miR-214在乌鳢水泡病毒感染SSN-1细胞中的调控机制研究

Snakehead vesiculovirus (SHVV) infection has caused great economic loss to snakehead fish culture in China, and there are not any efficient preventive and therapeutic ways. MicroRNA is a class of small non-coding RNA that plays an important role in the regulation of virus infection. Snakehead vesiculovirus (SHVV) infection has caused great economic loss to snakehead fish culture in China, and there are not any efficient preventive and therapeutic ways. MicroRNA is a class of small non-coding RNA that plays an important role in the regulation of virus infection. Our previous research indicates that cellular microRNA-214 (miR-214) was down regulated in striped snakehead fish (SSN-1) cells upon SHVV infection, and overexpression of miR-214 mimic inhibited the replication of SHVV. Further target-gene prediction of miR-214 in SHVV genome revealed that N and P genes were the potential target genes of miR-214. Therefore, this project aims at illuminating the regulatory role of miR-214 in SSN-1 cells upon SHVV infection. The main studies included: (1) Identify viral components that are involved in the down regulation of miR-214 in SSN-1 cells during the infection of SHVV; (2) Investigate whether miR-214 decreases the concentration of mRNAs of N and P genes via binding to these mRNAs, thus resulting in the inhibition of SHVV replication. The obtained results are highly important to reveal how miR-214 regulates SHVV infection and develop novel drugs against SHVV infection.

乌鳢水泡病毒(SHVV)感染导致我国乌鳢养殖业重大经济损失，目前还没有有效的防治措施。MicroRNA是一类非编码的小分子RNA，在病毒感染过程中发挥重要的调控作用。我们前期研究发现SHVV感染乌鳢细胞(SSN-1)导致细胞的microRNA-214 (miR-214)下调表达，而过表达miR-214类似物抑制SHVV的增殖。进一步从SHVV基因组搜索miR-214的靶基因发现N和P基因是miR-214的潜在靶基因。因此，本项目拟对miR-214在SHVV感染SSN-1细胞中的调控机制进行研究。主要内容包括：（1）在SHVV感染SSN-1细胞过程中，哪些病毒成分导致miR-214下调表达；（2）miR-214是否与潜在靶基因N和P的mRNA结合，降低N、P基因mRNA的含量，从而抑制SHVV的增殖。研究结果对认识miR-214如何调控SHVV的感染和研发新型抗SHVV的药物具有重要的意义。

乌鳢水泡病毒（SHVV）是从患病杂交鳢分离的一株弹状病毒，其感染对我国乌鳢养殖业造成了巨大经济损失。但是，我们对SHVV的致病机理知之甚少。.本项目围绕microRNA miR-214在SHVV感染条纹月鳢（SSN-1）细胞中的调控机制展开研究，取得了以下研究成果：1）SHVV感染SSN-1细胞导致细胞内miR-214下调表达，且呈现时间、剂量的依赖性；2）转染miR-214类似物可抑制SHVV增殖，而转染miR-214抑制剂可促进SHVV增殖，说明miR-214是SHVV增殖的负调控因子；3）靶基因预测及双荧光报告系统验证表明SHVV的N和P基因是miR-214的靶基因，过表达miR-214能抑制N和P基因的表达；4）通过转录组测序及双荧光报告系统验证发现AMP依赖的蛋白激酶（AMPK）是miR-214的靶基因，siRNA敲降AMPK可抑制SHVV的增殖，同时上调表达IFN-α。而siAMPK对SHVV增殖的抑制可以通过添加miR-214抑制剂得到一定程度的回补，表明miR-214可通过靶向AMPK抑制SHVV增殖；5）进一步研究发现糖原合成酶（GS）也是miR-214的靶基因，过表达miR-214降低细胞内GS基因的表达，siGS敲降细胞内GS基因的表达显著抑制SHVV增殖，这种抑制作用能通过添加miR-214抑制剂回补，表明miR-214也能通过靶向宿主的GS基因抑制SHVV增殖。.本项目的研究结果为揭示SHVV如何利用宿主miRNA调节自身增殖的分子机制奠定基础，也为药物靶点的发掘提供理论依据。