环状RNA作为阴道自取样HPV阳性者二次筛查标记物在宫颈癌发病中的分子机制研究

Human papillomavirus（HPV）testing with vaginal self-collection is the latest evolution trend of algorithm for cervical cancer screening.However, on account of lacking detectable hierarchical indicator in secondary screening, the HPV positives with no lesion actually are faced with overdiagnosis and overtreatment.This subject attempted to make a breakthrough on circRNAs. Through correlating and matching the ncRNA database and referring to the circRNA microarray results, we selected and identified partial candidate circRNAs. In our preceding work， we found the target circRNAs significantly changed in tissues with cervical intraepithelial neoplasia（CIN）,compared with normal control, and the expression level of target circRNAs in self-collection were connected with the CIN grades.The target circRNA interference would promote or hinder the cell cycle progress. In the current project, we plan to further observe the affection of target circRNA on the biological function of cervical carcinoma cells. Meanwhile，we will deeply research the possible molecular mechanism of target circRNAs may participate in the pathogenesis process of cervical cancer by directly or indirectly regulating the cell cycle pathway-associated proteins，aiming to providing theoretical basis for the application of target circRNAs in risk stratification of HPV positives and providing a novel idea to study the clinical intervention as well as diagnosis and treatment.

阴道自取样HPV检测是目前宫颈癌筛查模式的最新发展方向，但自取样本尚无可检测的二次筛查指标，HPV阳性实际无宫颈病变者仍面临过度诊疗问题。本项目拟从环状RNA（circRNA）方面寻求突破。前期我们利用非编码RNA数据库进行关联比对，结合高级别宫颈病变组织circRNA芯片结果，选取了部分circRNA进行PCR验证。发现与正常宫颈组织比较，宫颈上皮内瘤变（CIN）组织中目标circRNA表达显著改变；自取样本目标circRNA水平与CIN级别相关；干扰目标circRNA表达可促进或阻滞宫颈癌细胞的细胞周期转化。本课题拟：进一步观察目标circRNA对宫颈癌细胞生物功能的影响；阐明目标circRNA通过直接或间接调控细胞周期通路相关蛋白参与宫颈癌发病的过程。从而为目标circRNA可用于自取样HPV阳性分流提供理论依据，同时为宫颈癌的临床干预及诊治提供新思路。

项目背景：宫颈癌是由人乳头瘤病毒（HPV）持续感染所致，早期筛查可降低其发病率和死亡率。但针对HPV检测的低特异性常导致过度诊疗。目前无有效、稳定检测的二次筛查指标。因此，深入研究宫颈癌发病中的细胞和分子调控机制，对HPV阳性者进行危险分层和合理干预，有助减少医疗支出和民众负担。环状RNA（circRNA）是一种非编码RNA，因共价环状结构而具有稳定性高的特点。circRNA在不同细胞、组织中表达各异，并随着机体发育及各项病理生理过程发生显著变化。根据竞争性内源性RNA(ceRNA)调控假说，可预测环状RNA-miRNA—mRNA轴调控癌症信号通路，circRNA理论上具备作为疾病早期诊断指标的可能性，有应用于二次筛查的潜力。主要内容：（1）多路径明确所要研究的目标circRNA：联合测序及数据库筛选宫颈癌差异性表达的circRNA，并根据MREs原理及联合生物信息学分析手段，筛选出候选的目标circRNA,同时验证其在宫颈病变组织及细胞样本中的表达趋势，最终确定目标circRNA。（2）目标circRNA参与宫颈癌前病变进展或转归的分子机制研究。通过生物信息学分析预测目标circRNA可能结合的miRNAs靶点并构建相互作用的网络，分析其功能及可能参与的致病机制：1）从细胞增殖、周期等多个方面验证目标circRNA对宫颈癌细胞的影响；2） 建立并验证宫颈癌细胞基于circRNA-miRNA-mRNA网络的调控通路。重要结果：（1）目标circRNA的多维筛选结果及生物信息学分析及验证；（2）进行了目标circRNA的表达验证及细胞功能探索；（3）预测构建了目标circRNA参与细胞周期调控的ceRNA网络并深入研究了的分子作用机制。关键数据：（1）hsa_circ_0076194、hsa_circ_0023305、hsa_circ_0081030、hsa_circ_0005325均与宫颈癌发病相关，前者负相关，后三者正相关；（2）hsa_circ_0076194通过关联miR-93和miR-16分子的表达来双向调控细胞周期通路相关蛋白（CDKN1A&CDK6），来完成对宫颈癌细胞周期的调控。（3）hsa_circ_0005325通过关联miR-1206 和 miR-22的表达来双向调控泛素化通路E3连接酶（UBE3A）和CDK6参与宫颈癌发病过程。科学意义：本项目