HPV16 E6/E7调控长链非编码RNA促进宫颈癌进展的分子机制研究

High-risk human papillomaviruses (HR-HPVs) infection is considered the causative agents of cervical cancer and HPV genotypes 16 and 18 cause the majority of these cancers. It is well established that oncoproteins E6 and E7 are the major transforming agents, leading to carcinogenesis. Recently, some studies are carried out on long non-coding RNAs (lncRNAs) in cervical cancer with encouraging results. But there is scarcity of literature about the association of lncRNAs in HPV positive cervical cancer and their possible interactions. Considering that E6 and E7 have definite interactions with microRNAs (miRNAs) which can modulate the process of carcinogenesis, we hypothesized that HR-HPV infection may be a key factor inducing lncRNAs expression in cervical cancer, and some lncRNAs regulated by E6 or E7 can function as competing endogenous RNA (ceRNA) by competitively binding common miRNAs in cervical cancer development. .To test this hypothesis, we aim to identify lncRNAs regulated by HPV16 E6/E7 (named E6/E7 lncRNA) using microarray technology, and validate the relationship between E6/E7 lncRNAs and HPV16 E6/E7 expression. Further studies will be performed to clarify the biological functions of E6/E7 lncRNAs in cell proliferation, cell cycle, apoptosis, migration and invasion in vivo and in vitro through gain-of-function/loss-of-fuction experiments. Using dual-luciferase reporter assay, Ago2 immunoprecipitation and RNA pull-down, we will confirm the specific association between miRNA and E6/E7 lncRNA. In addition, we plan to examine the expression of E6/E7, lncRNA, miRNA and its target genes in sufficient series of well-defined clinical cervical tissues, tissue microarrays and different cervical cell lines by performing qRT-PCR, in situ hybridization (ISH) and immunohistochemical staining, and analyze the correlations between their expression and poor prognostic clinicopathologic parameters. Taken together, our study on HPV16 E6/E7 and host lncRNA-miRNA interactions will continue shedding more light on our understanding of the of the pathogenesis of cervcial tumorigenesis, and implicate its potential application for predicting prognosis or cancer therapy in the future.

高危人乳头瘤病毒E6、E7在细胞恶性转化中极为关键。近年来，长链非编码RNA在宫颈癌中的作用逐渐为人关注，但lncRNA与E6、E7的关系却鲜有报道。鉴于宫颈癌中E6、E7与miRNA的相互作用已有较多证据，我们大胆地猜想E6、E7通过调控lncRNA的表达，以竞争性内源RNA的机制影响宫颈癌的进展。为此，本课题拟在前期完成的HPV16阳性宫颈癌组织lncRNA表达谱基础上，采用芯片技术比较E6/E7沉默前后Caski细胞lncRNA表达谱的改变，从细胞水平鉴定受E6或E7调控的lncRNA。通过体内外实验观察其异常表达对宫颈癌肿瘤生物学效应的影响，结合双荧光素酶报告实验、免疫共沉淀、RNA pull-down等确定竞争结合的miRNA及下游靶基因。旨在进一步完善HPV致癌的分子机制，为从lncRNA、miRNA和HPV感染层面揭示宫颈癌的发病机理，探寻宫颈癌防治新策略提供科学依据。

高危人乳头瘤病毒E6、E7在细胞恶性转化中极为关键。近年来，长链非编码RNA（long non-coding RNA, lncRNA）在HPV相关肿瘤中的作用逐渐为人们所关注，但lncRNA与E6、E7的关系却鲜有报道。随着研究的深入，越来越多的报道提示，部分lncRNA在HPV感染后表达异常，其在病毒致癌过程中的作用及机制正被逐步揭晓。本项目利用lncRNA芯片技术比较了HPV16或HPV18阳性的宫颈鳞癌组织以及HPV阴性的正常宫颈上皮组织中的lncRNA表达谱，结合E6/E7干扰前后lncRNA的差异表达分析，通过临床样品及体外实验验证，筛选确定了受到E6/E7调控的候选lncRNA（SNHG12、HIF1A-AS2、CDKN2B-AS1等）。随后，体内外实验明确了SNHG12的表达受HPV16 E6/E7的调控，机制上E6/E7可能部分通过下调c-Myc进而抑制SNHG12的表达，沉默SNHG12可能通过ERK/Slug/E-cadherin信号通路抑制宫颈癌细胞的侵袭和迁移。进一步，我们探讨了CDKN2B-AS1、HIF1A-AS2、TINCR与HPV16 E6/E7的关系，发现CDKN2B-AS1并不受HPV16 E6/E7调控，但其在宫颈癌中发挥促癌效应，并可能通过激活MEK/ERK信号通路促进EMT，增强细胞侵袭和转移能力。HIF1A-AS2、TINCR的表达受HPV16 E6/E7调控，过表达和敲减实验初步明确了它们在宫颈癌细胞中的促癌效应。通过RNA pull-down、质谱、RIP等实验初步鉴定了lncRNA的互作蛋白。此外，还发现了肌球蛋白超家族成员Myo1b的表达随宫颈癌进展而逐步增加，并具有促癌效应，体内外实验证明沉默Myo1b可以抑制肿瘤细胞的增殖，机制上可能通过下调MMP-1/-9的表达而抑制细胞的侵袭迁移，在此基础上申请并获得了国家发明专利1项。项目实施过程中，负责人以通讯作者发表标注项目资助的研究论文7篇（SCI 6篇，lncRNA中文综述1篇），培养硕士研究生5人，指导八年制学生7人。本项目的开展，有助于完善HPV致癌的分子机制，为从lncRNA和HPV感染层面揭示宫颈癌的发病机理，探寻宫颈癌防治新策略提供了科学依据和实验基础。