放射线引起宫颈癌上皮间质转化（EMT）的机制研究：SPOP扮演的角色？

Radiotherapy is an important therapeutic tool for Cervical carcinoma, thus the effective regulation of epithelial mesenchymal transition (EMT) inducing by irradiation in Cervical carcinoma is one of the hot issues in Cervical carcinoma research. Irradiation activates DDR in tumor cells, and high level of DDR promotes tumor EMT, invasion and metastasis. However, the specific mechanism of how DDR regulating EMT in Cervical carcinoma after radiation is not clear. Our previous work showed SPOP played an important role in DDR pathway as a new DDR member. Combined with studies of SPOP regulated the invasion and metastasis in breast cancer cells, as well as the cause and development in gastric cancer, we hypothesize: SPOP regulates the EMT process, then promotes tumor invasion and metastasis in Cervical carcinoma after radiation. Based on former results, we will use diverse methods, such as CRISPR/Cas9, Quantitative Real-time PCR, Western Blotting, immunofluorescence and mass spectrometry, to perform the experiments in Cervical carcinoma tissues and cell lines respectively. We will determine the correlation between SPOP and EMT molecules. We will also explore the specific signal molecules and related signaling pathways by which SPOP involved in EMT after radiation. Finally, the function and mechanism of ‘irradiation - SPOP - EMT - invasion and metastasis’ in Cervical carcinoma will be realized. This work can provide new research idea in controlling tumor invasion and metastasisin in the course of radiotherapy for Cervical carcinoma.

放射治疗是宫颈癌治疗的重要手段之一。研究表明放射线可激活肿瘤DNA损伤应答机制（DDR），过高DDR能促进肿瘤上皮间质转化（EMT）及侵袭转移。但DDR参与宫颈癌EMT发生的机制未明。我们研究发现SPOP在放射线激活的DDR中发挥重要作用。结合文献SPOP调控乳腺癌细胞侵袭转移及胃癌发生发展。提出假说：放射线激活后，DDR成员SPOP参与调控宫颈癌EMT发生，进而影响宫颈癌侵袭转移?本项目拟采用CRISPR/Cas9、实时定量PCR、免疫印迹、蛋白质谱等技术分别在宫颈癌组织及细胞系水平展开研究。首先确定放射线激活后SPOP与EMT分子的相关性，随后筛选并验证SPOP下游关键分子在放射线引起宫颈癌EMT中的作用，最终通过明确放射线激活后SPOP调控宫颈癌EMT发生的信号分子及相关信号通路，探讨放射线激活的SPOP调控EMT发生的作用机制。为宫颈癌放射治疗过程中侵袭转移的控制提供新的研究思路。

本课题组既往研究发现泛素连接酶SPOP作为DNA损伤应答（DDR）新成员在放射线激活的DDR信号网络中发挥重要作用。在此基础上该项目进一步揭示了放射线引起DNA损伤后，SPOP通过参与DDR调控肿瘤细胞多种生物学行为的功能及机制。首先，我们明确了SPOP在肿瘤组织及肿瘤细胞系中的广泛表达。我们进一步发现DNA损伤后SPOP被招募至损伤位点，并出现mRNA水平、蛋白水平的表达升高。接着，基于SPOP knockdown及SPOP knockout肿瘤细胞系的成功构建，我们证实了SPOP在放射线诱导肿瘤细胞的DNA损伤修复、细胞周期阻滞、细胞凋亡、放疗敏感性、EMT及细胞迁移中的调控功能。在对机制的探究中，我们发现SPOP knockdown可提高DNA损伤修复通路关键蛋白Rad51表达水平，而对Ku80表达未见显著影响；同时发现SPOP knockdown可导致EMT标志物Snail的蛋白水平显著降低，且可逆转放射线诱导的肿瘤细胞迁移增加。最后，我们通过定量蛋白质谱检测及生物信息学分析，获得了放射线激活后SPOP的多个相关性分子，并验证了SPOP与下游分子的表达关系。综上所述，本项目的研究结果为临床放疗过程中肿瘤细胞残留及转移的控制提供了新的思路和策略，并为SPOP作为联合肿瘤放疗的基因治疗新靶点提供了重要的理论依据。