lincRNA-IFNL3P1在丙肝病毒感染中对III型干扰素基因表达调控的机制研究

LincRNAs play important roles in gene expression regulation. Genetic polymorphism near the IFNL3 gene is associated with spontaneous clearance of hepaptitis C virus and response to interferon-based therapy in hepatitis C patients. However, the mechanism is still of controversy. We found that one of the IFNL3 SNP was located in the promoter region of lincRNA IFNL3P1. Highly homologous to IFNL3, IFNL3P1 was upregulated upon virus infection as IFNL3. Moreover, the expression level of IFNL3P1 varied significantly among hepatits C patients carrying different IFNL3 SNP. Therefore, we hypothesize that IFNL3P1 can regulate the expression of IFNL3. The above-mentioned SNP changes the expression level of IFNL3P1 directly while IFNL3 level indirectly. In order to understand the role of IFNL3P1 in innate immunity against HCV, we will study the regulation of IFNL3P1 itself, its effect on IFNL3 gene expression and the relevance of IFNL3P1 level to the response to IFN-based therapy in hepatitis C patients. The study will provide us new insights into the genetic regulation of HCV clearance and its clinical management.

长链非编码RNA在调控基因转录表达方面发挥重要作用。III型干扰素基因IFNL3附近的SNP与丙肝患者自愈及对干扰素应答密切相关，但机制不明。我们发现其中一个SNP位于lincRNA-IFNL3P1的启动子区。IFNL3P1是与IFNL3高度同源的lincRNA，在病毒感染时，二者mRNA水平同步上升，且IFNL3P1的表达量在携带不同IFNL3-SNP基因型的丙肝患者中有差异。因此我们提出假说：IFNL3P1是调控IFNL3表达的lincRNA，前述SNP通过改变IFNL3P1的表达量，间接影响IFNL3的转录水平及宿主对丙肝病毒的免疫清除。本项目将研究IFNL3P1自身的转录调控和它对IFNL3基因表达的控制，以及IFNL3P1表达水平与丙肝患者对干扰素应答的相关性，深入探索IFNL3P1在抗病毒免疫中的作用机制，为理解丙肝病人对干扰素应答的个体差异和为临床上准确预测疗效提供实验依据。

长链非编码RNA在调控基因转录表达方面发挥重要作用。III型干扰素基因（IFNL1-L4）与丙肝患者自愈及对干扰素应答密切相关。通过生物信息学分析我们发现IFNL3P1是一个与三型干扰素基因IFNL3高度同源的长链非编码RNA。在病毒感染时，二者mRNA水平同步上升。我们提出假说：IFNL3P1是调控IFNL3表达的lincRNA，可影响IFNL3的转录水平及宿主对丙肝病毒的免疫清除。我们首先摸索了IFNL3P1的诱导条件，发现在A549细胞中，外源转染polyI:C或者感染仙台病毒均可大幅诱导IFNL3P1。然后，我们结合RT-PCR、Northern及RNAseq技术完成了IFNL3P1各种剪切变体的扩增、序列测定及丰度最高的转录本的鉴定。基于以上信息，我们应用CRISPR技术构建了IFNL3P1过表达细胞系和敲低细胞系。相比于sgRNA载体转染的对照细胞系，在sgRNA靶向过表达IFNL3P1的细胞系里，很多和IFN 信号相关的基因表达水平降低，即过表达IFNL3P1，会导致ISG降低，能促进病毒复制。这一结果已通过VSV感染实验验证。在过表达IFNL3P1的细胞系中，在感染VSV后的不同时间点，相比于对照细胞系，胞内VSV的复制水平及胞外VSV的滴度均有所提升。这一结果正在敲低IFNL3P1的细胞系中验证并进行机制研究。