剪接因子USP39通过调控HMGA2促进卵巢癌侵袭转移的机制

选择性剪接是产生蛋白质多样性和非编码RNA的重要机制。剪接因子的异常表达是导致肿瘤细胞发生异常剪接并参与肿瘤恶性行为的关键因素。课题组前期发现剪接因子USP39在卵巢癌组织中高表达并与患者预后相关，但其差异表达及调控选择性剪接的机制不明。预实验提示USP39可能调控HMGA2的选择性剪接，MYC高表达可上调USP39。我们提出假说：MYC过度活化转录激活USP39的表达，导致卵巢癌细胞发生剪接重编程，促进HMGA2短的剪接异构体产生，逃避Let-7的降解，HMGA2蛋白累积并促进卵巢癌的侵袭转移。根据该假说，课题组将运用CLIP-seq、RNA-seq等方法阐明USP39的RNA识别基序和调控的剪接事件；RIP、mini-gene等手段明确USP39调控HMGA2选择性剪接的机制；ChIP等方法探究MYC对USP39的转录调控机制。课题的实施有助于揭示剪接因子调控卵巢癌恶性行为的机制。

Alternative splicing (AS) is a regulated process during gene expression that results in a single gene coding for multiple proteins or noncoding RNAs. Dysregulated expression of splicing factors could cause abnormal splicing events which may promote tumor malignant behavior. Our previous study showed that USP39, a component of the U4/U6-U5 tri-snRNP, was overexpressed in high grade serous ovariancarcinoma(HGSOC) and was associated with poor prognosis. However, the roles and mechanisms of its dysregulation in HGSOC remains unclear. Pilot studies of this project showed that USP39 may regulate HMGA2 alternative splicing based on RIP-seq and RNA-seq data analysis. Preliminary findings of this project prompt us to propose the hypothesis that MYC amplification causes its aberrant expression and hence transcription activates expression of USP39. High level of USP39 will then cause mis-splicing of HMGA2 which play an important role in ovarian cancer invasion and metastasis.. To test our hypothesis, we will perform CLIP-seq and RNA-seq to elucidate USP39 affected AS events and binding motif. Then, RIP and mini-gene assays will be conducted to clarify the regulatory mechanisms connecting USP39 and HMGA2 by AS. To determine the molecular mechanisms that cause USP39 overexpression in HGSOC. ChIP-seq and luciferase assays will be used to identify the upstream regulator of USP39, MYC is a strong canditate.. The ultimate goal of this study is to clarify and characterize the functional role of USP39 in tumorigenesis and metastasis of high-grade serous ovarian carcinomas.