碱基切除修复通路与鼻咽癌的关联性分析及风险点功能探索

Nasopharyngeal carcinoma（NPC）is the most common malignant tumor in South China. Genetic susceptibility and environment factors including Epstein-Barr virus（EBV）infection contribute greatly to the occurrence and development of NPC, and gene-environment-EBV interactions have become the research hot spot in recent years. By an extensive literature review and a comprehensive bioinformatics search，we found genetic polymeriphormsims including single nucleotide polymorphisms(SNPs) in many key genes in the base excision repair（BER）pathway,which may correlate closely with the susceptibility to NPC. In our preliminary research，a large number of blood and tissue samples of NPC patients and healthy subjects were collected，and we found an apparent elevated concentration of 8-OHdG（a biomarker of DNA damage）but with a significant downregulation of mRNA of hOGG1(a key gene in BER pathway )in both serum and tumor tissue of NPC patients. In addition, we also found a increased frequence of Cys/Cys genotype in the hOGG1Ser326Cys，which is associated with a higher risk of NPC.To further investigate the association between NPC susceptibility and SNPs of key genes in BER pathway，bioinformatics methods were used to select candidate SNP sites within these genes and predict possible functions including their influence on protein activity,DNA methylation pattern,transcription factor and miRNA binding. The data of the bioinformatics analysis, in combination with the results of SNP genotyping, medical records and questionnair survey of the patients and healthy subjects, will be used to study the relation between NPC susceptibility and the possible SNP sites. Our results of this research would give a deeper insight into the occurrence mechanism of NPC and improve the effectiveness of early alert for this malignant tumor.

鼻咽癌是中国南方常见的恶性肿瘤,主要与环境、遗传因素及EBV感染有关，因此遗传因素与环境的交互作用成为近年来的研究热点。通过大量的文献阅读及生物信息学检索发现DNA碱基切除修复（BER）通路中多个关键基因存在着遗传多态性，与鼻咽癌的遗传易感性相关。本课题组前期收集了大量样本并研究发现鼻咽癌患者血清及组织样品中DNA损伤标志物8-OHdG的含量明显增高，而其特异性碱基切除修复基因hOGG1mRNA表达水平明显下调，且hOGG1Ser326Cys中Cys/Cys基因型增多，使患鼻咽癌的风险增加。为了进一步深入研究，本课题拟通过基因分型，结合临床病历及问卷调查资料进行BER通路中多个关键基因的风险位点与鼻咽癌易感性关联分析，并经生物信息学预测后验证不同区域风险点对蛋白活性、DNA甲基化模式、转录因子或miRNA结合的影响。为探索鼻咽癌的发病机制及早期预警奠定基础。

鼻咽癌（NPC）是广西地区高发的恶性肿瘤，主要与环境、遗传因素及EBV感染有关，因此遗传因素与环境交互作用的致病因素成为近年来的研究热点。DNA碱基切除修复通路（BER）在维持机体稳定，抵御外来损伤上起到了至关重要的作用，我们利用文献检索及生物信息学知识筛选了20个BER通路关键基因及60个潜在的功能性SNP位点，对约1000余例包括本地鼻咽癌及健康对照人群样本进行MALDI-TOP质谱分型，筛选出hOGG1 rs1052133、hMUTYH rs3219472、APEX1 rs1760944等潜在的差异性功能位点，经Taqman探针实时荧光定量PCR法进行分型验证，结果显示hOGG1 rs1052133、APEX1 rs1760944可能与鼻咽癌的发生发展相关。本课题组通过临床样本检测、患者预后分析、慢病毒载体构建检测不同基因型在正常及氧化应激情况下的功能差异、转录组测序功能分析及移植瘤小鼠模型等实验对hOGG1 rs1052133进行功能分析， 结果显示鼻咽癌患者中局部组织及外周血中氧化损伤产物8-OHdG含量明显高于粘膜慢性炎患者，由于组织样本偏少hOGG1 GG和CC型鼻咽癌患者中的差异尚无统计学差异。在纳入的371例鼻咽癌患者预后分析中发现CC型患者预后明显差于GG或CG型患者。感染了CC及GG型hOGG1基因慢病毒的鼻咽癌细胞株HK-1在正常培养情况下增殖活力与空载对照组细胞无明显差异，但在低剂量光动力治疗处理后的细胞中过载了hOGG1呈现出明显的抗损伤修复能力，且CC型强于GG型，通过转录组测序发现CC型在抵抗氧化损伤方面可以激活更多的抗损伤基因表达及减少凋亡相关基因的表达。通过移植瘤裸鼠实验也发现过载hOGG1的细胞形成的移植瘤更大。通过该项目的研究我们发现hOGG1 rs1052133多态性与鼻咽癌的发生发展密切相关，GG基因型可能在损伤早期更容易诱导凋亡而减少了肿瘤的产生，而在肿瘤治疗阶段由于氧化损伤修复能力较CC型弱，因此也更容易修复失败而诱导凋亡，形成更好的治疗效果，更具体的机制有待于我们进一步研究。