口腔黏膜上皮炎癌转化过程中TLR-9基因过表达的转录调控机制研究

It is well known that chronic inflammation has close relation with tumor occurrence and development.Our previous research identified an gradually evaluated expression of TLR-9 during the malignant transformation of oral mucosa epitheliitis to squamous cell carcinoma via stimulating the production and secretion of the AP-1 activated inflammatory factors，which contributed to the tumor cell proliferation, invasion, immune response increasing and tumor microenvironment formation. Thus, TLR-9 is strongly suspected to be one of the key factors in inflammation-related oral mucosa carcinogenesis. However,little is known about the regulating mechanism on overexpression of TLR-9 gene in detail during this procedure. By further literature review and priliminary experiment,we found that there are many GC box, GA repeated sequence and combining sites of inflammation-related transcription factors, including NF-kappaB and AP-1, in the transcription initiation site of TLR-9 gene. We also found an gradually increased TLR-9 transcription initiation site activity during the transformation of oral mucosa epitheliitis to squamous cell carcinoma. Thus we speculate that cis-acting elements such as GC box, GA repeated sequence, and trans-acting factors such as AP-1and NF-kappaB in the transcription initiation site of TLR-9 gene ,may play a key role in the initiation of oral mucosa inflammation-related carcinogenesis.And this abnormal regulation of TLR-9 gene transcription from mRNA level and subsequent cascade reaction of downstream inflammation-related signal pathway,including the TLR-9/AP-1 positive feedback loop, may involved in the whole procedure of malignant transformation of oral mucosa epitheliitis to squamous cell carcinoma. Based on combined utilization of bioinformatics,genomics,molecular biology,cytobiology and experimental zoology, the present study try to further explore the possible initial and promoting mechanisms in inflammation-related oral mucosa caicinogensis from gene transcription level.We believe this research would provide new information and inspiration in prevention and treatment of inflammation-related oral squamous cell carcinoma in future.

慢性炎症与恶性肿瘤的发生发展关系密切。课题组的前期研究明确了TLR-9可以通过活化AP-1炎性信号通路而促进口腔黏膜上皮的癌变，极有可能是口腔黏膜炎癌转化调控网络中的关键节点之一。然而，对于癌变过程中TLR-9基因过表达的始动调控机制仍未明确。结合文献检索及预初实验，课题组发现TLR-9基因的转录启动子含有多个GC盒、GA重复序列以及多种炎性转录因子结合位点；伴随癌变进程，TLR-9基因的转录启动子活性明显阶梯型增高。由此，课题组推测"由GC盒和GA重复序列为代表的顺式调控元件和以AP-1及NF-kB为代表的反式作用因子可能是口腔黏膜上皮炎癌转化过程中TLR-9基因过表达的转录起始调控关键。" ，并希望综合运用生物信息学、基因组学、分子生物学及实验动物学等学科交叉的研究方式，从基因转录调控的角度对口腔黏膜上皮炎癌转变的始动及促进机制进行深入研究，为口腔鳞癌炎性发病机制的探索提供新的启示。

慢性炎症与恶性肿瘤的发生发展关系密切。课题组前期研究发现TLR-9蛋白在口腔鳞癌中表达增高，且在正常口腔黏膜上皮、炎性上皮、不典型增生及早期癌变中的表达量呈现明显的上升趋势，过表达的TLR-9蛋白与口腔鳞癌患者的临床分期及预后显著相关。本课题进一步对癌变过程中TLR-9 基因过表达的始动调控机制进一步探讨。首先，本项目明确了TLR-9 启动子的核心位置（-1432至-725区间序列）及主要的转录调控元件（AP-1,STAT1, CREB,ATF-2等）；其次，体外联合体内实验表明口腔黏膜上皮炎癌转化过程中TLR-9mRNA表达量逐渐增高，且与TLR-9启动子活性逐渐增高相关；进一步地，AP-1过表达载体与TLR-9 启动子-荧光素酶报道基因共转染初步表明TLR-9/AP-1 正反馈环路的存在；随后，TLR-9全基因测序发现启动子区6个基因突变位点，突变率超过50%，尤其集中在核心区域上游250~310bp范围内,且MUT4可明显增高TLR-9启动子的活性；同时，对正常人、口腔癌变患者的外周血循环pDC及组织内浸润pDC进行了检测，结果显示循环外周血pDC在正常人与口腔癌变患者之间无明显差别，但组织内的浸润pDC在正常上皮组织、癌前病变组织及癌变组织内依次增高，提示癌变组织内的浸润pDC由外周循环血迁徙至癌变部位，且与癌变进程密切相关。本项目的研究结果显示TLR-9基因转录调控失衡在其过表达及其引发的下游炎性信号通路级联反应在口腔黏膜上皮炎癌转化过程中发挥了始动作用，且其启动子区特异的点突变可进一步增高启动子的活性并最终提高TLR-9的表达量。因此，应用特异的TLR-9启动子及特异位点（mut4）活性抑制剂及从转录水平抑制TLR-9基因的过表达，势必会在口腔癌患者精准治疗及口腔鳞状上皮细胞癌变的探讨中具有重要的提示作用。截止2018年12月，已经发表课题相关SCI论文2篇，均标注国家自然自然科学基金资助（Grant NO. 81472517），且上述研究进展已整理且文章已完成撰写，正在准备投稿 ( IF > 3.5 )，课题实施期间培养硕士研究生、博士士研究生及口腔肿瘤实验室技术骨干各1名，