Differential expression of plasma extracellular vesicle miRNAs as biomarkers for distinguishing psoriatic arthritis from psoriasis

To the Editor: Psoriatic arthritis (PsA) affects up to 30% of patients with psoriasis, contributing to a decreased quality of life. PsA can involve a variety of clinical manifestations, including peripheral and axial arthritis, enthesitis, dactylitis, and nail disease.[1] Early intervention is important for preventing loss of function and permanent disability; however, the diagnosis of PsA is often delayed by an average of five years.[1] Therefore, it is important to identify biomarkers that can detect PsA in patients with psoriasis. Circulating microRNAs (miRNAs) play an essential role in intercellular communication and can modulate gene expression. Several studies have supported the significant role of circulating miRNAs in psoriatic arthritis, while further studies are warranted to investigate their implications in the Chinese Han population. Herein, we performed next-generation sequencing of small RNAs from plasma extracellular vesicles (EVs) to investigate the differences in miRNAs profiles between psoriasis patients without psoriatic arthritis (PsO) and those with PsA. The study was approved by the Medical Ethics Committee of Huashan Hospital, Fudan University (approval number: MTX201501). Written informed consent was obtained from all participants before enrollment. The diagnosis of PsO and PsA was based on clinical and/or histopathological criteria and the Classification Criteria for Psoriatic Arthritis (CASPAR), respectively. The target genes of altered miRNAs were predicted using miRNA target-predicting tools and further validated using western blotting. A receiver operating characteristic (ROC) analysis was conducted to assess the diagnostic value of the selected miRNA candidates. The detailed methodology can be found in Supplementary Material, https://links.lww.com/CM9/C140. A total of 42 patients (25 with PsO and 17 with PsA) were recruited. The patients’ detailed demographic characteristics are listed in Supplementary Table 1, https://links.lww.com/CM9/C140. From these patients, 16 PsO cases and 8 PsA cases were randomly selected for exosome microRNA (miRNA) sequencing; of those, exosomes from one PsO case could not be isolated. Thus, 15 PsO cases and 8 PsA cases were included in the final analysis. We detected 597 miRNAs in the 23 samples after filtering noisy reads. Among these 597 miRNAs, 15 miRNAs exhibited significant differential expression between PsA and PsO samples based on a filter of P <0.05 and a fold change >1.2. Out of these 15 miRNAs, two (hsa-miR-219a-1-3p and hsa-miR-9-3p) showed incresed expression in PsA, while the remaining 13 (P <0.05), including hsa-miR-100-5p, hsa-miR-340-3p, hsa-miR-335-3p, hsa-miR-192-5p, hsa-miR-3591-3p, hsa-miR-183-5p, hsa-miR-218-5p, hsa-miR-4433b-3p, hsa-miR-184, hsa-miR-330-5p, hsa-miR-372-3p, hsa-miR-3177-3p, hsa-miR-191-3p showed decreased expression in PsA compared to PsO [Figure 1A, B and Supplementary Table 2, https://links.lww.com/CM9/C140].Figure 1: (A) Volcano plot of next-generation sequencing analysis detecting miRNAs from plasma EVs. The fold change of PsA/PsO was log2 transformed and plotted against the corresponding −log10 transformed P-values. Each spot represents an identified miRNA. The threshold P = 0.05 is indicated as a horizontal line. Two upregulated miRNAs are marked in red; 13 downregulated miRNAs are marked in blue. (B) Unsupervised hierarchical clustering and heatmap of the 15 significantly altered miRNAs in PsA vs. PsO. Distinct miRNA profiles were observed in some psoriatic arthritis-free patients, which exhibited a clustering pattern on the right side of the heatmap. (C) Protein expression levels of TNFAIP3 and ARF6. (D) The correlation between the expression of ARF6 and TNFAIP3 (n = 32). One-way ANOVA with Tukey’s multiple comparison test was used to compare the differences among the HC, PsO, and PsA groups. * P <0.05; † P <0.01. (E) ROC curve analysis was performed to demonstrate the diagnostic value of miR-218-5p (AUC = 0.758). The miRNA expression levels were quantified as RPM and then normalized with log2(RPM + 1). ANOVA: Analysis of variance; ARF6: ADP-ribosylation factor 6; AUC: Area under the curve; EVs: Extracellular vesicles; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; miRNA: MicroRNAs; NA: Invalid data; NS: No significant difference; N: Normal control; PsA: Psoriatic arthritis; PsO: Psoriasis patients without psoriatic arthritis; ROC: Receiver operating characteristic; RPM: Reads per million; TNFAIP3: Tumor necrosis factor alpha induced protein 3.To investigate the underlying mechanism of the 15 altered miRNAs in PsA, we used miRNA target-predicting tools (TargetScan, miRDB, and MIRDIP) to identify potential target genes. miR-218-5p was shown to potentially regulate ADP-ribosylation factor 6 (ARF6), one of the promising biomarkers identified through isobaric tags for relative and absolute quantification (iTRAQ) labeling and subsequent liquid chromatography tandem-mass spectrometry (LC–MS/MS) analysis.[2] As the initially collected extracellular samples were not enought for miRNA and western blotting analyses, we acquired additional samples based on the matching information (age, sex) for western blotting. Western blotting showed that the protein expression of ARF6 and tumor necrosis factor alpha induced protein 3 (TNFAIP3) was significantly higher in PsA than PsO (Figure 1C; P <0.01 and P <0.05, respectively). Furthermore, the expression of ARF6 was positively correlated with the expression of TNFAIP3 (P <0.01, r = 0.61, Figure 1D). In addition, to assess the diagnostic value of circulating miR-218-5p for PsA, we conducted ROC curve analysis. The area under the curve (AUC) of miR-218-5p was 0.758 (95% confidence interval [CI], 0.535–0.982; P = 0.023), with a sensitivity of 73.3% and a specificity of 87.5% [Figure 1E]. In this study, plasma EV miRNA isolation in combination with next-generation sequencing was adopted to identify differentially expressed EV miRNAs between psoriasis patients with and without arthritis. We detected a total of 597 miRNAs, among which 15 were significantly altered in PsA (two upregulated and 13 downregulated). However, these 15 miRNAs did not overlap with the profile reported by Pasquali et al,[3] who used a similar experimental approach based on RNA sequencing. This inconsistency may be attributed to differences in the severity of psoriasis and the ethnic backgrounds of the patients enrolled. In addition, miR-218-5p, which was downregulated in the plasma EVs of PsA patients and indicated as a potential diagnostic marker by ROC analysis, was identified as the most promising biomarker for PsA. miR-218-5p has been linked to both osteoarthritis (OA) and rheumatoid arthritis (RA) but exhibits different expression levels in each. Lu et al[4] observed significant upregulation of miR-218-5p in OA cartilage tissues, while Iwamoto et al[5] reported its marked reduction in RA fibroblast-like synovial cells after osteogenic induction. This discrepancy might be due to the distinct inflammatory environments in OA, which is less severe than in RA and PsA, where inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) differently modulate miR-218-5p levels.[5] Using miRNA target-predicting tools and proteomic data, we hypothesized that miR-218-5p may inhibit ARF6 by targeting its 3′ untranslated regions (UTR).[2] Western blotting confirmed significantly higher ARF6 levels in PsA patients. ARF6 is crucial in Toll-like receptor signaling and the nuclear factor-kappa B (NF-κB) pathway, and also contributes to IL-1β signaling pathway, thereby enhancing vascular stability and improving outcomes in inflammatory arthritis models.[6] Furthermore, we found TNFAIP3 significantly higher in PsA compared to PsO. While TNF-α inhibitors effectively alleviate arthritis symptoms, they are less effective for cutaneous psoriasis compared to IL-17 inhibitors, indicating immune-pathogenesis differences between PsA and PsO. Inhibition of miR-330-5p has been shown to increase TNF-α levels and TNFAIP3 expression in both in vivo and in vitro models.[7] Apart from the small sample size, this study also has a limitation that some PsO patients with a disease duration of less than 15 years might develop arthritis later, which could potentially affect the results. In conclusion, this pilot study identified a panel of candidate miRNAs in plasma EVs with the potential to discriminate PsA from PsO. Among the 15 significantly altered miRNAs, miR-218-5p represents the most promising biomarker and its potential target ARF6 was validated by Western blotting. The limitations of our study lie in its preliminary nature and small sample size. Expanding the study population would enhance the robustness of the results. Funding This research was funded by the National Key Research and Development Program of China (No. 2023YFC2508100), the National Natural Science Foundation of China (Nos. 81773322, 82173420, 81673054, 81673073, and 81974471), the Shanghai Municipal Commission of Health and Family Planning (No. 2023ZZ02018), the Shanghai Municipal Key Clinical Specialty (No. shslczdzk01002), the Natural Science Foundation of Shanghai (No. 20ZR1440100), the Key Project in Basic Research Advocated by Shanghai Science and Technology Commission (No. 13JC1402300), and the Clinical Research Plan of SHDC (Nos. SHDC2020CR6022, SHDC2020CR1014B, SHDC22022302, and SHDC2024CRI052). Conflicts of interest None.