阴道菌群变化调控TLR9对HPV16整合的影响及其在宫颈癌变中的作用

It is well known that the integration of human papillomavirus 16 (HPV 16) into the host genome is the causative agent of cervical cancer, but the mechanism is not clear precisely. The variation of bacterial vaginal microbiota and the expression of TLR9 are both closely related to HPV16 persistent infection and cervical carcinogenesis. Meanwhile, TLR9 could be activated by bacterial deoxycytidylate-phosphate -deoxyguanylate (CpG)-DNA. TLR9 has important roles in the progression of HPV-mediated cervical carcinogenesis, but there is no evidence to prove whether or not the effects were modulated by vaginal microbiome. Based on the expression of TLR9, which was activated by bacterial CpG-DNA, and was related to the methylation of HPV16 key integrative genes in the progression of cervical carcinogenesis, we hypothesize that the variation of vaginal microbiome may affect the integration of HPV16 and the development of cervical carcinoma by regulating TLR9. In order to verify this hypothesis, our study will focus on the interaction between TRL9 and key genes E2, L1, L2 of HPV16 integration. Women with different cervical lesions will be selected from the community and hospital cohorts that were previously established. Moreover, 16S rRNA sequencing, pyrosequencing, and other techniques will be applied. Firstly, we will investigate the interaction between characteristics of vaginal microbial flora, TLR9, and expression of HPV16 integrative key genes and effector genes in the process of cervical lesions. Furthermore, we will analyze the potential effects of TLR9 on methylation patterns of GpG sites in key genes of HPV16 integration. Meanwhile, the experiments in vitro will be carried out. TLR9 will be intervened by CpG-ODN agonist and siRNA technology, so as to confirm above biological functions. According to these results, this study aims to evaluate the effects of TLR9 regulated by vaginal microbial flora on HPV16 integration in cervical carcinogenesis, and further put forward a novel theoretical evidences for study on biological mechanism and risk prediction for cervical cancer as well as.

HPV16与宿主DNA整合是宫颈癌变的关键环节，但机制不明，TLR9与HPV16感染及宫颈病变进展关系密切，但是否受阴道菌群变化的影响缺乏证据。鉴于TLR9可被细菌CpG-DNA特异激活，且与HPV16整合基因的甲基化及宫颈病变紧密相关，提示，阴道菌群变化势必可通过调控TLR9影响HPV16整合及宫颈癌变的进展。为了验证该假设，本研究以TLR9与HPV16整合基因的相互作用为切入点，从建立的队列人群中选择不同宫颈病变者为研究对象，应用16S rRNA测序、焦磷酸测序等技术，分析阴道菌群及CpG-DNA变化与TLR9、HPV16基因表达在宫颈病变中的相互关系，探讨TLR9对整合基因甲基化模式的影响及其下游效应。同时结合体外实验，对TLR9实施双向调控，验证以上生物学功能。以此综合评价阴道菌群调控TLR9对HPV16整合及其在宫颈癌变中的效应，为宫颈癌生物学机制研究及风险预测提供新的理论依据。

阴道菌群结构、功能的改变和TLR9表达与HPV感染及宫颈病变的进展关系密切，但其相互关系及作用机制尚不明确。基于微生物基因CpG-DNA可特异激活TLR9的生物学基础，本研究依托前期建立的宫颈病变队列，形成从正常宫颈、宫颈癌前病变到宫颈癌的宫颈病变进展阶梯。利用16S rDNA测序、靶向DNA甲基化测序（TBS）及适宜的分子生物学技术，在系统分析阴道菌群特征及其CpG-DNA与HPV16感染及其整合关键基因表达的关系以及在宫颈病变进展中变化特点的基础上，深入探讨了阴道菌群诱导TLR9在HPV16致宫颈病变进展中的作用及可能机制。本次研究结果提示，阴道菌群结构功能改变及其导致的菌群CpG index的变化，可促进HPV16整合关键基因的表达和宫颈病变的进展，特别是在阴道微环境紊乱的情况下这种效应更为明显，维护阴道微生态的平衡对于预防和控制宫颈病变的进展意义重大；阴道菌群CpG基序的减少可诱导TLR9的异常高表达，促进宫颈病变的进展，TLR9作为阴道菌群未甲基化CpG基序的代表性生物标志，为宫颈病变的早期筛查和疾病进展的监测及预警提供了便捷手段，有极大的实用价值和推广前景；HPV16关键基因CpG位点的甲基化与HPV16 DNA与宿主整合及宫颈病变的进展密切相关，TLR9可能通过调节HPV16基因CpG位点甲基化影响HPV16的致癌效应及宫颈癌变的发生发展。本研究为宫颈病变病因和机制的研究提供了重要科学依据，从微生态-免疫学的角度为预测宫颈病变的进展和风险评估开拓了新思路。