MiR‐221‐5p is involved in the regulation of inflammatory responses in acute gouty arthritis by targeting IL‐1β

Gout is caused by the accumulation of deposited monosodium urate (MSU) crystals in the joints. Recent studies have shown that interleukin‐1β (IL‐1β) is a key inflammatory mediator of acute gouty arthritis (AGA), and its level is regulated by microRNAs (miRNAs). The purpose of this study was to study the role of miR‐221‐5p in the pathogenesis of AGA.


| INTRODUC TI ON
Gout attacks can be caused by hunger, trauma, surgery, ingestion of high-purine foods, excessive alcohol consumption, and medications that affect urate concentrations. 1 It is divided into 3 clinical stages: acute gouty arthritis (AGA), intercritical gout, and chronic gout. AGA is an acute inflammation caused by the precipitation of urate crystals in joints. 2 It is one of the most common types of auto-inflammatory arthritis, characterized by a sudden onset and significant pain that resolves spontaneously within a week. 3,4 Interleukin-1β (IL-1β) is a central cytokine in the initiation of the acute inflammatory response, which plays a key role in the pathogenesis of gout, 5 especially its role in the pathology of AGA.
Tongfengshu capsule, a Chinese patent medicine, is composed of radix et rhizoma rhei palmati, semen plantaginis, rhizoma alismatis, radix achyranthis bidentatae and radix stephaniae tetrandrae; it has been used for the treatment of AGA with the involvement of IL-1β and tumor necrosis factor (TNF)-α regulation, suggesting the potential role of IL-1β and TNF-α in the progression of AGA. 6 However, the mechanism of action of IL-1β in AGA is unclear.
MicroRNAs (miRNAs) are evolutionary conserved non-coding small RNA molecules that act as negative post-transcriptional gene regulators. 7 Since a single miRNA molecule can target 100s of messenger RNAs (mRNAs), the abnormal expression of miRNA is related to the occurrence of many diseases. 8 Recent research suggests that miRNAs may be involved in the development of arthritis. 9,10 For example, the expression level of miR-155 in patients with gout arthritis is significantly higher than that in healthy individuals, and the overexpressed miR-155 can promote the production of monosodium urate (MSU)-induced inflammatory cytokines by reducing SHIP-1 levels. 9 Considering the important role of miRNAs in inflammatory diseases, especially AGA, more studies on miRNAs are urgently needed. 11 MiR-221-5p has been widely reported to be aberrantly expressed in various metabolic diseases and involved in the diseases progression. For example, miR-221-5p is identified to be involved in the progression of diabetes. 12 Another study also confirmed that miR-221-5p participates in the development of osteoarthritis. In addition, after constructing a miRNA gene pathway network, miR-221-5p is identified to be enriched in various metabolic pathways. 13 Up to now, the molecular mechanism of miR-221-5p in AGA has been unclear.
In summary, miR-221-5p is critical for human cell inflammation and AGA. However, the functional role of miR-221-5p in AGA is not yet clear. Therefore, the purpose of this study was to study the role of miR-221-5p in the pathogenesis of AGA.

| Cell culture and transfection
The human monocyte THP-1 cell line was cultured in Roswell Park Memorial Institute 1640 medium (Life Technologies) and cultured in a 37°C, 5% CO 2 constant temperature incubator. THP-1 cells at 1.5 × 10 6 /mL were incubated in 96-well plates. The THP-1 cells were stimulated for 3 hours with 0.5 μmol/L phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) the day before stimulation. Then, the cells were stimulated with 250 μg/mL MSU crystals (Invitrogen) for 24 hours, causing inflammation, and presenting a variety of features of AGA. In order to regulate the expression level of miR-221-5p, cells were transfected with miR-221-5p mimic, miR-221-5p inhibitor, or their negative control (miR-NC), which was produced by Ribo Bio.
Liposome 2000 (Invitrogen) was used for transfection according to the manufacturer's instructions.

| Total RNA extraction and quantitative realtime polymerase chain reaction assay
Total RNA was extracted using TRIZOL reagent (Invitrogen). The miRNA bulge loop was reverse transcribed using the PrimeScript RT Reagent Kit (TaKaRa). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect gene expression using SYBR premix ExTaq M. II commercial kit (Takara) and the Applied Biosystems 7900 Real Time PCR System (Applied Biosystems). PCR parameters were as follows: 95°C for 3 minutes, followed by 40 cycles of 95°C for 10 seconds, 60°C for 20 seconds, and 72°C for 1 second. The relative gene expression was normalized to that of the internal control U6 according to the comparative delta CT (2 −ΔΔCt ) method.

| Evaluation of inflammatory cytokines
The concentrations of IL-1β, IL-8, and TNF-α proteins in THP-1 cell culture supernatant were determined using an enzyme-linked immunosorbent assay kit (UK Abeam) in accordance with the manufacturer's instructions. Each sample was analyzed 3 times.

| Luciferase reporter assay
The putative binding sites of miRNAs in the 3′-UTR (untranslated

| Statistical analysis
In our study, all statistical analyses were performed with Prism statistical software. The data were expressed as mean and standard deviation (SD). The differences between the 2 groups were compared by Student's t test or one-way analysis of variance. Receiver operating characteristic (ROC) curves were used to determine the specificity and sensitivity of the diagnostic value of miR-221-5p for AGA. Correlation analysis was performed using Pearson correlation coefficients. Table 1 reports the main characteristics of the study population and laboratory results. A total of 194 individuals were included, the age range was 27-65 years. Among them, 94 subjects were healthy controls (44 males/ 50 females), and 100 AGA patients (48 males/ 52 females). There was no difference in age, gender distribution, BMI, ESR, and lymphocytes count between the groups (P > .05). There were significant differences in SUA, leukocyte count, neutrophils counts (P < .001). The VAS score in patients was 6.16 ± 2.36.

| The expression level of miR-221-5p and its correlation with VAS in AGA
We first studied the serum levels of miR-221-5p in healthy controls and AGA groups. The results of the study are shown in Figure 1A. The expression level of miR-221-5p in the serum of patients with AGA (0.61 ± 0.23) was significantly lower than that of the healthy control group (1.00 ± 0.25) (P < .05). The results indicated that miR-221-5p may be a key biomolecule for AGA and play an important biological role in its disease progression. In addition, in order to further explore the relationship between miR-221-5p and AGA, we also made a correlation between VAS and miR-221-5p. As shown in Figure 1B, serum miR-221-5p was negatively correlated to VAS (r = −.7671, P < .0001) in AGA patients. We concluded that miR-221-5p might be associated with the occurrence and severity of AGA.

| Diagnostic value of miR-221-5p in patients with AGA
Receiver operating curve curves were drawn based on the expression level of miR-221-5p in AGA patients and the control group to evaluate the diagnostic value of miR-221-5p in AGA patients. As shown in

| Effect of miR-221-5p on inflammatory responses in THP-1 cells
As shown in Figure 3A, the transfection of miR-221-5p mimic/inhibitor had a significant effect on the expression of miR-221-5p (P < .001), transfection with miR-221-5p mimic significantly increased the expression of miR-221-5p, while transfection with miR-221-5p inhibitor had the opposite effect. As shown in Figure 3B

| MiR-221-5p directly targets IL-1β in THP-1 cell
miRNAs were known to function by inhibiting the expression of their target genes. According to the Target scan analysis results, the binding sites of miR-221-5p in IL-1β are shown in Figure 4A(a). Luciferase reporter assay results showed that miR-221-5p mimic significantly inhibited luciferase activity of IL-1β WT 3′-UTR ( Figure 4A(b), P < .001), while miR-221-5p inhibitor significantly increased luciferase activity. In addition, the luciferin activity of the mutant group was not affected by transfection with miR-221-5p mimic or miR-221-5p inhibitor. As shown in Figure 4B, the correlation between miR-221-5p expression and target gene IL-1β in AGA patients was also analyzed. The results showed that the expression of miR-221-5p in AGA patients was negatively correlated with IL-1β level (r = −.6762, P < .0001).

| D ISCUSS I ON
Gout is a common metabolic disease and AGA is one of the important complications. 14 AGA is a group of clinical syndromes caused by MSU crystal deposition on bone, joints, and subcutaneous tissues, which is the most common initial symptom of gout. 15 It is worth noting that while hyperuricemia has been classically associated with gouty arthritis, asymptomatic hyperuricemia is frequently found in metabolic syndrome, diabetes mellitus, chronic kidney disease, and osteoarthritis. 16,17 Osteoarthritis is the most common form of arthritis overall, and gout and osteoarthritis frequently coexist in the same patient. 18 Further, one study has confirmed the low expression of miR-221-5p in osteoarthritis. 19 Therefore, its role in AGA attracts our interest. In our study, it was proposed that the expression of miR-221-5p in AGA patients was significantly lower than in healthy subjects, which was consistent with the results reported in osteoarthritis, indicating the association of miR-221-5p with AGA. 19 Serum miRNA is stable in stored samples, 20 and it is more practical as a biomarker and easier to isolate than specific cell types of miRNAs, especially in AGA. Recent studies have suggested that miR-NAs including miR-155 and miR-146a may be involved in the development of AGA in humans. For instance, a study has confirmed that miR-155 was upregulated in synovial fluid mononuclear cells (SFMCs) from patients with AGA. 21 Another study confirmed that miR146a plays a negative regulatory role in AGA in humans. 22  arthritis. 26 It is demonstrated that IL-1β can be involved in the F I G U R E 1 The expression level of miR-221-5p and its correlation with visual analog scale (VAS) in acute gouty arthritis (AGA) patients. A, The expression level of miR-221-5p in the serum of patients with AGA was decreased compared with the control group (***P < .001). B, a negative correlation between serum miR-221-5p and VAS was found (r = −.7671, P < .0001)

F I G U R E 2
The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of miR-221-5p in acute gouty arthritis (AGA). The area under the curve (AUC) was 0.884, sensitivity was 82.0%, specificity was 80.9% treatment of AGA patients as a target gene. The identification of miRNAs possibly targeting IL-1β in gouty arthritis is another major goal for the future. 27 Therefore, targeting miRNAs may be an effective option for treating auto-inflammatory disease in the future. 28 In our results, the luciferase reporter assay analysis showed that IL-1β may be involved in the AGA process as a target gene of miR-221-5p. All evidence supported our conclusion that miR-221-5p might be involved in the development of AGA and inhibit inflammatory responses via targeting IL-1β. However, only part of the role of miR-221-5p in THP-1 cells is studied in this research. In the future, we can further verify its cell function and explore the specific mechanism of the role of miR-221-5p in the development of AGA patients. In addition, the study population should be expanded to better verify the current study effect.

| CON CLUS ION
It is commonly known that auto-inflammation plays a pivotal role in the pathology of AGA. However, the exact etiology and pathogenesis are poorly understood. In general, we found that miR-221-5p was confirmed. Therefore, it has potential as a therapeutic or biomarker for AGA. miR-221-5p may participate in the development of AGA patients by acting on target gene IL-1β to inhibit the release of inflammatory factors in THP-1 cells.

CO N FLI C T O F I NTE R E S T
The authors have declared that no conflict of interest exists.