Roles and Molecular Mechanisms of Serum Exosomal miRNA-223 and miRNA-132 in Juvenile Idiopathic Arthritis

Abstract

Introduction: The pathogenesis of juvenile idiopathic arthritis (JIA) has not yet been clarified and is closely related to persistent overactivation of the JAK/STAT signaling pathway. MicroRNA (miRNA)-223 (miR-223) and miRNA-132 (miR-132) might be involved in the development of JIA. However, the mechanism underlying the pathogenesis of JIA is unclear. In this study, we investigated the roles and molecular mechanisms of serum exosomal miR-223 and miR-132 in JIA. Methods: Patients with systemic JIA were selected as the systemic group (sJIA), patients with polyarticular JIA and oligoarticular JIA were selected as the articular group (aJIA), and healthy children who underwent physical examinations at the same time were selected as the normal control group (NC). Exosomes were extracted from the serum, and the purified exosomes were subjected to electron microscopy to observe their particle morphology. The particle size distribution and concentration of the exosomes were detected by an N30E particle size analyzer. The expression levels of miR-223 and miR-132 in exosomes were quantitatively detected by the SYBR green method. The protein levels of STAT3 and SOCS3 were detected by Western blot.

Results: The expression level of miR-223 in serum exosomes of the sJIA group was significantly higher than in the aJIA group and the NC group (4.04±0.34 vs. 1.52±0.30, 0.88±0.17), and the difference was statistically significant (P<0.001). However, the expression level of miR-132 in serum exosomes of the sJIA group was significantly lower than in the aJIA group and the NC group (0.09±0.01 vs. 0.17±0.02, 0.94±0.08), and the difference was statistically significant (P<0.001). The expression levels of serum interleukin (IL)-6, IL-8, and IL-10 in the sJIA group were significantly higher than those in the aJIA group and the NC group; the difference was statistically significant (P<0.001). The IL-17 expression in the sJIA group and aJIA group was significantly greater than the expression levels in the NC groups (P<0.001). The expression level of miR-223 in exosomes was positively correlated with the expression levels of the clinical inflammatory factors IL-6, IL-8, IL-10, and IL-17 (P<0.001). However, the expression level of miR-132 in exosomes was negatively correlated with the expression levels of clinical inflammatory factors of IL-6, IL-8, IL-10, and IL-17 (P<0.001). The expression level of SOCS3 protein in both the sJIA and aJIA groups was significantly higher than in the NC group (0.25±0.05 and 0.21±0.03 vs. 0.10±0.02, respectively), and the difference was statistically significant (P<0.05). Regrettably, there was no significant difference yet in the expression level of STAT3 in the three groups (P>0.05).

Conclusion: miR-223 and miR-132 may be two potential new markers of JIA, providing new ideas for diagnostic tests and therapeutic interventions. But larger studies are needed to confirm these findings and assess their generalizability across diverse populations. miR-223 may promote inflammation in JIA patients by enhancing the JAK/STAT signaling pathway, while miR-132 may reduce inflammation in JIA patients by inhibiting the JAK/STAT signaling pathway. Its inhibitory effect may be closely related to SOCS3 through the JAK/STAT signaling pathway.

Keywords: exosomes; juvenile idiopathic arthritis; mirna-132; mirna-223; socs3; stat3.

Figure 1. Electron microscopy images of exosomes

Exosomes were observed under electron microscopy. The extracted exosomes were membranous vesicles with diameters of approximately 30-150 nm. The magnification of all pictures was x40.0k, and the horizontal length of all pictures corresponded to the actual size of 100nm. A: electron microscopy images of exosomes in the sJIA group; B: electron microscopy images of exosomes in the aJIA group; C: electron microscopy images of exosomes in the NC group. sJIA: systemic juvenile idiopathic arthritis; aJIA: articular juvenile idiopathic arthritis; NC: normal control

Figure 2. Particle size distributions and concentrations of exosomes

The particle size and concentration of the exosomes were detected by an N30E particle size analyzer, and the exosomes were diluted from 10 μL to 30 μL and tested with standard products after the instrument performance was qualified. The horizontal coordinate of all the pictures in the figure represents the particle size of exosomes, and the vertical coordinate represents the concentration of exosomes. A: in the sJIA group, particle size distributions are respectively 87.3 nm, 87.7 nm, and 86.7 nm; concentrations of exosomes are respectively 4.07 × 10⁹ particles/mL, 2.17 × 10⁹ particles/mL, and 2.16 × 10⁹ particles/mL; B: in the aJIA group, particle size distributions are respectively 91.9 nm, 87.7 nm, and 86.5 nm; concentrations of exosomes are respectively 3.88 × 10⁹ particles/mL, 2.63 × 10⁹ particles/mL, and 2.09 × 10⁹ particles/mL; C: in the NC group, particle size distributions are respectively 88.6 nm, 90.4 nm, and 86.7 nm; concentrations of exosomes are respectively 2.63 × 10⁹ particles/mL, 4.48 × 10⁹ particles/mL, and 2.25 × 10⁹ particles/mL. sJIA: systemic juvenile idiopathic arthritis; aJIA: articular juvenile idiopathic arthritis; NC: normal control

Figure 3. Particle position distribution of exosomes

Particle position distributions were presented during particle size distributions and concentration of exosome detection by the N30E particle size analyzer. The horizontal coordinate represents lateral scattered light. All particles are in the negative fluorescence group because they are not fluorescently stained. A: particle position distribution of exosomes in the sJIA group; B: particle position distribution of exosomes in the aJIA group; C: particle position distribution of exosomes in the NC group. sJIA: systemic juvenile idiopathic arthritis; aJIA: articular juvenile idiopathic arthritis; NC: normal control

Figure 4. Specific markers of exosomes

Exosome-specific markers such as CD9, CD63, and CD81, and negative indicators such as calnexin, were detected by Western blot. The first sample in the sJIA group (sJIA-1); the second sample in the sJIA group (sJIA-2); the third sample in the sJIA group (sJIA-3); the first sample in the aJIA group (aJIA-1); the second sample in the aJIA group (aJIA-2); the third sample in the aJIA group (aJIA-3); the first sample in the NC group (NC-1); the second sample in the NC group (NC-2); and the third sample in the NC group (NC-3). sJIA: systemic juvenile idiopathic arthritis; aJIA: articular juvenile idiopathic arthritis; NC: normal control

Figure 5. Expression levels of miR-223 in exosomes

sJIA: systemic juvenile idiopathic arthritis; aJIA: articular juvenile idiopathic arthritis; NC: normal control **P<0.001

Figure 6. Expression levels of miR-132 in exosomes

sJIA: systemic juvenile idiopathic arthritis; aJIA: articular juvenile idiopathic arthritis; NC: normal control **P<0.001, *P<0.05

Figure 7. Correlations between miR-223 and IL-6, IL-8, IL-10, and IL-17

Spearman correlation analysis was used to analyze correlations between variables. The values of miR-223 and interleukin were sorted respectively and assigned ranks, with each point representing a rank pair of an observed value. A: the expression level of miR-223 in exosomes was positively correlated with the expression levels of IL-6, r value was 0.884, P<0.001; B: the expression level of miR-223 in exosomes was positively correlated with the expression levels of IL-8, r value was 0.899, P<0.001; C: the expression level of miR-223 in exosomes was positively correlated with the expression levels of IL-10, r value was 0.869, P<0.001; D: the expression level of miR-223 in exosomes was positively correlated with the expression levels of IL-17, r value was 0.885, P<0.001. miRNA-223: miR-223

Figure 8. Correlations between miR-132 and IL-6, IL-8, IL-10, and IL-17

Spearman correlation analysis was used to analyze correlations between variables. The values of miR-132 and interleukin were sorted respectively and assigned ranks, with each point representing a rank pair of an observed value. A: the expression level of miR-132 in exosomes was negatively correlated with the expression levels of IL-6; the r value was -0.857, P<0.001; B: the expression level of miR-132 in exosomes was negatively correlated with the expression levels of IL-8, r value was -0.860, P<0.001; C: the expression level of miR-132 in exosomes was negatively correlated with the expression levels of IL-10, r value was -0.877, P<0.001; D: the expression level of miR-132 in exosomes was negatively correlated with the expression levels of IL-17, r value was -0.912, P<0.001. miRNA-132: miR-132

Figure 9. SOCS3 protein relative expression levels

The relative expression of SOCS3 protein is represented by the ratio of gray values, which is the ratio of the gray value of SOCS3 to the gray value of CD63. sJIA: systemic juvenile idiopathic arthritis; aJIA: articular juvenile idiopathic arthritis; NC: normal control *P<0.05.

Figure 10. Western blot image of SOCS3

Since CD63 was found to be stably expressed in exosomes in this study, CD63 was chosen as the loading control. The first sample in the sJIA group(sJIA-1); the second sample in the sJIA group(sJIA-2); the third sample in the sJIA group(sJIA-3); the first sample in the aJIA group(aJIA-1); the second sample in the aJIA group(aJIA-2); the third sample in the aJIA group(aJIA-3); the first sample in the NC group(NC-1); the second sample in the NC group(NC-2); the third sample in the NC group(NC-3). sJIA: systemic juvenile idiopathic arthritis; aJIA: articular juvenile idiopathic arthritis; NC: normal control

Figure 11. Melt curve of PCR

A: the melt curve of miR-223; B: the melt curve of miR-132; C: the melt curve of U6 PCR: polymerase chain reaction