易感基因CLPTM1L抑制鼻咽癌细胞凋亡的作用及机制研究

Nasopharyngeal carcinoma (NPC) is a malignant tumor prevalent in South China, with obvious genetic susceptibility. By large-scale multicenter genetic studies of NPC, we recently identified a susceptibility gene, CLPTM1L, to be associated with NPC risk. However, its functions and the mechanisms involved in NPC have yet to be defined. Analyzing the transcriptome sequencing data of NPC, we identified CLPTM1L gene was overexpressed in NPC tissues and high expression was associated with poor outcomes of NPC patients. After the CLPTM1L gene was knocked down, the cell apoptosis rate was increased and the expression of the BCL-2 was downregulated. We therefore hypothesize that CLPTM1L participate in the carcinogenesis and development of NPC through cell apoptosis. This study aims to further characterize the oncogenic capacity of CLPTM1L through the experiments in vitro and in vivo, and explore the novel CLPTM1L-related signaling pathways involved in NPC cell apoptosis by protein mass spectrometry and co-immunoprecipitation analysis. Besides, we will confirm the clinical value and prognosis evaluation of the CLPTM1L using the NPC specimens. By revealing the molecular mechanism underlying CLPTM1L and NPC pathogenesis, we will provide more biological evidence for the involvement of susceptible gene regions and more insights to the identification of biomarkers for early diagnosis, prognosis and targeted therapy as well as the development of novel diagnostic and therapeutic strategies for NPC.

鼻咽癌是我国特色恶性肿瘤，高发于华南地区，具有明显的遗传易感性。本课题组前期完成了大规模多中心的鼻咽癌遗传学分析，明确CLPTM1L基因与鼻咽癌发病风险显著关联（已发表）。目前，关于CLPTM1L在鼻咽癌中的功能、致癌机制及临床意义未见报道。我们初步研究显示CLPTM1L在鼻咽癌组织中高表达，且其高表达与鼻咽癌患者的预后显著相关；敲低CLPTM1L基因，鼻咽癌细胞凋亡增加，BCL2显著下调，提示CLPTM1L可能通过调控鼻咽癌细胞凋亡进而参与鼻咽癌的发生发展。在此基础上，本项目拟利用细胞及动物实验进一步证明CLPTM1L在鼻咽癌细胞中的功能，通过蛋白质谱及Co-IP分析确定其互作蛋白，阐释CLPTM1L下游信号轴参与鼻咽癌细胞凋亡的分子机制，并明确CLPTM1L在临床预后评价中的价值，为易感基因区域参与鼻咽癌致病提供生物学证据，为筛选鼻咽癌诊疗相关分子标志物及其相关诊疗手段提供必要依据。

鼻咽癌发病存在明显的地区聚集、家族聚集和种族差异，具有遗传易感性。前期全基因组关联研究分析发现5p15.33区域CLPTM1L基因的rs31489、rs401681位点和鼻咽癌的发病具有显著关联。eQTL和sQTL分析发现易感位点与CLPTM1L的表达和剪切相关。鼻咽癌转录组学数据、鼻咽癌单细胞测序、荧光定量PCR、免疫组化等方法分析结果表明CLPTM1L在鼻咽癌中表达显著高于鼻咽正常上皮细胞，且CLPTM1L基因在浆细胞中、EBV阳性的细胞群中表达水平较高。建立敲低CLPTM1L的鼻咽癌细胞模型，发现敲低CLPTM1L抑制肿瘤细胞增殖和迁移能力、促进肿瘤细胞凋亡，且提示CLPTM1L参与鼻咽癌细胞凋亡的分子机制与其引起PARP裂解有关。通过裸鼠皮下成瘤实验发现敲低CLPTM1L基因的两组裸鼠皮下肿瘤生长速度较Scramble对照组明显减慢，且瘤体体积均显著小于对照组。免疫荧光显示敲低组相比对照组Ki67荧光强度较弱，C-PARP荧光强度较强。皮下成瘤实验结果与细胞结果一致。转录组测序、荧光定量PCR验证差异表达基因，GO功能富集分析提示差异表达基因的功能涉及代谢、细胞应激、细胞凋亡和EBV感染等。IP及质谱分析探究CLPTM1L在发挥功能中可能的互作蛋白。临床病例预后分析CLPTM1L蛋白表达对鼻咽癌患者的预后影响。本研究明确了易感基因CLPTM1L在鼻咽癌发生发展中的作用，揭示CLPTM1L影响肿瘤细胞恶性行为的分子机制、阐释遗传易感因素的病因学机制。