口腔鳞癌恶性进程中circRFWD3/miR27/PPARγ正反馈循环形成的机制及意义

The circRNA-miRNA-mRNA signaling axis plays a regulatory role in disease progression (Science, 2017), but there is insufficient research on the positive feedback regulation of target mRNA and the upstream cirRNA. The applicant found earlier that circRFD3 can sponge miR-27 in the malignant process of oral squamous cell carcinoma, miR-27 can target PPAR, which in turn regulates circRFWD3 transcription. Therefore, we hypothesized that there is a circRFWD3/miR-27/PPARγ positive feedback loop in the malignant progress of oral squamous cell carcinoma promotes. For this purpose, firstly verify the expression and significance of the positive feedback loop-related molecules on the clinical cohort; then utilize genetic modification techniques to detect the circularization mechanism of circRFWD, and use RNA interaction techniques to explore regulation mechanism of the positive feedback loop; then adopt genetically modified cell lines to detect the biological function of non-coding RNA in the positive feedback loop; finally employ animal models to evaluate the intervention effects by target circRFWD3 and miR-27. The study not only elucidates the role and mechanism of this positive feedback loop in the malignant process of OSCC, but also provides a basis for the feedback regulation of circRNA and its downstream target molecules.

circRNA-miRNA-mRNA信号轴在疾病进程中发挥调控作用（Science,2017），但对靶mRNA正反馈调控上游circRNA研究不足。申请人发现口腔鳞癌（OSCC）恶性进程中circRFWD3吸附miR-27上调其靶基因PPARγ，后者又调控circRFWD3转录。因此，假定：circRFWD3/miR27/PPARγ形成正反馈循环参与OSCC恶性进程。为验证此假说，拟首先在临床队列上验证该正反馈循环相关分子的表达规律及意义；然后采用基因修饰技术检测circRFWD成环机制，并采用RNA互作技术探究该正反馈循环调控机制；继之采用基因改造细胞系检测正反馈循环中非编码RNA的生物学功能；最后采用动物模型评估circRFWD3和miR-27为靶标的干预效果。研究不仅阐明此正反馈循环在OSCC恶变进程中的作用机制及意义，也为circRNA与下游靶分子形成反馈调控的理论提供依据。

环状RNA（circRNA）通过复杂的信号通路影响肿瘤的发生、转移以及耐药等各个方面，但circRNA在口腔鳞状细胞癌（OSCC）中的表达规律及核心功能尚不明确。本项目首先对OSCC组织和癌旁组织样本进行测序，分析二者中差异表达circRNAs表达规律及类别，筛选并验证circRFWD3在OSCC组织和细胞中表达均升高，并发现其高表达的OSCC患者转移率较高；其次，采用 circBASE数据库、sanger测序、RNase R耐受实验、放线菌素D干扰实验和荧光原位杂交实验分析发现circRFWD3是由RFWD3的第7和第8个外显子反向剪切而成的典型的环状RNA。继之，采用Transwell实验检测发现circRFWD3可促进OSCC细胞的侵袭迁移能力；同时，体内研究发现敲低circRFWD可以抑制OSCC细胞远处转移比率。再次，采用荧光素酶报告实验、RNA免疫共沉淀、RNA pull down、荧光原位杂交实验和RNAseq的机制分析发现circRFWD3 是通过 miR27a/b/PPAR-γ/NF-κb/MMP13 信号轴促进OSCC转移。最后，采用OSCC临床队列样本及TCGA数据库中头颈部鳞癌（HNSCC）数据分析发现该信号通路中 circRFWD3、miR-27b和PPAR-γ均可作为预测OSCC患者转移或预后的分子标志物。除此以外，还发现circTPST2通过miR-770-5p和Nucleolin双通路调控HNSCC细胞化疗敏感性，具有成为经HNSCC患者化疗预后标志物和治疗靶点的潜能；还发现circFANCA 可通过miR-34a/PA28γ信号通路调OSCC的侵袭迁移能力。该项目的执行及相关数据可加深我们对OSCC中circRNA表达规律的认识，以及对OSCC中核心circRNA分子作用机制的了解，更可为寻找以circRNA为靶向的肿瘤治疗提供新靶点，为发展新的OSCC治疗策略提供新的思路。