Liu Jiqing, Zhao Xuelin, Duan Xuemei, Zhang Weiwei, Li Chenghua
State Key Laboratory for Quality and Safety of Agro-products, Ningbo University, Ningbo, China.
Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University, Ningbo, China.
Front Immunol. 2021 Nov 17;12:770055. doi: 10.3389/fimmu.2021.770055. eCollection 2021.
Circular RNAs (circRNAs) act as essential regulators in many biological processes, especially in mammalian immune response. Nonetheless, the functions and mechanisms of circRNAs in the invertebrate immune system are largely unclarified. In our previous work, 261 differentially expressed circRNAs potentially related to the development of skin ulceration syndrome (SUS), which is a major problem restricting the sea cucumber breeding industry, were identified by genome-wide screening. In this study, miRanda analysis, both circRNA75 and circrRNA72 were shown to share the miR-200 binding site, a key microRNA in the SUS. The two circRNAs were verified to be increased significantly in LPS-exposed primary coelomocytes, similar to the results of circRNA-seq in sea cucumber under -challenged conditions. A dual-luciferase assay indicated that both circRNA75 and circRNA72 could bind miR-200 , in which circRNA75 had four binding sites of miR-200 and only one for circRNA72. Furthermore, we found that miR-200 could bind the 3'-UTR of Toll interacting protein (Tollip) to negatively mediate the expression of Tollip. Silencing Tollip increased primary coelomocyte apoptosis. Consistently, inference of circRNA75 and circRNA72 could also downregulate Tollip expression, thereby increasing the apoptosis of primary coelomocytes, which could be blocked by miR-200 inhibitor treatment. Moreover, the rate of si-circRNA75-downregulated Tollip expression was higher than that of si-circRNA72 under an equivalent amount. CircRNA75 and circRNA72 suppressed coelomocyte apoptosis by sponging miR-200 to promote Tollip expression. The ability of circRNA to adsorb miRNA might be positively related to the number of binding sites for miRNA.
环状RNA（circRNAs）在许多生物学过程中发挥着重要的调节作用，尤其是在哺乳动物的免疫反应中。然而，circRNAs在无脊椎动物免疫系统中的功能和机制在很大程度上仍不清楚。在我们之前的工作中，通过全基因组筛选，鉴定出261个可能与皮肤溃疡综合征（SUS）发展相关的差异表达circRNAs，SUS是限制海参养殖业发展的一个主要问题。在本研究中，通过miRanda分析发现，circRNA75和circRNA72都共享miR-200结合位点，miR-200是SUS中的一个关键微小RNA。这两种circRNAs在暴露于脂多糖（LPS）的原代体腔细胞中被证实显著增加，这与在应激条件下海参的circRNA测序结果相似。双荧光素酶报告基因检测表明，circRNA75和circRNA72都能与miR-200结合，其中circRNA75有四个miR-200结合位点，而circRNA72只有一个。此外，我们发现miR-200可以与Toll相互作用蛋白（Tollip）的3'-非翻译区（3'-UTR）结合，从而负向调节Tollip的表达。沉默Tollip会增加原代体腔细胞凋亡。一致的是，circRNA75和circRNA72的干扰也会下调Tollip的表达，从而增加原代体腔细胞的凋亡，而miR-200抑制剂处理可以阻断这种凋亡。此外，在等量情况下，si-circRNA75下调Tollip表达的速率高于si-circRNA72。CircRNA75和circRNA72通过海绵吸附miR-200来促进Tollip表达，从而抑制体腔细胞凋亡。circRNA吸附miRNA的能力可能与miRNA的结合位点数量呈正相关。
