环状RNA调控肝癌转移的分子机理研究

Circular RNAs (circRNAs) are a novel type of RNA that, unlike linear RNAs, form a covalently closed continuous loop and are highly represented in the eukaryotic transcriptome. Although circRNAs are generally expressed at low levels, recent findings have shed new light on their cell type-specific and tissue-specific expression and on the regulation of their biogenesis..Furthermore, recent researches also have revealed that circRNAs can function as microRNA (miRNA) sponges, regulators of splicing and transcription, and modifiers of parental gene expression, especially in neurological disorders and cancer development. For cancer, aberrant expression of circRNAs have been reported in colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) and hepatocellular carcinoma (HCC), which may serve as diagnostic or predictive biomarkers. Whereas, the potential role of circRNAs during the development and metastasis of HCC are still unreported. .Previously, we have applied next generation sequencing methods detecting the whole expression profile of 21 paired-match HCC samples. Hence, we firstly plan to use real-time PCR and siRNA specifically targeting circRNAs to filter the metastatic-related circRNAs. Subsequently, in vitro and in vivo experiments will be performed to further verify the effect of circRNAs on cell metastasis. Thirdly, signal pathway screening, RNA pull-down and RIP/PAR-CLIP methods will be applied to explore the potential interaction between cicrRNAs and miRNAs or RNA binding proteins, which will help us calrify the molecular mechanism of circRNA-mediated modulation of cell metastasis. Finally, mono- and multi-factors analysis will be used to further evaluate the potential role of circRNAs in HCC prognosis and prevention.

环状RNA（circRNAs）是一类不同于线性RNA分子的内源性环状非编码RNA。近年来已成为继miRNA和LncRNA之后，RNA研究领域的又一全新热点。已经发现某些环状RNA可能参与肿瘤进展，然而其在肝癌中的生物学功能尚未见报道。前期我们已经完成了21例肝癌配对样本mRNA和环状RNA的二代测序分析。本课题拟在此基础上首先通过大样本定量检测及针对环状接头序列的干扰实验从高低转移潜能肝癌样本中筛选具有调控转移能力的环状RNA；之后在细胞和动物水平分析其调控肝癌转移侵袭的主要作用方式；进一步通过信号通路干预、RNA捕获、RIP/PAR-CLIP技术，以潜在互作RNA结合蛋白及miRNA分子为切入点开展环状RNA作用靶点及分子机理的研究。从而阐明环状RNA-靶基因-信号通路-细胞表型的分子调控机制。最后结合临床随访样本组化及原位杂交分析探讨其作为肝癌复发转移独立预警分子和治疗靶点的应用价值。

环状RNA（circRNA）是多种癌症中临床前诊断的潜在标志物和治疗的潜在靶点。然而circRNA与癌症干细胞（CSC）之间的潜在相关性尚未有报道。 体内、体外的实验结果表明circZKSCAN1敲低显著增强了肝癌细胞的恶性表型，尤其是细胞干性，并且与肝癌患者的总体生存率和无复发生存率紧密相关。生物信息学分析和RNA免疫共沉淀测序（RIP-seq）结果表明，circZKSCAN1通过竞争性结合FMRP发挥其抑制作用。circZKSCAN1能够阻断FMRP与β-catenin结合蛋白（CCAR1）mRNA的结合，并随后抑制Wnt信号传导的转录活性。此外在肝癌组织中，RNA剪接蛋白Quaking 5（Qki5）被下调，这一变化导致了circZKSCAN1的减少。本项研究揭示了circZKSCAN1在肝癌干细胞中发挥调节作用的潜在机制，并确定了Qki5–circZKSCAN1–FMRP–CCAR1–Wnt信号轴是HCC治疗的潜在重要治疗靶点。