Ficolin-2下调的LncRNA介导HCV免疫逃逸的分子机制研究

Currently, the mechanisms underlying the expression and regulation of LncRNAs related to human important viruses remain elusive. We firstly found that infection of hepatitis C virus (HCV) caused liver cells to secrete high levels of Ficolin-2. In previous work we further detected that Ficolin-2 negatively regulated critical LncRNA-D expression; While LnRNA-D bound to PKM2 in liver cells, inhibited the expression of type I interferon and promoted HCV replication. We propose that Ficolin-2, which negatively regulates LncRNA-D, may promote HCV replication by interacting with its downstream PKM2 protein and complementary miR-124-3p, thereby mediating viral chronic infection, and immune escape. This project aims to: 1. Through CHIP-Seq, determine the molecular mechanism underlying how Ficolin-2 regulated critical LncRNA-D expression at the transcriptional level during HCV infection; 2. Reveal the inter molecular regulatory network and molecular mechanism underlying HCV-Ficolin-2-(miR124-3p)LncRNA-D-PKM2-IFN-I interaction; 3. Verify the effects of HCV-associated LnRNA-D on promoting HCV replication and its interaction with other molecules using animal models and clinical samples, thus exploring the the function of LncRNA-D in negative regulation of viral innate immunity, and laying the foundation to provide novel targets for HCV diagnosis and treatment.

目前与人类重要病毒相关的LncRNA报道甚少。我们首次发现丙型肝炎病毒-HCV感染引发肝细胞分泌高水平的Ficolin-2；前期工作表明Ficolin-2负调控关键LncRNA-D的表达；LnRNA-D结合肝细胞中PKM2，并抑制IFN-I表达，显著促进HCV复制。因而我们提出假设：Ficolin-2负调控的LncRNA-D可能通过与下游PKM2蛋白以及其互补的miR-124-3p互作而调控HCV复制，从而介导病毒免疫逃逸。本项目拟：1. 通过CHIP-Seq等确定Ficolin-2在转录水平调控HCV感染中LncRNA-D表达的分子机制；2.在细胞水平深入揭示HCV-Ficolin-2-(miR124-3p)LncRNA-D-PKM2-IFN-I互调机制；3.在动物模型和临床样本中验证LnRNA-D及其互作分子的功能，发掘LncRNA-D的负调控机制，为HCV提供新型诊疗靶标。

长链非编码RNA（long non-coding RNAs，LncRNA）是抗病毒天然免疫的关键调节分子，丙肝病毒（HCV）感染肝细胞中LncRNA的表达和调控机制还不很清楚，且至今仍无针对抗HCV感染的有效疫苗。因此很有必要深入开展LncRNA参与HCV免疫逃逸和致病机制的研究，以发掘新的防治靶点。我们前期工作中首次发现HCV感染引发肝细胞分泌高水平的Ficolin-2 （纤维胶凝蛋白-2）；发现Ficolin-2负调控关键LncRNA--Lnc-Far的表达；Lnc-Far结合肝细胞中PKM2，并抑制IFN-I表达，显著促进HCV复制。因而本研究着重阐明Ficolin-2下调LncRNA--Lnc-Far的分子机制，并阐释Lnc-Far调控抗病毒天然免疫等介导HCV病毒免疫逃逸的机制。.项目实施过程中，发现Ficolin-2下调Lnc-Far的分子机制，并发现Lnc-Far通过糖代谢途径和调控抗病毒天然免疫，介导HCV病毒免疫逃逸：即Ficolin-2/A通过增强H3K4me3修饰促进miR-124表达，miR-124能够结合Lnc-Far导致其发生的降解，进而负调控Lnc-Far表达；发现Lnc-Far可调控I型干扰素信号通路重要接头蛋白VISA（MAVS/IPS-1/Cardif）与E3泛素连接酶MARCH5结合，促进VISA发生K48多聚泛素化，促进VISA的蛋白酶体降解，从而抑制I型干扰素表达，进而显著促进RNA病毒的复制；Lnc-Far还可促进糖代谢中糖酵解关键酶PKM2的O-GlcNAc修饰和四体聚形成，促进乳酸生成，使糖酵解增加，抑制IFN-I，从而促进HCV复制；相关成果从另一个角度观察到LncRNA通过调控糖代谢途径促进HCV病毒免疫逃逸的机制。发现HCV的RNA表观遗传调控新机制--G四链体核酸，并发现宿主细胞蛋白-核仁素 (nucleolin, NCL) 参与的靶向HCV G4结构的抗病毒天然免疫机制，相关成果以通讯作者发表在核酸研究领域权威期刊Nucleic Acids Research (2019, IF: 11.561，通讯作者) 上。还提出了一种以苯并噻唑为基础的G4靶向荧光探针ThT-NE，丰富了病毒RNA基因组的活细胞成像化学工具箱，相关成果发表在J Am Chem Soc (2019)。