circRNA1149调控灿烂弧菌介导的刺参体腔细胞凋亡的分子机制研究

The participation of circRNA as miRNA sponges in the regulation of the occurrence and development of various diseases is an international research hotspot in recent years. Our previous studies found that circRNA1149 was significantly expressed in Vibrio splendins induced skin ulcer syndrome (SUS), and bioinformatic analysis revealed that circRNA1149 may mediate coelomocytes apoptosis through regulating the miR-92a/miR-33-Bax axis. However, it is not clear whether circRNA1149 can regulate coelomocytes apoptosis induced by V. splendins and whether the regulation is realized through the axis. Our project intends to determine the existence of the regulatory axis in space and structure through fluorescene in situ hybridization and dual-luciferase reporter assay. Through the overexpression, interference and recovery of circRNA1149, qPCR and western blot will be applied to detect the expression changes of miR-92a, miR-33 and Bax, and the changes of apoptosis will be detected by flow cytometry, which would further reveal the molecular pathway of circRNA1149 regulating the coelomocytes apoptosis induced by V. splendins. The results will further expand the regulatory network in the immune response of invertebrates and provide new ideas for understanding and targeting control of SUS disease.

circRNA作为miRNA海绵体参与调控多种疾病的发生和发展过程是近年来国际上的研究热点。申请人前期发现circRNA1149在灿烂弧菌诱导的刺参腐皮综合症中显著表达，生物信息学分析揭示circRNA1149可能通过调控miR-92a/miR-33-Bax轴介导刺参体腔细胞凋亡。但对于circRNA1149能否调控该病原诱导的细胞凋亡以及该调控是否经由该轴实现等关键科学问题尚不清楚。本项目拟通过荧光原位杂交和双荧光素酶报告基因实验从空间和结构上确定该调控轴的存在；通过circRNA1149的过表达、干扰及拯救，借助qPCR、免疫印迹技术检测miR-92a、miR-33、Bax的表达变化，并结合流式细胞术检测细胞凋亡变化，从功能上进一步揭示circRNA1149调控灿烂弧菌诱导的刺参体腔细胞凋亡分子途径。旨在进一步拓展无脊椎动物免疫应答的调控网络，为认识和定向防治刺参腐皮综合症提供新思路。

刺参作为海洋无脊椎动物，没有适应性免疫系统，仅依靠先天性免疫抵御病原侵染。非编码RNA（ncRNAs）已被证实广泛参与调控多种疾病的发生和发展进程，然而关于ncRNAs对于刺参先天免疫调节机制的研究还非常欠缺。本项目在课题组前期获得的患腐皮综合症刺参体腔细胞circRNA和miRNA转录组数据的基础上，筛选并鉴定了其中的多个关键circRNAs和miRNAs，分析了其在病原灿烂弧菌感染前后的刺参体腔细胞中的表达分布规律，并应用生物信息学以及ncRNAs及其靶基因功能缺失分析等技术构建了circRNA-miRNA-mRNA以及miRNA-mRNA的表达调控网络。通过构建的表达调控网络，明确了多个关键ncRNAs介导病原诱导的刺参体腔细胞先天免疫，并通过后续详细鉴定、双荧光素酶报告系统、敲降功能以及荧光原位杂交等研究，筛选并鉴定出四条关键调控途径：（1）阐明了circ1149-miR-92a/miR-33-Bax轴介导刺参体腔细胞凋亡的分子机制；（2）揭示了circ254调控灿烂弧菌诱导的刺参体腔细胞吞噬的分子途径；（3）解析了miR-375调控BAG2介导的刺参体腔细胞凋亡研究。相关研究结果已发表论文8篇，申请专利1项，培养研究生4名。通过项目研究，在国内建立了较为成熟的ncRNAs研究平台和技术，极大丰富和拓展了刺参先天免疫学的研究广度和深度，促进了我国刺参产业的绿色健康发展。