剪接因子USP39促进卵巢癌恶性行为的作用及机制

Alternative splicing (AS) is an important process during gene expression regulation. To date, more than 90% of human precursor messenger RNAs (pre-mRNAs) are reported to undergo AS based on RNA-seq and EST analysis. The immense majority of genes are alternatively spliced and there are many isoforms specifically associated with cancer metastasis, angiogenesis and immune escape, etc. Several studies have demonstrated specific alterations in the expression of splicing factors in cancer. This may cause alternative splicing patterns in cancer. . Our previous study showed that USP39, a component of the U4/U6-U5 tri-snRNP, was overexpressed in high grade serous ovarian carcinoma (HGSOC) and was associated with poor prognosis. Pilot studies showed that USP39 overexpression promotes colony formation and cell invasion of ovarian cancer cells. Global profiling of alternative splicing in USP39 knockdown ovarian cancer cells showed a lot of changes of alternative spliced events. We also validated some candidate downstream target genes for USP39 by QPCR and western blot. All these preliminary findings prompt us to propose that USP39 may play an important role in the tumorigenesis and metastasis of HGSOC.. To test our hypothesis, we will first determine the effect of USP39 on tumorigenesis and metastasis of ovarian cancer in vitro and in vivo. Then, mini gene, northern blot, CLIP-seq and in vitro splicing assays will be used to identify downstream targets of USP39. Finally, we will examine the expressions of USP39 and its targets in HGSOC patient samples to determine whether their expressions are correlated with tumor progression and overall survival. The ultimate goal of this study is to clarify and characterize the functional role of USP39 in tumorigenesis and metastasis of HGSOC.

选择性剪接是基因表达的重要调控机制，RNA-seq等分析发现95%以上的人类基因发生选择性剪接。肿瘤相关基因的异常剪接与肿瘤的发生和转移密切相关，而剪接因子的异常表达是导致肿瘤相关基因发生异常剪接的主要原因。我们前期研究发现剪接因子USP39在高级别浆液性卵巢癌组织中高表达并与预后相关，初步功能实验显示USP39促进卵巢癌细胞的克隆形成和侵袭力，选择性剪接表达谱分析发现了USP39的数个候选靶基因。本课题拟通过体内外实验系统研究USP39对卵巢癌细胞的增殖、侵袭及转移等恶性生物学行为的影响；利用小基因模型（mini-gene）、Northern blot、CLIP-Seq及体外剪接实验等方法筛选和鉴定USP39下游关键分子；利用临床组织标本，通过免疫组化方法分析USP39及下游分子的表达情况，以明确其与肿瘤发生发展及临床预后的相关性，为该病的诊断和治疗提供新的分子靶标。

选择性剪接是基因表达中的重要调控机制。肿瘤相关基因的异常剪接与肿瘤的转移、血管新生及免疫逃逸等密切相关，而剪接因子的异常表达是导致肿瘤相关基因发生异常剪接的主要原因。本课题发现剪接因子USP39在高级别浆液性卵巢癌中高表达并与患者预后差密切相关；进一步研究发现USP39促进卵巢癌细胞的增殖、侵袭等恶性生物学行为；利用Rip- Seq结合RNA-seq方法分析发现USP39敲低细胞的剪接效率降低，并发现了包括HMGA2在内的数个靶基因。本课题圆满完成任务书规定研究目标。在该课题的资助下发表SCI论文5篇，其中4篇是第一标注，1篇是第二标注，另有1篇文章发表在Cell death &differ, 中科院1区，影响因子10.5。另外，授权1项国家发明专利，公开1项发明专利。