Genome-wide analysis of differentially expressed profiles of mRNAs, lncRNAs and circRNAs in chickens during Eimeria necatrix infection

Abstract

Background: Eimeria necatrix, the most highly pathogenic coccidian in chicken small intestines, can cause high morbidity and mortality in susceptible birds and devastating economic losses in poultry production, but the underlying molecular mechanisms in interaction between chicken and E. necatrix are not entirely revealed. Accumulating evidence shows that the long-non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are key regulators in various infectious diseases. However, the expression profiles and roles of these two non-coding RNAs (ncRNAs) during E. necatrix infection are still unclear.

Methods: The expression profiles of mRNAs, lncRNAs and circRNAs in mid-segments of chicken small intestines at 108 h post-infection (pi) with E. necatrix were analyzed by using the RNA-seq technique.

Results: After strict filtering of raw data, we putatively identified 49,183 mRNAs, 818 lncRNAs and 4153 circRNAs. The obtained lncRNAs were classified into four types, including 228 (27.87%) intergenic, 67 (8.19%) intronic, 166 (20.29%) anti-sense and 357 (43.64%) sense-overlapping lncRNAs; of these, 571 were found to be novel. Five types were also predicted for putative circRNAs, including 180 exonic, 54 intronic, 113 antisense, 109 intergenic and 3697 sense-overlapping circRNAs. Eimeria necatrix infection significantly altered the expression of 1543 mRNAs (707 upregulated and 836 downregulated), 95 lncRNAs (49 upregulated and 46 downregulated) and 13 circRNAs (9 upregulated and 4 downregulated). Target predictions revealed that 38 aberrantly expressed lncRNAs would cis-regulate 73 mRNAs, and 1453 mRNAs could be trans-regulated by 87 differentially regulated lncRNAs. Additionally, 109 potential sponging miRNAs were also identified for 9 circRNAs. GO and KEGG enrichment analysis of target mRNAs for lncRNAs, and sponging miRNA targets and source genes for circRNAs identified associations of both lncRNAs and circRNAs with host immune defense and pathogenesis during E. necatrix infection.

Conclusions: To the best of our knowledge, the present study provides the first genome-wide analysis of mRNAs, lncRNAs and circRNAs in chicken small intestines infected with E. necatrix. The obtained data will offer novel clues for exploring the interaction mechanisms between chickens and Eimeria spp.

Keywords: Chicken small intestine; Eimeria necatrix; Expression profile; circRNAs; lncRNAs; mRNAs.

Fig. 1

Screening, exon numbers and classification of the candidate lncRNAs in chicken small intestines. a Venn diagram of coding potential analysis according to strict criteria. Four tools (CPC, CNCI, Pfam and PLEK) were used to analyze the coding potential of lncRNAs. b The distribution of exon numbers of lncRNAs. c Classification of the four subtypes of lncRNAs

Fig. 2

Exon numbers and classification of the candidate circRNAs in chicken small intestines. a The distribution of exon numbers of circRNAs. b Classification of the five subtypes of circRNAs

Fig. 3

Expression patterns of differentially expressed mRNAs, lncRNAs and circRNAs in mid-segments of chicken small intestines infected with E. necatrix oocysts. a Hierarchical clustering plot showing the expression profiles of mRNAs. b Hierarchical clustering plot showing the expression profiles of lncRNAs. c Hierarchical clustering plot showing the expression profiles of circRNAs. In a–c, S1-S3 represent samples infected with E. necatrix oocysts and N1-N3 represent samples without infection. d Volcano plot showing the distributions of mRNAs. e Volcano plot showing the distributions of lncRNAs. f Volcano plot showing the distributions of circRNAs. In d–f, the significantly upregulated and downregulated mRNAs are presented as red and green dots, respectively, and the expression of mRNAs not significantly differentially expressed are presented as blue dots (FC > 2.0 and P-value < 0.05)

Fig. 4

Validation of the differentially expressed genes using qRT-PCR. Three biological repeats were included for each gene. a The validation result for mRNAs. b The validation result for lncRNAs. c The validation result for circRNAs. *P< 0.05, **P< 0.01, ***P< 0.001

Fig. 5

Co-repression network of significantly differentially expressed lncRNAs with their targets, with statistical relevance (Pearson’s correlation coefficient ≥ 0.8 and P-value ≤ 0.05) for considering co-repression

Fig. 6

circRNA-miRNA-targets network generated using Cytoscape 3.6.1. The network consists of 4 circRNAs, 2 miRNAs and 56 targets

Fig. 7

The top 10 GO terms enriched for the targets of differentially expressed lncRNAs in three categories (biological process, cellular component and molecular function), with the P-value ≤ 0.05 indicating significant enrichment

Fig. 8

The top 20 KEGG pathway terms enriched for the targets of differentially expressed lncRNAs, with the P-value ≤ 0.05 indicating significant enrichment

Fig. 9

The top 10 GO terms enriched for the sponging miRNA targets of differentially expressed circRNAs in three categories (biological process, cellular component and molecular function), with the P-value ≤ 0.05 indicating significant enrichment

Fig. 10

The top 20 KEGG pathway terms enriched for the sponging miRNA targets of differentially expressed circRNAs, with the P-value ≤ 0.05 indicating significant enrichment