Fu X et al. (2023), circRNA432 enhances the coelomocyte phagocytosi...

ECB-ART-51644

Commun Biol 2023 Jan 28;61:115. doi: 10.1038/s42003-023-04516-8.

Show Gene links Show Anatomy links

circRNA432 enhances the coelomocyte phagocytosis via regulating the miR-2008-ELMO1 axis in Vibrio splendidus-challenged Apostichopus japonicus.

Fu X , Guo M , Liu J , Li C .

???displayArticle.abstract???
Circular RNAs (circRNAs) are a kind of extensive and diverse covalently closed circular endogenous RNA, which exert crucial functions in immune regulation in mammals. However, the functions and mechanisms of circRNAs in invertebrates are largely unclarified. In our previous work, 261 differentially expressed circRNAs including circRNA432 (circ432) were identified from skin ulcer syndrome (SUS) diseased sea cucumber Apostichopus japonicus by RNA-seq. To better address the functional role of sea cucumber circRNAs, circ432 was first found to be significantly induced by Vibrio splendidus challenge and LPS exposure in this study. Knock-down circ432 could depress the V. splendidus-induced coelomocytes phagocytosis. Moreover, circ432 is validated to serve as the sponge of miR-2008, a differential expressed miRNA in SUS-diseased sea cucumbers, by Argonaute 2-RNA immunoprecipitation (AGO2-RIP) assay, luciferase reporter assay and RNA fluorescence in situ hybridization (FISH) in vitro. Engulfment and cell motility protein 1 (AjELMO1) is further demonstrated to be the target of miR-2008, and silencing AjELMO1 inhibits the V. splendidus-induced coelomocytes phagocytosis, and this phenomenon could be further suppressed by supplementing with miR-2008 mimics, suggesting that circ432 might regulate coelomocytes phagocytosis via miR-2008-AjELMO1 axis. We further confirm that the depressed coelomocytes' phagocytosis by circ432 silencing is consistent with the decreased abundance of AjELMO1, and could be recovered by miR-2008 inhibitors transfection. All our results provide the evidence that circ432 is involved in regulating pathogen-induced coelomocyte phagocytosis via sponge miR-2008 and promotes the abundance of AjELMO1. These findings will enrich the regulatory mechanism of phagocytosis in echinoderm and provide theoretical data for SUS disease prevention and control in sea cucumbers.

???displayArticle.pubmedLink??? 36709365
???displayArticle.pmcLink??? PMC9884281
???displayArticle.link??? Commun Biol

???attribute.lit??? ???displayArticles.show???

	Fig. 1. The characteristics of circ432.a The junction splice site of circ432 was confirmed by Sanger sequencing. b RT-PCR with divergent (◀▶) and convergent primers (▶◀) showing the amplification of circ432 from cDNA or gDNA of coelomocytes. c The divergent and convergent primers of circ432 in the presence or absence of RNase R was validated by RT-PCR. d RNA extracted from the cytoplasm and nuclear of coelomocytes were performed to detect circ432 abundance by RT-PCR. Ajβ-actin, and RNU6B served as the controls, n = 3. e circ432 mainly located in the cytoplasm was verified by RNA FISH. Scale bar, 10 μm. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01.
	Fig. 2. circ432 participates in the phagocytosis of coelomocytes induced by V. splendidus.a Tissue abundance distribution of circ432 in coelomocytes, intestine, tentacles, respiratory tree, and muscle was measured by qPCR. b The time-course abundance patterns of circ432 in coelomocytes of V. splendidus-challenged A. japonicus and the LPS-exposed primary cultured cell as measured by qPCR. c The relative abundance level of circ432 and the host gene of circ432 after circ432 siRNA transfection in vivo and in vitro were detected by qPCR. d The phagocytosis ratio of coelomocytes in circ432 siRNA transfection under the induction of V. splendidus was detected by flow cytometry. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01. Different letters above each bar indicate significant differences: P < 0.05, whereas bars with the same letter indicates non-significant differences.
	Fig. 3. circ432 functions as a miRNA sponge of miR-2008.a The combination of circ432 and miR-2008 was predicted by miRanda. b The divergent primers of circ432 of AGO2-RIP were validated by RT-PCR. c The relative luciferase activities were detected after co-transfection with circ432-wt or circ432-mut and modified miR-2008 mimics or NCM, and the circ432 relative luciferase activity was detected at different concentration gradients of miR-2008 mimics, including 50, 75, and 100 nM. d That circ432 can adsorb miR-2008 was detected by RNA FISH. Scale bar, 10 μm. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01.
	Fig. 4. Inhibitory effect of miR-2008 on phagocytosis of coelomocytes.a The relative miR-2008 abundance level after transfection with modified miR-2008 mimics and inhibitor in vivo and in vitro was detected by qPCR. b, c The phagocytosis ratio of coelomocytes in modified miR-2008 mimics/inhibitor under the induction of V. splendidus was detected by flow cytometry. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01. Different letters above each bar indicate significant differences: P < 0.05, whereas bars with the same letter indicates non-significant differences.
	Fig. 5. miR-2008 inhibits the V. splendidus-induced coelomocytes phagocytosis via targeting AjELMO1.a The combination of miR-2008 and AjELMO1, cell division cycle 42 was predicted by the miRanda program. b The relative luciferase activities were detected in Hela cells after co-transfection with cell division cycle 42-wt or cell division cycle 42-mut plasmids and modified miR-2008 mimics or NCM. c The relative luciferase activities were detected after co-transfection with AjELMO1-wt or AjELMO1-mut plasmids and modified miR-2008 mimics or NCM, and the AjELMO1 relative luciferase activity was detected at different concentration gradients of miR-2008 mimics, including 50, 75, and 100 nM. d Hela cells were co-transfected with pcGFP-MS2, pcGFP-MS2-AjELMO1, or pcGFP-MS2-AjELMO1-mut plasmids and miR-2008 mimics for 48 h, and then incubated with anti-GFP antibody and further detect the enrichment of miR-2008 by qPCR. e, f The relative abundance level of AjELMO1 after transfection with modified miR-2008 mimics and inhibitor in vivo and in vitro was detected by qPCR and western blotting. g The phagocytosis ratio of coelomocytes in AjELMO1 siRNA transfection and modified miR-2008 mimics under the induction of V. splendidus was detected by flow cytometry. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01. Different letters above each bar indicate significant differences: P < 0.05, whereas bars with the same letter indicates non-significant differences.
	Fig. 6. AjELMO1 promotes phagocytosis of responses upon V. splendidus infection.a The time-course abundance patterns of AjELMO1 in the V. splendidus-challenged A. japonicus and the LPS-exposed primary cultured cell as measured by qPCR. b The relative abundance level of AjELMO1 after AjELMO1 siRNA transfection in vivo and in vitro was detected by qPCR and western blotting. c The phagocytosis ratio of coelomocytes in AjELMO1 siRNA transfection under the induction of V. splendidus was detected by flow cytometry. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01. Different letters above each bar indicate significant differences: P < 0.05, whereas bars with the same letter indicates non-significant differences.
	Fig. 7. circ432 as a sponge for miR-2008 enhances phagocytosis of AjELMO1.a The relative abundance level of AjELMO1 and miR-2008 after circ432 siRNA transfection in vivo and in vitro was detected by qPCR and western blotting. b The relative abundance level of AjELMO1 after circ432 siRNA transfection and modified miR-2008 inhibitor in vivo and in vitro was detected by qPCR and western blotting. c The phagocytosis ratio of coelomocytes in circ432 siRNA transfection and modified miR-2008 inhibitor under the induction of V. splendidus was detected by flow cytometry. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01. Different letters above each bar indicate significant differences: P < 0.05, whereas bars with the same letter indicates non-significant differences.
	Fig. 8. The schematic diagram shows the mechanism underlying circ432 as a ceRNA for miR-2008 to regulate V. splendidus-induced coelomocytes phagocytosis by targeting AjELMO1.miR-2008 targets AjELMO1 and represses AjELMO1-mediated coelomocytes phagocytosis. circ432 acts as a molecular sponge regulating miR-2008 to enhance AjELMO1 abundance to maintain the phagocytosis balance.

References [+] :

Aderem, Mechanisms of phagocytosis in macrophages. 1999, Pubmed

Aderem, Mechanisms of phagocytosis in macrophages. 1999, Pubmed
Andersen, Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. 2004, Pubmed
Brugnera, Unconventional Rac-GEF activity is mediated through the Dock180-ELMO complex. 2002, Pubmed
Bustin, The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. 2009, Pubmed
Chen, Circular RNA WDR77 target FGF-2 to regulate vascular smooth muscle cells proliferation and migration by sponging miR-124. 2017, Pubmed
Chen, A unique NLRC4 receptor from echinoderms mediates Vibrio phagocytosis via rearrangement of the cytoskeleton and polymerization of F-actin. 2021, Pubmed , Echinobase
Chu, A Highly Conserved Circular RNA circRasGEF1B Enhances Antiviral Immunity by Regulating miR-21-3p/MITA Pathway in Lower Vertebrates. 2021, Pubmed
Csaba, Phylogeny and ontogeny of chemical signaling: origin and development of hormone receptors. 1994, Pubmed
Dong, The Expression, Functions and Mechanisms of Circular RNAs in Gynecological Cancers. 2020, Pubmed
Elcheva, CRD-BP protects the coding region of betaTrCP1 mRNA from miR-183-mediated degradation. 2009, Pubmed
Forman, A search for conserved sequences in coding regions reveals that the let-7 microRNA targets Dicer within its coding sequence. 2008, Pubmed
Grimsley, Dock180 and ELMO1 proteins cooperate to promote evolutionarily conserved Rac-dependent cell migration. 2004, Pubmed
Gumienny, CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration. 2001, Pubmed
Guo, BAG2 mediates coelomocyte apoptosis in Vibrio splendidus challenged sea cucumber Apostichopus japonicus. 2021, Pubmed , Echinobase
Hellemans, qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. 2007, Pubmed
Huang, MicroRNA-181a modulates gene expression of zinc finger family members by directly targeting their coding regions. 2010, Pubmed
Karapetyan, Regulatory Roles for Long ncRNA and mRNA. 2013, Pubmed
Kulcheski, Circular RNAs are miRNA sponges and can be used as a new class of biomarker. 2016, Pubmed
Li, miR210 modulates respiratory burst in Apostichopus japonicus coelomocytes via targeting Toll-like receptor. 2016, Pubmed , Echinobase
Li, Production, characterization and application of monoclonal antibody to spherulocytes: a subpopulation of coelomocytes of Apostichopus japonicus. 2010, Pubmed , Echinobase
Liu, CircRNA75 and CircRNA72 Function as the Sponge of MicroRNA-200 to Suppress Coelomocyte Apoptosis Via Targeting Tollip in Apostichopus japonicus. 2021, Pubmed , Echinobase
Liu, Circular RNA hsa_circRNA_103809 promotes lung cancer progression via facilitating ZNF121-dependent MYC expression by sequestering miR-4302. 2018, Pubmed
Lu, MiR-31 modulates coelomocytes ROS production via targeting p105 in Vibrio splendidus challenged sea cucumber Apostichopus japonicus in vitro and in vivo. 2015, Pubmed , Echinobase
Lu, PH domain of ELMO functions in trans to regulate Rac activation via Dock180. 2004, Pubmed
Lu, miRNA-133 augments coelomocyte phagocytosis in bacteria-challenged Apostichopus japonicus via targeting the TLR component of IRAK-1 in vitro and in vivo. 2015, Pubmed , Echinobase
Lv, miR-92a regulates coelomocytes apoptosis in sea cucumber Apostichopus japonicus via targeting Aj14-3-3ζ in vivo. 2017, Pubmed , Echinobase
Lv, MiR-200 modulates coelomocytes antibacterial activities and LPS priming via targeting Tollip in Apostichopus japonicus. 2015, Pubmed , Echinobase
McCormick, ELMO1: More Than Just a Director of Phagocytosis. 2015, Pubmed
Na, Astragalus extract inhibits destruction of gastric cancer cells to mesothelial cells by anti-apoptosis. 2009, Pubmed
Ørom, MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. 2008, Pubmed
Pfaffl, Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. 2004, Pubmed
Piddini, Modulation of developmental signals by endocytosis: different means and many ends. 2003, Pubmed
Qu, The emerging landscape of circular RNA in life processes. 2017, Pubmed
Salmena, A ceRNA hypothesis: the Rosetta Stone of a hidden RNA language? 2011, Pubmed
Salzman, Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. 2012, Pubmed
Schnall-Levin, Conserved microRNA targeting in Drosophila is as widespread in coding regions as in 3'UTRs. 2010, Pubmed
Shao, miR-31 Links Lipid Metabolism and Cell Apoptosis in Bacteria-Challenged Apostichopus japonicus via Targeting CTRP9. 2017, Pubmed , Echinobase
Shu, The role of shrimp miR-965 in virus infection. 2016, Pubmed
Song, Expression profile of circular RNAs in oral lichen planus. 2021, Pubmed
Stark, Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures. 2007, Pubmed
Underhill, Phagocytosis of microbes: complexity in action. 2002, Pubmed
Wang, An invertebrate β-integrin mediates coelomocyte phagocytosis via activation of septin2 and 7 but not septin10. 2018, Pubmed , Echinobase
Wang, Effects of acute temperature or salinity stress on the immune response in sea cucumber, Apostichopus japonicus. 2008, Pubmed , Echinobase
Yokoyama, Identification of tyrosine residues on ELMO1 that are phosphorylated by the Src-family kinase Hck. 2005, Pubmed
Zhang, The sea cucumber genome provides insights into morphological evolution and visceral regeneration. 2017, Pubmed , Echinobase
Zhang, Identification and characterization of miR-92a and its targets modulating Vibrio splendidus challenged Apostichopus japonicus. 2014, Pubmed , Echinobase
Zhang, The Roles of Two miRNAs in Regulating the Immune Response of Sea Cucumber. 2015, Pubmed , Echinobase
Zhao, Genome-Wide Identification of Circular RNAs Revealed the Dominant Intergenic Region Circularization Model in Apostichopus japonicus. 2019, Pubmed , Echinobase
Zheng, Circular RNA circDtx1 regulates IRF3-mediated antiviral immune responses through suppression of miR-15a-5p-dependent TRIF downregulation in teleost fish. 2021, Pubmed
Zheng, CircMYOF triggers progression and facilitates glycolysis via the VEGFA/PI3K/AKT axis by absorbing miR-4739 in pancreatic ductal adenocarcinoma. 2021, Pubmed
Zwollo, Polystyrene microplastics reduce abundance of developing B cells in rainbow trout (Oncorhynchus mykiss) primary cultures. 2021, Pubmed

	Fig. 1. The characteristics of circ432.a The junction splice site of circ432 was confirmed by Sanger sequencing. b RT-PCR with divergent (◀▶) and convergent primers (▶◀) showing the amplification of circ432 from cDNA or gDNA of coelomocytes. c The divergent and convergent primers of circ432 in the presence or absence of RNase R was validated by RT-PCR. d RNA extracted from the cytoplasm and nuclear of coelomocytes were performed to detect circ432 abundance by RT-PCR. Ajβ-actin, and RNU6B served as the controls, n = 3. e circ432 mainly located in the cytoplasm was verified by RNA FISH. Scale bar, 10 μm. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01.
	Fig. 2. circ432 participates in the phagocytosis of coelomocytes induced by V. splendidus.a Tissue abundance distribution of circ432 in coelomocytes, intestine, tentacles, respiratory tree, and muscle was measured by qPCR. b The time-course abundance patterns of circ432 in coelomocytes of V. splendidus-challenged A. japonicus and the LPS-exposed primary cultured cell as measured by qPCR. c The relative abundance level of circ432 and the host gene of circ432 after circ432 siRNA transfection in vivo and in vitro were detected by qPCR. d The phagocytosis ratio of coelomocytes in circ432 siRNA transfection under the induction of V. splendidus was detected by flow cytometry. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01. Different letters above each bar indicate significant differences: P < 0.05, whereas bars with the same letter indicates non-significant differences.
	Fig. 3. circ432 functions as a miRNA sponge of miR-2008.a The combination of circ432 and miR-2008 was predicted by miRanda. b The divergent primers of circ432 of AGO2-RIP were validated by RT-PCR. c The relative luciferase activities were detected after co-transfection with circ432-wt or circ432-mut and modified miR-2008 mimics or NCM, and the circ432 relative luciferase activity was detected at different concentration gradients of miR-2008 mimics, including 50, 75, and 100 nM. d That circ432 can adsorb miR-2008 was detected by RNA FISH. Scale bar, 10 μm. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01.
	Fig. 4. Inhibitory effect of miR-2008 on phagocytosis of coelomocytes.a The relative miR-2008 abundance level after transfection with modified miR-2008 mimics and inhibitor in vivo and in vitro was detected by qPCR. b, c The phagocytosis ratio of coelomocytes in modified miR-2008 mimics/inhibitor under the induction of V. splendidus was detected by flow cytometry. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01. Different letters above each bar indicate significant differences: P < 0.05, whereas bars with the same letter indicates non-significant differences.
	Fig. 5. miR-2008 inhibits the V. splendidus-induced coelomocytes phagocytosis via targeting AjELMO1.a The combination of miR-2008 and AjELMO1, cell division cycle 42 was predicted by the miRanda program. b The relative luciferase activities were detected in Hela cells after co-transfection with cell division cycle 42-wt or cell division cycle 42-mut plasmids and modified miR-2008 mimics or NCM. c The relative luciferase activities were detected after co-transfection with AjELMO1-wt or AjELMO1-mut plasmids and modified miR-2008 mimics or NCM, and the AjELMO1 relative luciferase activity was detected at different concentration gradients of miR-2008 mimics, including 50, 75, and 100 nM. d Hela cells were co-transfected with pcGFP-MS2, pcGFP-MS2-AjELMO1, or pcGFP-MS2-AjELMO1-mut plasmids and miR-2008 mimics for 48 h, and then incubated with anti-GFP antibody and further detect the enrichment of miR-2008 by qPCR. e, f The relative abundance level of AjELMO1 after transfection with modified miR-2008 mimics and inhibitor in vivo and in vitro was detected by qPCR and western blotting. g The phagocytosis ratio of coelomocytes in AjELMO1 siRNA transfection and modified miR-2008 mimics under the induction of V. splendidus was detected by flow cytometry. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01. Different letters above each bar indicate significant differences: P < 0.05, whereas bars with the same letter indicates non-significant differences.
	Fig. 6. AjELMO1 promotes phagocytosis of responses upon V. splendidus infection.a The time-course abundance patterns of AjELMO1 in the V. splendidus-challenged A. japonicus and the LPS-exposed primary cultured cell as measured by qPCR. b The relative abundance level of AjELMO1 after AjELMO1 siRNA transfection in vivo and in vitro was detected by qPCR and western blotting. c The phagocytosis ratio of coelomocytes in AjELMO1 siRNA transfection under the induction of V. splendidus was detected by flow cytometry. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01. Different letters above each bar indicate significant differences: P < 0.05, whereas bars with the same letter indicates non-significant differences.
	Fig. 7. circ432 as a sponge for miR-2008 enhances phagocytosis of AjELMO1.a The relative abundance level of AjELMO1 and miR-2008 after circ432 siRNA transfection in vivo and in vitro was detected by qPCR and western blotting. b The relative abundance level of AjELMO1 after circ432 siRNA transfection and modified miR-2008 inhibitor in vivo and in vitro was detected by qPCR and western blotting. c The phagocytosis ratio of coelomocytes in circ432 siRNA transfection and modified miR-2008 inhibitor under the induction of V. splendidus was detected by flow cytometry. All data represented the mean ± SD from three independent triplicated experiments. P < 0.05, *P < 0.01. Different letters above each bar indicate significant differences: P < 0.05, whereas bars with the same letter indicates non-significant differences.
	Fig. 8. The schematic diagram shows the mechanism underlying circ432 as a ceRNA for miR-2008 to regulate V. splendidus-induced coelomocytes phagocytosis by targeting AjELMO1.miR-2008 targets AjELMO1 and represses AjELMO1-mediated coelomocytes phagocytosis. circ432 acts as a molecular sponge regulating miR-2008 to enhance AjELMO1 abundance to maintain the phagocytosis balance.