Differential expression analysis of mRNAs, lncRNAs, and miRNAs expression profiles and construction of ceRNA networks in PEDV infection

Abstract

Background: Porcine Epidemic Diarrhea Virus (PEDV) is a coronavirus that seriously affects the swine industry. MicroRNAs and long noncoding RNAs are two relevant non-coding RNAs (ncRNAs) class and play crucial roles in a variety of physiological processes. Increased evidence indicates a complex interaction between mRNA and ncRNA. However, our understanding of the function of ncRNA involved in host-PEDV interaction is limited.

Results: A total of 1,197 mRNA transcripts, 539 lncRNA transcripts, and 208 miRNA transcripts were differentially regulated at 24 h and 48 h post-infection. Gene ontology (GO) and KEGG pathway enrichment analysis showed that DE mRNAs and DE lncRNAs were mainly involved in biosynthesis, innate immunity, and lipid metabolism. Moreover, we constructed a miRNA-mRNA-pathway network using bioinformatics, including 12 DE mRNAs, 120 DE miRNAs, and 11 pathways. Finally, the target genes of DE miRNAs were screened by bioinformatics, and we constructed immune-related lncRNA-miRNA-mRNA ceRNA networks. Then, the selected DE genes were validated by qRT-PCR, which were consistent with the results from RNA-Seq data.

Conclusions: This study provides the comprehensive analysis of the expression profiles of mRNAs, lncRNAs, and miRNAs during PEDV infection. We characterize the ceRNA networks which can provide new insights into the pathogenesis of PEDV.

Keywords: Functional enrichment; PEDV; Signaling pathway; ceRNA network; lncRNA.

Fig. 1

Overview of RNA sequencing. The distribution of mRNA (a), lncRNA (b), and miRNA (c) expressions at different times following viral infection were represented by histograms. (d) Exon number distribution of mRNAs and lncRNAs. Length distribution of mRNAs, lncRNAs (e), and miRNAs (f)

Fig. 2

Identification and characterization of mRNAs. (a, b) Volcano plots of DE mRNAs between the mock-infected and PEDV-infected groups. The red and green dots represent up-regulated and down-regulated genes, respectively. (c, d) Heat map showing DE mRNAs expression levels. Individual samples are represented by columns, while genes with significant expression differences between the two groups are represented by rows. (e) Venn diagram for the intersection of DE mRNAs between the two groups

Fig. 3

Functional enrichment analysis of DE mRNAs. (a, b) The top 30 significantly enriched GO terms. (c, d) The top 20 significantly enriched KEGG pathways

Fig. 4

Identification and characterization of miRNAs. (a) Portions of small RNA types in the clean reads. (b) Venn diagram for the intersection of DE miRNAs between the two groups. (c, d) Volcano plots of DE miRNAs between the mock-infected and PEDV-infected groups. Up-regulated and down-regulated genes are represented by red and green dots, respectively

Fig. 5

The miRNA-mRNA-pathway network. The ellipse, round rectangle, and diamond symbols represent mRNA, miRNA, and the pathway, respectively. Up-regulated and down-regulated genes are represented by red and green, respectively

Fig. 6

Identification and characterization of lncRNAs. (a) Screening of non-coding lncRNAs by using CPC, Pfam, PLEK, and CNCI. (b) Distribution of the four types of lncRNAs. (c) Chromosome distribution of lncRNAs. (d) Venn diagram for the intersection of DE lncRNAs between the two groups. (e, f) Volcano plots of DE lncRNAs between the mock-infected and PEDV-infected groups. Up-regulated and down-regulated genes are represented by red and green dots, respectively

Fig. 7

An overview of the lncRNA-miRNA-mRNA ceRNA network. The ellipse, triangle, and round rectangle symbols represent mRNA, lncRNA, and miRNA respectively

Fig. 8

Validation of RNA-seq results by qRT-PCR analysis. (a) The expression levels of DE mRNAs were validated by qRT-PCR. (b) The expression levels of DE lncRNAs were validated by qRT-PCR. (c) The expression levels of DE miRNAs were validated by qRT-PCR. Log₂ fold change was expressed as mean ± SD