Transcriptome Analysis of miRNA and mRNA in Porcine Skeletal Muscle following Glaesserella parasuis Challenge

Abstract

Glaesserella parasuis (G. parasuis) causes systemic infection in pigs, but its effects on skeletal muscle and underlying mechanisms are poorly understood. We investigated G. parasuis infection in colostrum-deprived piglets, observing decreased daily weight gain and upregulation of inflammatory factors in skeletal muscle. Muscle fiber area and diameter were significantly reduced in the treated group (n = 3) compared to the control group (n = 3), accompanied by increased expression of FOXO1, FBXO32, TRIM63, CTSL, and BNIP3. Based on mRNA and microRNA (miRNA) sequencing, we identified 1642 differentially expressed (DE) mRNAs and 19 known DE miRNAs in skeletal muscle tissues between the two groups. We predicted target genes with opposite expression patterns to the 19 miRNAs and found significant enrichment and activation of the FoxO signaling pathway. We found that the upregulated core effectors FOXO1 and FOXO4 were targeted by downregulated ssc-miR-486, ssc-miR-370, ssc-miR-615, and ssc-miR-224. Further investigation showed that their downstream upregulated genes involved in protein degradation were also targeted by the downregulated ssc-miR-370, ssc-miR-615, ssc-miR-194a-5p, and ssc-miR-194b-5p. These findings suggest that G. parasuis infection causes skeletal muscle atrophy in piglets through accelerated protein degradation mediated by the "miRNAs-FOXO1/4" axis, while further research is necessary to validate the regulatory relationships. Our results provide new insights into the understanding of systemic inflammation growth mechanisms caused by G. parasuis and the role of miRNAs in bacterial infection pathogenesis.

Keywords: G. parasuis; miRNAs; pig; skeletal muscle atrophy; transcriptome.

Figure 1

Assessment of G. parasuis infection in colostrum-deprived (CD) piglets. (A) Schematic diagram illustrating the construction of G. parasuis-infected CD piglet model. (B) Changes in average daily gain of piglets from birth to slaughter at three different stages in the treated and control groups. The stages include 0–20 days (birth to pre-weaning), 21–30 days (post-weaning to pre-infection), and 31–32 days (post-infection to slaughter). The number of piglets per group is indicated as “n = 3”. (C) Expression levels of inflammatory cytokines in skeletal muscle determined by Q-PCR. The fold change in expression is relative to the control group. * p < 0.05; ** p < 0.01.

Figure 2

G. parasuis infection leading to skeletal muscle atrophy. (A) Representative H&E staining images of skeletal muscle isolated from control or treated piglets. Magnification: 400×; scale bars: 25 μm. (B) Average diameter and cross-sectional area of myofiber measured using ImageJ 1.46r software. Data are presented as mean ± SD. Statistical significance was determined using Student’s t-test. (C) Expression levels of FOXO1, FBXO32, TRIM63, CTSL, and BNIP3 in skeletal muscle via Q-PCR. The expression fold change is relative to the control group. * p < 0.05; ** p < 0.01.

Figure 3

Analysis of DE mRNAs between the treated and control groups. (A) Volcano plot of DE mRNAs in skeletal muscle comparing the treated and control groups, determined by RNA-seq data. (B) Heat map of DE mRNAs generated by biclustering analysis. The values displayed represent the FPKM of the DE genes. (C) Verification of differentially expressed mRNAs using Q-PCR. (D) Pearson’s correlation analysis between Q-PCR and RNA-seq data. The x-axis and y-axis represent the log₂(FC) data of the 8 genes between the groups, as determined by Q-PCR and RNA-seq methodologies, respectively. * p < 0.05; ** p < 0.01.

Figure 4

Functional annotation and enrichment analysis of DE mRNAs between the two groups. (A) Top 20 significant GO terms of DE mRNAs. The y-axis represents the GO enrichment in biological process, and the x-axis represents the enrichment factor. (B) Top 20 significantly enriched KEGG pathways of DE mRNAs. The y-axis represents the pathway, and the x-axis represents the enrichment factor.

Figure 5

Differential miRNA expression analysis between the treated and control groups by miRNA-seq. (A) Volcano plot illustrating the differential expression of miRNAs in skeletal muscle between treated and control. (B) Biclustering analysis of the 19 differentially expressed (DE) miRNA expression profiles (CPM) in the two groups. (C) Expression levels of miRNAs as quantified by Q-PCR, represented as fold change relative to the control group. (D) Comparison of miRNA expression patterns evaluated by miRNA-seq and Q-PCR, presented as log2(FC). * p < 0.05; ** p < 0.01.

Figure 6

Function and pathway enrichment analysis of DE miRNA target mRNAs in skeletal muscle between the control and treated groups. (A) Volcano plot showing all target mRNAs of the 8 upregulated miRNAs. The red shading represents the final set of miRNA targets that exhibited an expression pattern opposite to that of the corresponding upregulated miRNAs. (B) All target mRNAs of the 11 downregulated miRNAs displayed by volcano plot. The red shading indicates the final set of miRNA targets of the downregulated miRNAs. (C) GO annotation results of DE miRNA targets. The y-axis represents the top 20 significantly enriched biological processes. (D) Top 20 significant pathways identified through the KEGG enrichment analysis of DE miRNA targets.

Figure 7

GSEA analysis of the representative significantly enriched gene set. (A) GSEA results of FoxO signaling pathway, comparing the treated group to the control group. (B) The core enrichment genes of FoxO signaling pathway identified through GSEA. The p-value was determined by nominal p-value; the q-value was determined by FDR. ES, enrichment score; NES, normalized enrichment score.

Figure 8

Construction of the potential miRNA-target negative correlation regulatory network involved in the FoxO signaling pathway following G. parasuis infection.