microRNAs 协同抑制生物钟基因RORɑ在口腔鳞癌发生中的作用

Recent studies showed that the tumorigenesis of oral squamous cell carcinoma (OSCC) is closely related to the lost of circadian gene. Based on our pre-experiment data of microRNA and mRNA deep sequencing on five pairs of OSCC and adjacent normal tissues, it was found that the circadian gene RORα was inactivation in oral cancer, and was a highly potential target of a group of co-overexpressed oncomiRs (mirRNAs act as oncogene). The latest science view showed that the microRNAs cooperativity is an efficient mechanism for an enhanced target repression. As a result, we speculated that multiple of microRNAs increased in OSCC suppressing RORα cooperatively might be an important mechanism of RORα downregulation. So what is the potential mechanism to explicate the role of repressing RORα in OSCC tumorigenesis? By comprehensively analysis of high-throughput sequencing result, KEGG database and the current research progress, we Hypothesizes that the RORα may activate the P53 signaling pathway through RORα/HIPK2/P53 "positive feedback triangle". On the contrary, multiple microRNAs cooperative inactivation of RORα will breakdown this "positive feedback triangle" chain, leading to apoptosis signal inhibition, thus promotes OSCC. This project will try to discover RORα regulatory mechanism through exploring its upstream or downstream signaling pathway. The technology of anti-miRNA sponges, gene expression and knockdown, animal tumor-burdened model， etc. will be applied to further study of cooperative effects of microRNAs on suppressing circadian genes RORα and its potential signaling downstream pathways, thus further reveal " cooperative microRNA-mRNA network" and provide new targets for OSCC treatment.

口腔鳞癌的发生与生物钟基因紊乱密切相关。本课题组前期实验通过miRNA和mRNA高通量测序，发现生物钟基因RORα在口腔癌中失活，且是多个上调的癌基因型miRNA的潜在靶点。最新观点认为多个mRNAs叠加协同性的调节模式可极大强化对靶基因的抑制。因此我们推测：口腔癌中多个上调的癌基因型miRNAs协同抑制RORα的表达，是RORα下调的一个重要机制。那么RORα下调与口腔鳞癌发生的潜在机制是什么呢？综合测序结果、数据库分析和新的研究进展，我们推测RORα可能通过RORα/HIPK2/P53“正反馈三角”活化P53信号通路。而多个miRNAs协同性失活RORα将导致 RORα/HIPK2/P53三角链断裂、凋亡信号受抑制，从而促进肿瘤的发生。本项目将从探讨RORα上、下游调控机制入手，深入研究miRNAs对生物钟基因RORα及其下游潜在信号通路的协同抑制作用，为口腔癌的治疗提供新的靶点。

口腔鳞状细胞癌（口腔鳞癌）的发生是一个从“正常上皮→异常增生→鳞癌”的演变过程。在这个演变过程中，正常口腔黏膜上皮的节律性自我更新的平衡被打破，细胞生长脱离了正常上皮细胞的生物钟控制、进入快速增殖状态。有研究显示，这与生物钟基因调控紊乱密切相关。本课题组前期实验通过miRNA和mRNA高通量测序，发现生物钟基因RORα在口腔癌中失活，且是多个上调的癌基因型miRNA的潜在靶点。最新观点认为多个mRNAs叠加协同性的调节模式可极大强化对靶基因的抑制。因此我们推测：口腔癌中多个上调的癌基因型miRNAs协同抑制RORα的表达，是RORα下调的一个重要机制。并可能通过RORα/HIPK2/P53“正反馈三角”活化P53信号通路。本研究发现OSCC中异常上调的213个miRNA主要通过靶向下调2172个转录因子mRNA构成网状调控关系，从而影响肿瘤的发生发展。并对该网络中生物钟基因RORA及相关25个miRNA组成的子网进行了进一步的研究：验证了其中5个肿瘤高度相关miRNA（miR-503-5p、miR-450b-5p、miR-27a-3p、miR-181a-5p和miR-183-5p）对RORA的靶向作用和协同抑制作用；利用CCK-8实验和裸鼠植瘤实验检测了RORA对口腔鳞癌增殖的抑制作用；并进一步通过体内、体外和分子生物学实验探索了RORA影响增殖的分子机制。研究证实，下调的RORA可能是促进OSCC增殖的重要因素之一。RORα可通过影响p53和HIPK2来发挥抑癌作用。多个miRNAs协同性失活RORα将导致 RORα/HIPK2/P53三角链断裂、凋亡信号受抑制，从而促进肿瘤的发生。本项目从探讨RORα上、下游调控机制入手，深入研究miRNAs对生物钟基因RORα及其下游潜在信号通路的协同抑制作用，将可能为揭示生物钟节律与癌症治疗之间的临床联系提供进一步的线索。