Transcriptome analysis of differentially expressed circRNAs miRNAs and mRNAs during the challenge of coccidiosis

Abstract

Avian coccidiosis is a common enzootic disease caused by infection of Eimeria species parasites. It causes huge economic losses in the global poultry industry. Current control using anticoccidial drugs or vaccination is limited due to drug resistance and the relatively high cost of vaccines. Improving host genetic resistance to Eimeria species is considered an effective strategy for improved control of coccidiosis. Circular RNAs (circRNAs) have been found to function as biomarkers or diagnoses of various kinds of diseases. The molecular biological functions of circRNAs, miRNAs, and mRNAs related to Sasso chicken have not yet been described during Eimeria species challenge. In this study, RNA-seq was used to profile the expression pattern of circRNAs, miRNAs, and mRNAs in spleens from Eimeria tenella-infected and non-infected commercial dual-purpose Sasso T445 breed chickens. Results showed a total of 40 differentially expressed circRNAs (DEcircRNAs), 31 differentially expressed miRNAs (DEmiRNAs), and 820 differentially expressed genes (DEmRNAs) between infected and non-infected chickens. Regulatory networks were constructed between differentially expressed circRNAs, miRNAs, and mRNAs to offer insights into the interaction mechanisms between chickens and Eimeria spp. Functional validation of a significantly differentially expressed circRNA, circMGAT5, revealed that circMGAT5 could sponge miR-132c-5p to promote the expression of the miR-132c-5p target gene monocyte to macrophage differentiation-associated (MMD) during the infection of E. tenella sporozoites or LPS stimulation. Pathologically, knockdown of circMGAT5 significantly upregulated the expression of macrophage surface markers and the macrophage activation marker, F4/80 and MHC-II, which indicated that circMGAT5 might inhibit the activation of macrophage. miR-132c-5p markedly facilitated the expression of F4/80 and MHC-II while circMGAT5 could attenuate the increase of F4/80 and MHC-II induced by miR-132c-5p, indicating that circMGAT5 exhibited function through the circMGAT5-miR-132c-5p-MMD axis. Together, our results indicate that circRNAs exhibit their resistance or susceptive roles during E. tenella infection. Among these, circMGAT5 may inhibit the activation of macrophages through the circMGAT5-miR-132c-5p-MMD axis to participate in the immune response induced by Eimeria infection.

Keywords: MMD; avian coccidiosis; circMGAT5; circRNAs; sasso chicken.

Figure 1

Volcano map of differentially expressed circRNAs, miRNAs and mRNAs associated with E. tenella infection. (A) Volcano map of differentially expressed circRNAs. (B) Volcano map of differentially expressed miRNAs. (C) Volcano map of differentially expressed mRNAs. For (A-C), threshold used to define differentially expressed genes is(fold change)> 0 and P < 0.05. (D) Read count of the selected DEcircRNAs from the sequence data. (E) RT-qPCR quantification of the selected DEcircRNAs. (F) TPM of the selected DEmiRNAs from the sequence data. (G) RT-qPCR quantification of the selected DEmiRNAs. (H) FPKM of DEmRNAs from the sequence data. (I) RT-qPCR quantification of the selected DEmRNAs. For D-I, results are shown as mean ± SEM. Statistical significance of differences between means was assessed using unpaired Student’s t-test. (*P < 0.05; **P < 0.01).

Figure 2

Interaction networks of DEcircRNA-DEmiRNA pairs involved in the immune response to E. tenella infection with threshold parameters (single-residue-pair match scores> 140, ΔG< -10 kcal/mol). (A) Networks of up_DEcircRNA-down_DEmiRNA generated using Cytoscape 3.7.2. (B) Networks of down_DEcircRNA-up_DEmiRNA generated using Cytoscape 3.7.2.

Figure 3

Networks of DEmiRNA-DEmRNA involved in the immune response to E. tenella infection predicted by miRDB. (A) Networks of up_DEmiRNA-down_DEmRNA generated using Cytoscape 3.7.2. (B) Networks of down_DEmiRNA-up_DEmRNA generated using Cytoscape 3.7.2.

Figure 4

DEcircRNA-DEmiRNA-DEmRNA ceRNA regulatory networks generated by Cytoscape 3.7.2. (A) up_DEcircRNA-down_DEmiRNA-up_DEmRNA ceRNA networks generated using Cytoscape 3.7.2. (B) down_DEcircRNA-up_DEmiRNA-down_DEmRNA ceRNA networks generated using Cytoscape 3.7.2.

Figure 5

Identification of circMGAT5-miR-132c-5p-MMD axis. (A) Amplification of the junction sequence of circMGAT5. (B) Divergent primers amplify circMGAT5 in cDNA but not genomic DNA (gDNA). (C) RT-qPCR quantification of circMGAT5 after RNase R treatment. (D) The potential interaction model between circMGAT5 and miR-132c-5p predicted by RNAhybird. (E) The wild and mutant binding site sequence between circMGAT5 and miR-132c-5p in the pmirGLO vector. (F) Dual-luciferase reporter assay measuring the binding of circMGAT5 to miR-132c-5p. (G) RT-qPCR quantification of circMGAT5 enriched in the biotinylated miR-132c-5p mimic pull down RNA. (H) qPCR product of circMGAT5 in the biotinylated miR-132c-5p mimic pull down RNA and biotinylated mimic NC. (I-M) miR-132c-5p interacts with MMD. (I) The potential interaction model between miR-132c-5p and MMD predicted by RNAhybird; (J) The wild and mutant binding site sequence between miR-132c-5p and MMD in the pmirGLO vector; (K) Dual-luciferase reporter assay measuring the binding of miR-132c-5p to MMD; (L) miR-132c-5p mimic inhibits the expression of MMD; (M) miR-132c-5p inhibitor promotes the expression of MMD. For (C, F, G, K, L, M), results are shown as mean ± SEM. Statistical significance of differences between means was assessed using unpaired Student’s t-test. (*P < 0.05; **P < 0.01; N.S. no significant difference).

Figure 6

circMGAT5 inhibits macrophage activation and differentiation through the circMGAT5-miR-132c-5p-MMD axis. (A-D) miR-132c-5p inhibits the expression of MMD during E. tenella infection or LPS stimulation in HD11 cells. (A) Expression of MMD was inhibited by miR-132c-5p mimic during E. tenella infection; (B) miR-132c-5p inhibitor promoted the expression of MMD during E. tenella infection; (C) Expression of MMD was decreased by miR-132c-5p mimic during LPS stimulation of HD11 cells; (D) miR-132c-5p inhibitor increased expression of MMD during LPS stimulation of HD11 cells. (E-J) circMGAT5 promotes MMD expression during E. tenella sporozoite infection or LPS stimulation of HD11 cells. (E) Overexpression of circMGAT5. (F) Knockdown of circMGAT5. (G) Overexpression of circMGAT5 promoted MMD expression during E. tenella sporozoite infection. (H) Knockdown of circMGAT5 inhibited MMD expression during E. tenella sporozoite infection. (I) Overexpression of circMGAT5 promoted MMD expression of during LPS stimulation of HD11 cells. (J) Knockdown of circMGAT5 inhibited MMD expression during LPS stimulation of HD11 cells. (K, L) circMGAT5 inhibits macrophage activation and differentiation through the circMGAT5-miR-132c-5p-MMD axis. (K) Knockdown of circMGAT5 markedly increased F4/80 and MHC-II expression, indicating that downregulation of circMGAT5 may result in enhanced macrophage activation and differentiation. (L) miR-132c-5p markedly facilitated expression of F4/80 and MHC-II while circMGAT5 could attenuate the increase induced by miR-132c-5p. In addition, the expression of MMD in the miR-132c-5p and circMGAT5 co-transfected groups was significantly higher than that in the miR-132c-5p overexpressing group, indicating that circMGAT5 attenuates the inhibitory effect of miR-132c-5p on MMD and circMGAT5 may inhibits macrophage activation and differentiation through the circMGAT5-miR-132c-5p-MMD axis. In all panels, results are shown as mean ± SEM. Statistical significance of differences between means was assessed using unpaired Student’s t-test. (*P < 0.05; **P < 0.01; N.S. no significant difference).