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, 18 (3), 3219-3228

MicroRNA‑143‑3p Contributes to the Regulation of Pain Responses in Collagen‑induced Arthritis

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MicroRNA‑143‑3p Contributes to the Regulation of Pain Responses in Collagen‑induced Arthritis

Ling-Ling Zhou et al. Mol Med Rep.

Abstract

Patients with rheumatoid arthritis (RA) suffer from pain, which is associated with inflammation, peripheral and central pain processing, and joint structure damage. The aim of the present study was to investigate a key microRNA (miR) and its target genes that are involved in the pain responses of RA, and to clarify the mechanism of pain regulation. Collagen‑induced arthritis (CIA) was induced in DBA/1 and C57BL/6 mice. The paw swelling, mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), and expression levels of tumor necrosis factor (TNF)‑α and prostaglandin (PG)E2 in the sera were investigated. Decreased MWT and TWL, and increased TNF‑α and PGE2, in the CIA model group were observed in DBA/1 and C57BL/6 mice. DBA/1 mice exhibited greater hyperalgesia and higher levels of inflammatory mediators. miR‑143‑3p expression in the blood and the dorsal root ganglion (DRG) were detected, and low miR‑143‑3p expression was demonstrated in the blood and DRG tissue of CIA mice. The target genes of miR‑143 were predicted and analyzed. A total of 1,305 genes were predicted and 55 pain‑associated genes were obtained. Prostaglandin‑endoperoxide synthase 2 (Ptgs2), MAS related GPR family member E (Mrgpre), prostaglandin D2 receptor and Tnf were selected as target genes of miR‑143. DRG cells were cultured and transfected with miR‑143‑3p inhibitor or mimic. The expression of Mrgpre, Ptgs2 and Tnf was significantly inhibited following miR‑143‑3p mimic transfection, while the expression of Mrgpre, Ptgs2 and Tnf was increased following inhibitor transfection. Additionally, the expression of pain‑associated genes in the DRG of mice was investigated and the expression of Ptgs2, Mrgpre and Tnf in the DRG of CIA mice was also significantly upregulated. These results revealed that CIA mice exhibited marked hyperalgesia and high levels of inflammatory pain mediators. Low expression of miR‑143‑3p negatively regulated the pain‑associated target genes, including Mrgpre, Ptgs2 and Tnf, thereby affecting chronic inflammatory pain and neuropathic pain in RA.

Figures

Figure 1.
Figure 1.
Inflammatory reactions and pain responses in DBA/1 or C57BL/6 mice with CIA. Paw thickness was measured between d21 and d49. TWL and MWT were detected between d20 and d48. The levels of TNF-α and PGE2 in the sera were determined by ELISA on d49. (A) Footpad diameter of DBA/1 mice. (B) Footpad diameter of C57BL/6 mice. (C) TWL of DBA/1 mice. (D) TWL of C57BL/6 mice. (E) MWT of DBA/1 mice. (F) MWT of C57BL/6 mice. (G) TNF-α levels in the sera. (H) PGE2 levels in the sera. *P<0.05, **P<0.01 vs. respective control. TWL, thermal withdrawal latency; MWT, mechanical withdrawal threshold; TNF, tumor necrosis factor; PGE2, prostaglandin E2; CIA, collagen-induced arthritis.
Figure 2.
Figure 2.
miR-143-3p expression measured by reverse transcription quantitative polymerase chain reaction in DBA/1 mice. miR-143-3p expression in (A) the peripheral blood and (B) DRG of mice.*P<0.05 vs. control. CIA, collagen-induced arthritis; miR, microRNA; DRG, dorsal root ganglion.
Figure 3.
Figure 3.
miR-143 target gene regulatory network. The red square represents the miR, the blue circles represent the target genes and the lines with grey arrowheads indicate their interactions. miR, microRNA.
Figure 4.
Figure 4.
GO and KEGG analysis of miR-143 target pain-associated genes. (A) GO analysis of cell components. (B) GO analysis of molecular functions. (C) KEGG analysis of miR-143. GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; miR, microRNA; VEGF, vascular endothelial growth factor; MAPK, mitogen-activated protein kinase; ATP, adenosine triphosphate.
Figure 5.
Figure 5.
Effects of miR-143 on pain-associated target genes. Mouse DRG cells were transfected with Lipofectamine RNAiMAX transfection reagent and the miR-143-3p mimic NC-carboxyfluorescein. (A) Bright field (magnification, ×100). (B) Fluorescence microscopy (magnification, ×100). RNA was extracted from the DRG cells 48 h following transfection with an miR-143-3p inhibitor or mimic. (C) miR-143-3p expression following transfection with the mimic. (D) miR-143-3p expression following transfection with the inhibitor. (E) Ptgdr expression, (F) Mrgpre expression, (G) Ptgs2 expression and (H) Tnf expression. *P<0.05, **P<0.01 vs. NC group. miR, microRNA; TNF, tumor necrosis factor; DRG, dorsal root ganglion; Mrgpre, MAS related GPR family member E; Ptgdr, prostaglandin D2 receptor; Ptgs2, prostaglandin-endoperoxide synthase 2; NC, negative control.
Figure 6.
Figure 6.
Expression of pain-associated genes in the dorsal root ganglia of CIA mice. (A) Ptgs2 expression, (B) Mrgpre expression and (C) Tnf expression. *P<0.05, **P<0.01 vs. control. Mrgpre, MAS related GPR family member E; Ptgs2, prostaglandin-endoperoxide synthase 2; TNF, tumor necrosis factor; CIA, collagen-induced arthritis.

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