Liu J et al. (2021), CircRNA75 and CircRNA72 Function as the Sponge ...

ECB-ART-50645

Front Immunol 2021 Jan 01;12:770055. doi: 10.3389/fimmu.2021.770055.

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

Liu J , Zhao X , Duan X , Zhang W , Li C .

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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.

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	Figure 1. Identification of the two circRNAs related to SUS development. (A) The back-splicing junctions of circRNA75 and circRNA72 were verified by RT-PCR and Sanger sequencing, respectively. (B) Schematic illustration exhibited the formation of the two circRNAs through the circularization of their host genes. (C) Relative expressions of circRNAs and linear RNA in coelomocytes were detected by RT-PCR upon RNase R treatment. (D) The expression patterns of circRNA75 and circRNA72 under LPS stimulation. p < 0.05 and *p < 0.01.
	Figure 2. CircRNA75 and circRNA72 could bind to miR-200 as seen in the dual-luciferase reporter assay. (A) Schematic illustration of the two circRNAs and mutant luciferase reporter vectors. (B, C) Relative luciferase activities were measured after transfection in EPC with circRNA-WT or circRNA-Mut and miR-200 mimic or miR-NC. p < 0.05 and *p < 0.01.
	Figure 3. Tollip is a direct target of miR-200 to attenuate LPS-induced coelomocyte apoptosis. (A) Schematic illustration of target genes and Mut luciferase reporter vectors. (B) Relative luciferase activities were measured in EPC cells after transfection with WT or Mut and a miR-200 mimic or miR-NC. (C, D) qRT-PCR was applied to detect the regulation of miR-200 mimics and inhibitors on the mRNA levels of Tollip, circRNA75, and circRNA72. (E) Western blot and gray value analysis were used to detect the regulation of miR-200 mimics and inhibitors on the protein levels of Tollip. (F) qRT-PCR detected the mRNA level of Tollip after the transfection of si-Tollip. (G) Western blot and gray value analyses detected the protein level of Tollip after the transfection of si-Tollip. (H) Coelomocyte apoptosis assay after Tollip knockdown in vitro. (I) Statistical analysis of apoptosis rate after Tollip knockdown. p < 0.05 and *p < 0.01.
	Figure 4. CircRNA75 and circRNA72 could regulate Tollip-mediated coelomocyte apoptosis by sponging miR-200. (A, B) After circRNA75 or circRNA72 knockdown, qRT-PCR detected the regulatory effect on Tollip and miR-200 mRNA levels. (C) After circRNA75 or circRNA72 knockdown, Western blot and gray value analyses detected the regulatory effects on Tollip protein levels. (D, E) Under knockdown conditions of circRNA75 or circRNA72, qRT-PCR detected the effect of miR-200 mimics or inhibitors on the mRNA expression of Tollip. (F, G) Under knockdown conditions of circRNA75 or circRNA72, Western blot and gray value analyses detected the effect of miR-200 mimics or inhibitors on the protein expression of Tollip. (H) Coelomocyte apoptosis assay after circRNA75 or circRNA72 knockdown and in circRNA knockdown after the addition of the miR-200 inhibitor. (I, J) Statistical analysis of apoptosis rate after circRNA75 or circRNA72 knockdown and in the circRNA knockdown of the added miR-200 inhibitor. p < 0.05 and *p < 0.01.
	Figure 5. Schematic diagram displaying the mechanism underlying the two circRNAs as endogenous ceRNA for miR-200.

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	Figure 1. Identification of the two circRNAs related to SUS development. (A) The back-splicing junctions of circRNA75 and circRNA72 were verified by RT-PCR and Sanger sequencing, respectively. (B) Schematic illustration exhibited the formation of the two circRNAs through the circularization of their host genes. (C) Relative expressions of circRNAs and linear RNA in coelomocytes were detected by RT-PCR upon RNase R treatment. (D) The expression patterns of circRNA75 and circRNA72 under LPS stimulation. p < 0.05 and *p < 0.01.
	Figure 2. CircRNA75 and circRNA72 could bind to miR-200 as seen in the dual-luciferase reporter assay. (A) Schematic illustration of the two circRNAs and mutant luciferase reporter vectors. (B, C) Relative luciferase activities were measured after transfection in EPC with circRNA-WT or circRNA-Mut and miR-200 mimic or miR-NC. p < 0.05 and *p < 0.01.
	Figure 3. Tollip is a direct target of miR-200 to attenuate LPS-induced coelomocyte apoptosis. (A) Schematic illustration of target genes and Mut luciferase reporter vectors. (B) Relative luciferase activities were measured in EPC cells after transfection with WT or Mut and a miR-200 mimic or miR-NC. (C, D) qRT-PCR was applied to detect the regulation of miR-200 mimics and inhibitors on the mRNA levels of Tollip, circRNA75, and circRNA72. (E) Western blot and gray value analysis were used to detect the regulation of miR-200 mimics and inhibitors on the protein levels of Tollip. (F) qRT-PCR detected the mRNA level of Tollip after the transfection of si-Tollip. (G) Western blot and gray value analyses detected the protein level of Tollip after the transfection of si-Tollip. (H) Coelomocyte apoptosis assay after Tollip knockdown in vitro. (I) Statistical analysis of apoptosis rate after Tollip knockdown. p < 0.05 and *p < 0.01.
	Figure 4. CircRNA75 and circRNA72 could regulate Tollip-mediated coelomocyte apoptosis by sponging miR-200. (A, B) After circRNA75 or circRNA72 knockdown, qRT-PCR detected the regulatory effect on Tollip and miR-200 mRNA levels. (C) After circRNA75 or circRNA72 knockdown, Western blot and gray value analyses detected the regulatory effects on Tollip protein levels. (D, E) Under knockdown conditions of circRNA75 or circRNA72, qRT-PCR detected the effect of miR-200 mimics or inhibitors on the mRNA expression of Tollip. (F, G) Under knockdown conditions of circRNA75 or circRNA72, Western blot and gray value analyses detected the effect of miR-200 mimics or inhibitors on the protein expression of Tollip. (H) Coelomocyte apoptosis assay after circRNA75 or circRNA72 knockdown and in circRNA knockdown after the addition of the miR-200 inhibitor. (I, J) Statistical analysis of apoptosis rate after circRNA75 or circRNA72 knockdown and in the circRNA knockdown of the added miR-200 inhibitor. p < 0.05 and *p < 0.01.
	Figure 5. Schematic diagram displaying the mechanism underlying the two circRNAs as endogenous ceRNA for miR-200.