DNA/RNA结合蛋白PURα与食管癌变机理研究

Purine-rich element binding protein alpha (PURα) is a multifunctional single-stranded DNA- and RNA-binding protein which is highly conserved in eukaryotic cell evolutionarily. As a kind of transcription factor and RNA binding protein (RBP), PURα has been implicated in diverse cellular functions, including transcriptional activation and repression, translation, cell growth and proliferation, cell cycle and oncogenic transformation. Our previous work found that PURα was overexpressed in tissues of esophageal squamous cell carcinoma (ESCC), and ESCC cells were found to be led to the epithelial mesenchymal transition (EMT) after PURα over-expressed. It was known that transcription, post-transcriptional regulation, and LncRNA-RBP interaction play important roles during the development and progression of tumor and hereditary diseases. This study will identify PURα-targeted genes and network at whole genome level using chromatin immunoprecipitation (ChIP)-sequencing, and investigate PURα interaction mRNA, lncRNA and their binding sites using UV-crosslinking and immunoprecipitation (CLIP)-sequencing via the comparison of wild-type with PURα overexpressed ESCC cells. Subsequently, the expression of mRNA and lncRNA was compared in PURα knockdown and wildtype cells using RNA-sequencing. The integrative analysis of CLIP-seq and previous GWAS data will also facilitate the discovery of lncRNAs-RBP binding sites affected by ESCC-related single nucleotide polymorphism (SNP). The overall results will reveal the roles of PURα on pre-mRNA splicing, capping, polyadenylation, nuclear exportion, localization and translation of EMT related genes, and thus deepen our understanding on the molecular mechanisms of PURα underlying EMT, invasion and metastasis processes in ESCC.

富含嘌呤单链DNA结合蛋白α (PURα) 是能与单链DNA/RNA结合的多效转录因子和RNA结合蛋白 (RBP)，在进化中高度保守并与细胞周期调控、DNA复制和RNA转录与翻译等生物学进程，以及肿瘤发生发展密切相关。曾发现食管鳞状细胞癌 (ESCC) 组织PURα蛋白表达增高，过表达PURα导致ESCC细胞上皮间叶转化 (EMT)。本研究拟对高表达PURα的食管癌细胞进行ChiP-seq和CLIP-seq分析，对比PURα敲除和野生型细胞的RNA-seq水平，在全基因组范围鉴定PURα调控的靶基因及其网络；在全转录组范围发现PURα结合的mRNA和lncRNA及其结合位点，整合CLIP-Seq与GWAS分析数据发现食管癌相关单核苷酸多态性 (SNP) 的lncRNAs-RBP结合位点，揭示PURα转录和转录后调控食管癌细胞EMT改变，促进肿瘤侵袭转移的分子机理。

单链核苷酸结合蛋白PURα，作为多效转录因子，通过与特定基因启动子区域DNA结合或与不同转录因子结合而调控基因转录；作为RNA结合蛋白（RBP）与单链RNA结合参与特定基因mRNA的出核转运与翻译。PURα参与DNA复制、RNA转录与翻译和细胞周期调控等多种生物学进程，在神经系统发育中具有重要作用。近年研究发现PURα功能异常与肿瘤发生发展密切相关，本研究系统探讨了食管鳞状细胞癌中PURα功能异常与细胞癌变的关系，发现PURα通过改变与RNA和DNA的结合模式，诱导食管上皮细胞的上皮间质转化（epithelial–mesenchymal transition, EMT），促进肿瘤侵袭转移的分子机理。课题完成了预期考核指标，发表SCI论文13篇，核心期刊论文5篇，综述4篇。投稿国际会议摘要23篇，国内会议摘要17篇，分会场报告9次。参编/译专著2部。授权国家发明专利4项。培养博士和硕士研究生10名。