Differential expression and correlation analysis of miRNA-mRNA profiles in swine testicular cells infected with porcine epidemic diarrhea virus

Abstract

The variant virulent porcine epidemic diarrhea virus (PEDV) strain (YN15) can cause severe porcine epidemic diarrhea (PED); however, the attenuated vaccine-like PEDV strain (YN144) can induce immunity in piglets. To investigate the differences in pathogenesis and epigenetic mechanisms between the two strains, differential expression and correlation analyses of the microRNA (miRNA) and mRNA in swine testicular (ST) cells infected with YN15, YN144, and mock were performed on three comparison groups (YN15 vs Control, YN144 vs Control, and YN15 vs YN144). The mRNA and miRNA expression profiles were obtained using next-generation sequencing (NGS), and the differentially expressed (DE) (p-value < 0.05) mRNA and miRNA were obtained using DESeq R package. mRNAs targeted by DE miRNAs were predicted using the miRanda algortithm. 8039, 8631 and 3310 DE mRNAs, and 36, 36, and 22 DE miRNAs were identified in the three comparison groups, respectively. 14,140, 15,367 and 3771 DE miRNA-mRNA (targeted by DE miRNAs) interaction pairs with negatively correlated expression patterns were identified, and interaction networks were constructed using Cytoscape. Six DE miRNAs and six DE mRNAs were randomly selected to verify the sequencing data by real-time relative quantitative reverse transcription polymerase chain reaction (qRT-PCR). Based on bioinformatics analysis, we discovered the differences were mostly involved in host immune responses and viral pathogenicity, including NF-κB signaling pathway and bacterial invasion of epithelial cells, etc. This is the first comprehensive comparison of DE miRNA-mRNA pairs in YN15 and YN144 infection in vitro, which could provide novel strategies for the prevention and control of PED.

Figure 1

The growth curves of the two PEDV strains (YN15 and YN144) in ST cells (0.001 multiplicity of infection (MOI)). (a) The growth curves of the two PEDV strains (YN15 and YN144) in ST cells. (b) Confirmation of YN15 and YN144 replication in ST cells at 12, 24, and 36 h pi by IIFA. Mock-infected ST cells at 36 h pi were set as control. Scale bar = 100 μm.

Figure 2

mRNAs expression profiles in PEDV-infected ST cells. (a) The bar graph shows the number of DE mRNAs in each one of the three comparison groups, red represents the amount of up-regulated DE mRNAs, green represents the count of down-regulated DE mRNAs (p-value < 0.05). (b) GO analysis of the DE mRNAs in PEDV infection ST. The three panels show the GO enrichment analysis of each of the top 30 significantly most DE mRNAs from the three comparison groups (YN15 vs Control, YN144 vs Control, and YN15 vs YN144), respectively. (c) KEGG analysis of the DE mRNAs in ST cells infected with PEDV. The three panels show the KEGG enrichment analysis of each of the top 20 significantly most DE mRNAs between YN15, YN144 and control groups, respectively. (d) Results of relative quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis (alongside RNA-seq analysis) of ATF3, CRAMP, NKTR, RND1, SMAGP and TNFSF10 in YN15- and YN144-infected ST cells, respectively. The relative expression of each mRNA was calculated using the 2^-ΔΔCt method. The data are presented as mean ± SEM from three independent experiments.

Figure 3

miRNA expression profiles in PEDV-infected ST cells. (a) Length of distribution of small RNA-seq reads. (b) Pie charts of small RNA-seq results showing the percentage of small RNA components in PEDV YN15-, YN144-, and mock-infected (Control) ST cells. (c) Volcano plots of up-regulated and down-regulated differentially expressed microRNAs (DE miRNAs) in three comparison groups (YN15 vs Control, YN144 vs Control, and YN15 vs YN144) (p-value < 0.05). (d) Venn diagram of the number of the commonly and uniquely DE miRNAs between the two comparison groups (YN15 vs Control and YN144 vs Control). Each number in a circle represents the number of the DE miRNAs found in each comparison group. The number in the overlapping area represents miRNAs commonly DE between the two comparison groups. The number in each non-overlapping area indicates the unique DE miRNAs in each comparison group. (p-value < 0.05). (e) Results of real-time relative qRT-PCR analysis (alongside RNA-seq analysis) of ssc-miR-9–1, ssc-miR-27a, ssc-miR-32, ssc-miR-192, ssc-miR-671-5p and ssc-miR-1306-5p fold-change in the two comparison groups (YN15 vs Control and YN144 vs Control). The relative expression of each miRNA was calculated using the 2^-ΔΔCt method. The data are presented as mean ± SEM from three independent experiments.

Figure 4

DE miRNAs and their predicted target mRNAs in PEDV-infected ST cells. (a) GO analysis of the predicted target genes of the DE miRNAs from the three comparison groups. (b) KEGG analysis of the predicted target genes of the DE miRNAs from the three comparison groups. Each panels indicates the top 20 significantly enriched pathways identifed by KEGG analysis of each of the three comparison groups (Left, YN15 vs Control; Middle, YN144 vs Control; Right, YN15 vs YN144).

Figure 5

Joint functional analysis of DE miRNA and inversely DE target mRNA in PEDV-infected ST cells. (a) Flow diagram of joint analysis of DE miRNA–mRNA pairs in the three comparison groups. (b) GO analysis of DE miRNA–mRNA pairs between YN15 infection and YN144 infection. The top panel shows the GO enrichment analysis of up-regulated miRNA-down-regulated mRNA pairs, and the lower panel shows the GO enrichment analysis of down-regulated miRNA-up-regulated mRNA pairs during YN15 infection compared with YN14 infection. (c) KEGG analysis of the genes of the DE miRNA–mRNA pairs between YN15 infection and YN144 infection. The left panel shows the KEGG pathways of the top 20 significantly up-regulated miRNA-down-regulated mRNA pairs, and the right panel shows the KEGG pathways of the top 20 significantly down-regulated miRNA-up-regulated mRNA pairs during YN15 infection compared with YN144 infection. (d) The DE miRNA–mRNA interaction networks in PEDV-infected ST cells. The left panel shows the significant interactions of the up-regulated miRNA-down-regulated mRNA pairs, and the right panel shows the significant interactions of the down-regulated miRNA-up-regulated mRNA pairs during YN144 infection in comparison with YN15 infection. p-value < 0.05.