外泌体蛋白PPP1CB介导的食管癌放疗抵抗机制研究

Radiochemoresistance of patients with locally advanced esophageal cancer （ESCC）is still a challenge for the clinical treatment. A noninvasive liquid biomarker is urgently needed to accurately predict the sensitivity of patients with ESCC before treatment. Previous studies showed that radiotherapy can promote the release of exosomes in cancer cells. However, the mechanism of exosomal protein mediating radioresistance of esophageal cancer remains unclear. We used the radioresistant esophageal cancer cell model, and established methods of exosome isolation and purification. Protein spectrum（ITRAQ/TMT）is used to determine the type of exosome-derived proteins, and identify the differentially expressed exosomal proteins. In this study, we explore the biological function of PPP1CB. PPP1CB interaction proteins (PIPs) will be screened using protein spectrum and determine the model of PPP1CB-PIPs interaction. Moreover, serum PPP1CB will be detected in patients with ESCC using EILSA kit，and identify the correlation of PPP1CB protein levels with the pathological response or prognosis. The exosome-based delivery systems (PPP1CB siRNA-iExosome or PPP1CB regulated protein-exosome) will be used to explore whether or not PPP1CB affect the sensitivity of radiotherapy. This study, focusing on the role of PPP1CB in regulating radiosensitivity in ESCC, could be a promising non-invasive biomarker for facilitating personalized treatments.

局部晚期食管癌患者放化疗抵抗仍然是临床治疗所面临的一个挑战，迫切需要一种无创的液体标志物在治疗前精确地预测食管癌患者对放化疗是否敏感。研究表明放疗可以促进肿瘤细胞外泌体释放，至于外泌体蛋白通过何种机制介导食管癌放疗抵抗尚不清楚。我们前期以食管癌耐放射细胞为模型，建立和优化外泌体分离、纯化及检测平台；利用蛋白质谱iTRAQ/TMT方法确定差异表达的外泌体蛋白；本课题以外泌体蛋白PPP1CB为研究对象，探讨PPP1CB的生物学功能；筛选PPP1CB相互作用蛋白(PIPs)，确定PPP1CB-PIPs作用模式；检测血清PPP1CB蛋白水平，确定其与食管癌放疗疗效及预后的关系；应用外泌体递送系统(PPP1CB siRNA-iExosome 或PPP1CB调节蛋白-Exosome)，确定其是否影响使放疗敏感性；深入阐述外泌体蛋白PPP1CB介导的食管癌放疗抵抗机制，为临床个体化精准治疗提供依据。

局部晚期食管癌患者放化疗抵抗仍然是临床治疗所面临的一个挑战，研究表明放疗可以促进肿瘤细胞外泌体释放，至于外泌体蛋白通过何种机制介导食管癌放疗抵抗尚不清楚。我们以前期构建的食管癌耐放射细胞为模型，建立和优化外泌体分离、纯化及检测平台；利用蛋白质谱iTRAQ/TMT方法筛选差异表达的外泌体蛋白；本项目以外泌体蛋白PPP1CB为研究对象，探讨食管癌耐放射细胞与PPP1CB的生物学功能及其与放疗抵抗的相关性，利用蛋白组学与磷酸化组学方法探究PPP1CB调控的蛋白网络，深入阐述外泌体蛋白PPP1CB 介导的食管癌放疗抵抗机制，为临床食管癌个体化精准治疗提供依据。.通过本项目研究发现食管癌耐放射细胞KYSE30R干性、侵袭与转移能力显著增强；KYSE30R分泌外泌体可以显著促进食管癌细胞迁移与侵袭、放疗抵抗；外泌体蛋白PPP1CB在食管癌耐放射细胞与食管癌组织中表达显著上调，PPP1CB主要位于细胞核和细胞浆，细胞膜也可见少了表达。过表达PPP1CB可以促进食管癌细胞增殖、转移及放疗抵抗, 相反地，敲降PPP1CB可以促进食管癌细胞放疗敏感性。PPP1CB可能通过影响剪切复合体多个成员的磷酸化修饰，进而促进食管癌放疗抵抗。此外，我们在外泌体多组学数据分析中，将本项目研究外衍，利用交叉学科互融的优势，开发了细胞外囊泡药物递送体系和GDY-CeO2纳米酶递送体系实现了食管癌放化疗增敏。本研究构建外泌体分离纯化平台、细胞外囊泡和GDY-CeO2纳米酶药物递送体系，有望为肿瘤药物高效递送提供理想地工具。