CircRNA75 and CircRNA72 Function as the Sponge of MicroRNA-200 to Suppress Coelomocyte Apoptosis Via Targeting Tollip in Apostichopus japonicus

Jiqing Liu,Weiwei Zhang,Chenghua Li,Xuelin Zhao,Xuemei Duan

doi:10.3389/fimmu.2021.770055

Abstract

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 Apostichopus japonicus skin ulceration syndrome (SUS), which is a major problem restricting the sea cucumber breeding industry, were identified by genome-wide screening. In this study, via 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 Vibrio splendidus-challenged conditions. A dual-luciferase assay indicated that both circRNA75 and circRNA72 could bind miR-200 in vivo, 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.

Highlights

CircRNAs are a new type of non-coding RNA with neither 5’ to 3’ polarity nor a polyadenylation tail and with covalently closed circular structure [1]
We found that miR-200 was abundant in coelomocytes of skin ulceration syndrome (SUS) and might play important roles in the immune response of A. japonicus [28]
The results showed that circRNA75 and circRNA72 transcripts were significantly upregulated with a 1.73- and 7.18-fold increase at 6 h (p

Summary

Introduction

CircRNAs are a new type of non-coding RNA (ncRNA) with neither 5’ to 3’ polarity nor a polyadenylation tail and with covalently closed circular structure [1]. With the development of high-throughput sequencing and bioinformatics, researchers have discovered more than 10,000 circRNAs in many animals, such as fruit fly [2], fish [3], mouse [4], monkey [5], and human [6]. The presence of circRNAs has been confirmed in plants [7], fungi [8], and protists [9]. Emerging studies suggested that some circRNAs played crucial roles under physiological and pathological conditions [10]. Knockout experiments proved that neuronal function and development may be mediated by circRNAs [11]. The onset of many diseases or worsening of tumor formation is due to abnormal circRNA expression in the internal environment [12, 13]

Methods

Results

Conclusion