DNA修复蛋白Ku在人甲状腺乳头状癌发生发展中的作用及其分子机制研究

Papillary thyroid carcinoma (PTC) is the most common malignant tumor of the endocrine system. The incidence of PTC is rising. Recent reports suggest that there is a positive relationship between Ku and the development of cancer, however, the relationship between Ku and PTC still unclear. Our prior studies suggest that the expressions of Ku80 were increased in PTC, and the NF-κB were activated. Further work is required to investigate whether and how Ku participates in the pathogenesis of PTC. Based on our prior results, we intend to detect the expressions of Ku, RET/PTC, NF-κB and P53 in PTC and normal thyroid tissues and cells to identify the link between Ku and PTC in this study. The correlation between the expession of Ku and the characters of clinic, pathology and biology will also be analysised. In addition, we will use RNAi plasmid and Ku expression plasmid transfection of PTC cells to inhibit or promote Ku expression to investigate the effect of Ku on PTC cancer cells and its transplanted models in nude mice. The purpose of this project is to clarify the possible role and pathogenesis of Ku and RET/PTC/NF-kB/Ku/P53 signal pathway in the occurrence and development of PTC. This will provide new thinking to the etiopathogenisis of PTC and will provide new rational target molecule treatment for PTC.

甲状腺乳头状癌(PTC)是最常见的内分泌系统恶性肿瘤，其发病呈上升趋势。最近报道DNA修复蛋白Ku与多种恶性肿瘤的发生发展密切相关，但与PTC的关系尚不清楚。我们前期研究发现PTC癌组织中Ku80表达增高且核转录因子NF-κB活化，为研究Ku与PTC的关系奠定了一定的基础。本研究拟通过检测PTC癌组织及多株癌细胞内Ku、RET/PTC、NF-κB及P53的表达明确Ku与PTC的相关性及RET/PTC/NF-κB/Ku/P53信号通路蛋白间交互作用；通过RNAi抑制Ku表达试验及Ku转染试验，研究Ku对PTC癌细胞生物学特性及裸鼠移植瘤生长的影响，结合分析研究Ku表达与PTC临床病理参数的关系，明确Ku在PTC发生发展中的作用。本研究旨在阐明Ku及RET/PTC/NF-κB/Ku/P53信号通路在PTC发生发展中的作用及分子机制，为PTC病因机制研究提供新思路，为PTC治疗提供新的分子靶点。

甲状腺乳头状癌(PTC)是最常见的甲状腺恶性肿瘤，好发于儿童和年轻女性，易发生淋巴结转移，部分患者还多次复发、手术，严重危害人类健康。Ku蛋白是DNA双链断裂的主要修复蛋白，由Ku80与Ku70异源二聚体构成。有研究发现Ku表达异常与多种肿瘤的发病机制相关，但与PTC的关系尚未见文献报道。本研究从组织、细胞两个层面研究PTC癌组织及细胞内Ku的表达及其作用，探讨Ku与PTC发生、发展的相关性及其作用机制。用免疫组织化学方法（IHC）检测47例PTC及18例正常甲状腺组织（NTT）内Ku蛋白的表达，用Real time PCR方法检测目的基因Ku在PTC细胞株中mRNA表达情况。20例桥本甲状腺炎(HT）、15例HT合并PTC、20例PTC及18例NTT，用聚合酶链反应PCR方法检测RET/PTC重排，用IHC方法检测RET/PTC、NF-κB、Ki67、P53蛋白表达。回顾性分析53例PTC临床病理资料。用配对卡方检验研究甲状腺组织中Ku与各分子表达及临床参数之间的相关性。结果显示，Ku在PTC癌组织及多个PTC癌细胞株中均高表达，而在癌旁及NTT中，阳性信号明显减少；RET/PTC、NF-κB、Ki67、P53在PTC中阳性率分别为65%，95%，33.3%和34.5%，在NTT中阳性率分别为0，27.8%，0和0，差异有统计学意义（均P<0.05）。PCR结果显示PTC病人中60%，HT中35%发生RET/PTC重排，NTT中无重排，IHC检测结果与PCR 结果一致。Ku表达与RET/PTC、NF-κB、P53、Ki67表达相关，且Ku表达与肿瘤TNM分期、肿瘤个数、肿瘤大小正相关；CRISPR/CAS9技术敲除Ku基因后观察细胞增殖、凋亡及细胞周期的变化，结果发现，Ku基因敲除后，PTC癌细胞增殖、侵袭、转移能力以及增殖成瘤能力均显著下降，而凋亡率明显上升，细胞周期阻滞。用抗体芯片方法研究Ku相关信号通路。结果发现，Ku基因敲除后，PTC癌细胞MAPK信号通路相关分子及增殖、凋亡相关分子蛋白表达水平均显著下调。这些研究结果提示Ku高表达与PTC的发生、发展密切相关。Ku表达水平上调，调控多种基因转录，通过MAPK通路活化RET/PTC和NF-κB，抑制凋亡、促进细胞增殖、侵袭、转移，导致PTC发生和发展。我们的研究结果为PTC靶向治疗新靶点提供了理论依据。