNA干扰、免疫沉淀、ChIP及EMSA等技术，研究干预ATM和/或TGFβ1信号通路对上述通路关键节点分子的表达、活性、亚细胞定位及细胞放射敏感性的影响；采用基因微阵列技术检测基因表达谱改变，运用生物信息学工具分析差异表达基因的功能和信号通路网络，并利用裸鼠移植瘤模型和患者标本进一步验证体外实验结果。本项目拟从分子、细胞及整体水平揭示分割照射下ATM和TGFβ1/smad信号通路cross-talk对宫颈癌的放射拮抗的调控作用及机制，为逆转分割照射下宫颈癌获得性放射拮抗提供理论基础和实验依据。

宫颈癌是严重威胁妇女身心健康的恶性肿瘤之一，放射治疗是中晚期宫颈癌的主要治疗手段之一，而放射拮抗是宫颈癌患者局部治疗失败的重要原因。Rad21是黏连蛋白的核心亚单位，在DNA损伤修复、染色体凝集和细胞凋亡中具有一定的作用。但其在宫颈癌放射拮抗和EMT方面的作用尚不清楚。本研究通过长期分割照射从亲代细胞系(HeLa和Siha)中建立了两种耐辐射宫颈癌细胞系(HeLa-R和Siha-R)。与亲代细胞相比耐辐射的细胞表现出显著的获得性放射拮抗和EMT表型。.研究发现：（1）耐辐射宫颈癌细胞对放疗的敏感性有显著改变，通过对照射后细胞存活率进行分析，Siha-R和Hela-R细胞在单次剂量（0-8Gy）照射下，和亲代细胞相比表现出明显的放疗抵抗性；（2）放射拮抗的宫颈癌细胞株呈现EMT表型并增强肿瘤的恶性程度，在耐辐射细胞中，mRNA和蛋白表达水平上N-cadherin和Vimentin均有较高的上调，Transwell结果显示耐辐射细胞具有更强的迁移和侵袭能力；（3）RAD21参与放疗抵抗和EMT，RAD21过表达能显著促进HeLa细胞和Siha细胞的辐射抗性和增强DNA损伤修复，沉默RAD21时可以逆向改变耐辐射细胞（Hela-R和Siha-R）的放射敏感性以及EMT；（4）RAD21与TGF-β1启动子结合调节TGF-β1的表达,从而促进宫颈癌细胞照射后产生放疗抵抗和EMT。.在耐辐射宫颈癌细胞中RAD21呈高表达状态。RAD21可以通过与TGF-β1启动子结合调节TGF-β1的表达增加，从而促进宫颈癌细胞照射后产生的放疗抵抗和EMT。这些结果表明RAD21在宫颈癌获得性放疗抵抗中发挥了重要作用，这可能是患者放疗后局部复发和预后不良的因素，也是作为增加宫颈癌放疗敏感性及改善患者生存的一个潜在治疗靶点。