miR-31靶向调控刺参腐皮综合症发生的分子机制研究

With the financial support by previous NFSC, miR-31 had been identified and linked to Apostichopus japonicus skin ulcer syndrome (SUS ) explosion under natural conditions. Accordingly, it is interesting to further investigate the concrete cellular and molecular mechanism of miR-31 and determine its putative target genes in this host-pathogen interaction. In this work, we intend to clone the genomic sequence of miR-31 through genome walking, then identify and characterize its promising regulatory elements in response to pathogen infection. Subsequently, miR-31 targets are computationally predicted by RNA-seq method combined with hybrid PCR and expression profiles analysis. Functional validation of these targets will be performed by loss- and gain-function of miR-31 in vitro and in vivo, and at least 3 target are finally functional annotated in this non-model organism. To thoroughly interpret the regulatory roles of miR-31, the changes of cell immune indices , including phagocytosis, apoptosis, autophagy and respiratory burst, will be assayed after target gene silencing or miR-31 aberrant expression to address the connection between cell and humoral immunity. Overall, the present work will enhance our understanding in the context of miR-31 modulating host-pathogen interaction, and provide promising candidates for controlling SUS outbreak in practice.

在已完成的国家自然科学基金的资助下，申请人证实了miR-31参与了刺参腐皮综合症发生的免疫应答过程，但对于该调控分子是如何被病原激活的？其调控的靶基因又如何介导刺参的防御机制等科学问题不甚明确。本项目拟通过构建刺参基因组文库，克隆刺参miR-31基因上游调控序列，识别和鉴定关键调控元件响应病原感染的方式或途径。随后融合高通量转录组测序、杂交PCR、基因定量表达分析和双荧光素报告测定等手段筛选和鉴定刺参体腔细胞miR-31的靶基因；进而在个体和培养体腔细胞两个层次上功能确认miR-31靶向调控的目标基因3个以上。最后，阐明miR-31和靶基因异常表达对刺参体腔细胞吞噬、凋亡、呼吸爆发或自噬等细胞免疫指标的影响，初步掌握miR-31介导的刺参免疫应答调控途径，为从根本上控制刺参腐皮综合症发生提供新思路。

本研究构建了miR-31过表达或抑制表达不同时间点（12h、24h）刺参体腔细胞高通量转录组文库，发现36个unigene在不同时间点均呈现一致表达，表明其可能为miR-31的靶基因。为明确miR-31和靶基因的调控关系，我们建立了刺参个体和体外培养细胞两个层次上的miRNA和靶基因功能鉴定体系，证实了miR-31通过靶向双功能分子CTRP9调控脂代谢进而介导细胞凋亡的过程。确立了miR-31靶向Ajp105基因调控呼吸爆发的分子过程；构建了刺参基因组文库四个，利用基因组步移方法分别获得了刺参精氨酸代谢的NOS、arginase和agmatinase等3个关键基因的5’调控序列。发现当截短的AjNOS的-375 bp到-366 bp是NF-κB的关键结合位点，正调控NOS基因转录。同时发现共转染NF-κB/Rel或STAT5后，转染有截短的arginase启动子报告基因的荧光素酶转录活性显著降低，而共转染各截短的agmatinase启动子质粒的双荧光素酶活性都无明显变化。借助启动子克隆和双荧光素报告测定，阐明了转录因子MYC通过结合Bax启动子序列的第一个E-box序列调控Bax表达，进而促进病原胁迫的刺参体腔细胞凋亡的分子过程.