miRNA Expression and Interaction with Genes Involved in Susceptibility to Pristane-Induced Arthritis

Abstract

Pristane-induced arthritis (PIA) in mice is an experimental model that resembles human rheumatoid arthritis, a chronic autoimmune disease that affects joints and is characterized by synovial inflammation and articular cartilage and bone destruction. AIRmax and AIRmin mouse lines differ in their susceptibility to PIA, and linkage analysis in this model mapped arthritis severity QTLs in chromosomes 5 and 8. miRNAs are a class of small RNA molecules that have been extensively studied in the development of arthritis. We analyzed miRNA and gene expression profiles in peritoneal cells of AIRmax and AIRmin lines, in order to evaluate the genetic architecture in this model. Susceptible AIRmax mice showed higher gene (2025 vs 1043) and miRNA (240 vs 59) modulation than resistant AIRmin mice at the onset of disease symptoms. miR-132-3p/212-3p, miR-106-5p, miR-27b-3p, and miR-25-3p were among the miRNAs with the highest expression in susceptible animals, showing a negative correlation with the expression of predicted target genes (Il10, Cd69, and Sp1r1). Our study showed that global gene and miRNA expression profiles in peritoneal cells of susceptible AIRmax and resistant AIRmin lines during pristane-induced arthritis are distinct, evidencing interesting targets for further validation.

Figure 1

Gene expression modulation in AIRmax and AIRmin mice after pristane injection. (a–d) Upregulated (red) and downregulated (green) genes 120 and 170 days after pristane injection in peritoneum. (a) AIRmax 120 days, (b) AIRmax 170 days, (c) AIRmin 120 days, (d) AIRmin 170 days, and (e) exclusive and common DEGs in AIRmax and AIRmin lines injected with pristane and compared to controls. Differentially expressed genes (DEGs) were detected using unpaired one-way ANOVA in the Transcriptome Analysis Console 3.0 (TAC) software, with FDR < 0.05 considering a minimum difference of 3 times for differentially expressed genes. N = 5 animals per group.

Figure 2

Up- and downregulated genes in AIRmax lines, located in the 1-LOD score confidence interval (CI) of Prtia2 (Chr 5) and Prtia3 QTL (chr 8) (>3-fold). (a) Chromosome 5: left—120 days; right—170 days. (b) Chromosome 8: left—120 days; right—170 days. Differentially expressed genes (DEGs) were detected using unpaired one-way ANOVA, with FDR < 0.05.

Figure 3

Upregulated (red) and downregulated (green) miRNAs after pristane injection in peritoneum. (a) AIRmax 120 days, (b) AIRmax 170 days, (c) AIRmin 120 days, (d) AIRmin 170 days, and (e) exclusives and commons miRDEGs in AIRmax and AIRmin lines injected with pristane and compared to controls. Differentially expressed miRNAs (miRDEGs) were detected using unpaired one-way ANOVA in the Transcriptome Analysis Console 3.0 (TAC) software, with FDR < 0.05 considering a minimum difference of 2 times for differentially expressed genes. N = 5 animals per group.

Figure 4

(a) Up- and downregulated miRNAs after pristane injection. Only the miRNAs predicted to interact with their targets in at least 3 different databases were shown. (a) miRNAs exclusively modulated in AIRmax mice and (b) miRNAs exclusively modulated in AIRmin mice. Differentially expressed miRNAs (miRDEGs) were detected using unpaired one-way ANOVA, with FDR < 0.05 and considering a minimum 2-fold difference for miRDEGs.

Figure 5

mRNA-miRNA interaction network. (a) miRNAs upregulated in AIRmax mice and their interaction with predicted target genes; (b) miRNAs upregulated in AIRmin mice and their interaction with predicted target genes. Red and blue = upregulated genes; green and purple = downregulated genes. The interaction network was built with Cytoscape 3.4.0.

Figure 6

Relative gene expression determined by qRT-PCR in peritoneal cells of AIRmax and AIRmin mice 120 and 170 days after pristane injection (n = 5 mice/group). Data expressed as mean ± standard error of two independent experiments; statistical analysis by ANOVA followed by Tukey's post hoc tests. ^∗p < 0.05 between AIRmax and AIRmin; ^#p < 0.05 between control and pristane-injected animals.