Introduction: Rheumatoid arthritis (RA), a chronic autoimmune disorder, is currently a severe health threat. Previous studies have documented the altered expression of various miRNAs in RA patients. This study determined the expression of miR-124a in RA patients and estimated its diagnostic value for RA. Methods: A total of 80 RA patients were enrolled as the study subjects, and 36 patients with osteoarthritis were included, with another 36 healthy people as the controls. miR-124a expression levels in peripheral blood plasma, peripheral blood mononuclear cells (PBMCs), and synovial fluid were measured using reverse transcription quantitative polymerase chain reaction, followed by Pearson correlation analysis. Additionally, the association between miR-124a and major clinical indicators was assessed, such as rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), and disease activity score of 28 joints (DAS28). The diagnostic efficacy of miR-124a expression in plasma, PBMCs, and synovial fluid for RA was evaluated by the receiver operating characteristic curve, and the difference in the area under the curve (AUC) was analyzed. Results: miR-124a was downregulated in RA patients, and the expression levels of miR-124a in plasma, PBMCs, and synovial fluid showed a certain degree of positive correlation. miR-124a was inversely linked with RF, ESR, and DAS28. For the diagnosis of RA patients, the AUC of plasma miR-124a was 0.899 and the cut-off value was 0.800, with 68.75% sensitivity and 94.44% specificity; the AUC of miR-124a in PBMCs was 0.937 and the cut-off value was 0.805, with 82.50% sensitivity and 91.67% specificity; the AUC of miR-124a in plasma combined with PBMCs was 0.961, with a higher diagnostic value than independent plasma or PBMCs; the AUC of miR-124a in synovial fluid was 0.929 and the cut-off value was 0.835, with 80.00% sensitivity and 88.89% specificity. Conclusion: miR-124a expression is downregulated in the plasma, PBMCs, and synovial fluid of RA patients and has a high diagnostic value for RA.
Rheumatoid arthritis (RA), an autoimmune disorder, is construed as the slow, progressive destruction of the joints, which is attributed to autoantibodies that target multiple organs, thereby causing auto-destruction . RA significantly afflicts 0.3–1% of the world population, and it is more prevalent in North America and northern Europe, with predominance among females and the elderly . The pathogenesis of RA is related to the interaction of epigenetic, genetic, metabolic, immune, environmental, and microbial factors . The main risk factors for RA include age, female gender, silica exposure, tobacco use, and obesity . As a multifactorial disease, RA often leads to joint injury as a result of the chronic inflammation of synovial joints, causing disability, loss of mobility, and increased mortality . At present, imaging combined with related markers is mostly used for the diagnosis and evaluation of RA in clinical practice, but the diagnostic efficacy is still low [6, 7]. Herein, it is of paramount significance to explore novel and effective biomarkers for RA.
microRNAs (miRNAs) are non-coding single-stranded RNA sequences, with approximately 18–25 nucleotides in length, and miRNAs possess the ability to change gene expression by post-transcriptional modification and to mediate about 30% of protein-coding proteins [2, 8]. miRNAs are widely accepted as potential biomarkers for the diagnosis and prognosis of autoimmune diseases . Importantly, accumulating studies have suggested the involvement of miRNAs in the progression of RA; for instance, miR-129 is downregulated in synovial tissues of RA patients, and overexpression of miR-129 can reduce the extent of synovitis in RA ; miR-137 is under-expressed in RA tissues and cells, and it is negatively correlated with inflammatory factors . In addition, miR-146a and miR-499 are highly expressed in RA individuals and can be used as diagnostic markers for RA . miR-451 expression is increased in peripheral blood mononuclear cells (PBMCs) from RA-risk individuals . Currently, the function of miR-124a in inhibiting inflammatory responses has gained increasing attention, and miR-124a can restrain the proliferation and inflammation in fibroblast-like synoviocytes from RA patients by targeting the PIK3/NF-κB pathway , which can also suppress the invasion of RA synovial fibroblasts . Notably, miR-124a is a key miRNA in the post-transcriptional regulatory mechanism of RA synoviocytes and possesses therapeutic potential . Moreover, miR-124 is weakly expressed in autoimmune diseases, such as RA and systemic lupus erythematosus . Nevertheless, whether the expression of miR-124a in the peripheral blood and synovial fluid of RA patients can be used as a diagnostic marker for RA still needs to be explored. Therefore, this study investigated the expression level and clinical significance of miR-124a in the peripheral blood and synovial fluid of RA patients to provide potential effective indicators for monitoring RA and a more reliable basis for the clinical diagnosis of RA.
Materials and Methods
A total of 80 RA patients who were treated in the Department of Rheumatology and Immunology of The Third Hospital of Hebei Medical University from October 2020 to September 2021 were prospectively enrolled as the study subjects, including 17 males and 63 females (aged 30–75 years, with an average age of 46.17 ± 7.12 years), all of whom were in concert with the RA diagnostic criteria issued by the American College of Rheumatology (ACR) in 2010 . Moreover, 36 hospitalized patients with osteoarthritis (OA) during the same period were prospectively selected as the OA group , including 9 males and 27 females (aged 26–71 years, with an average age of 48.54 ± 7.56 years), and the patients all met the ACR criteria for OA of the knee . Another 36 healthy people in the same period were included as the control group, including 11 males and 25 females (aged 23–70 years, with an average age of 45.67 ± 6.78 years).
Inclusion and Exclusion Criteria
The included patients all met the following inclusion criteria: diagnosed with RA or OA based on the ACR criteria; without the use of glucocorticoids and other immunosuppressive drugs for clinical treatment in the last 3 months; with complete clinical data; and with good compliance. The subjects were excluded if they met the following exclusion criteria: with a history of severe cardiovascular diseases; with liver and kidney dysfunction; with inflammatory diseases; with malignant tumors; combined with hypertension, diabetes, infectious diseases, and other autoimmune diseases (such as systemic lupus erythematosus and ankylosing spondylitis); and with prior treatment.
The following clinical data were recorded through questionnaires and medical records of patients: age, gender, weight, and height of all participants; disease duration of RA and OA patients; swollen joint count in 28 joints (SW28), tender joint count in 28 joints (T28), and general health (GH) of RA patients. Finally, the disease activity score of 28 joints (DAS28) was calculated according to the following formula: DAS28 = 0.56 × sqrt (T28) + 0.28 × sqrt (SW28) + 0.70 × Ln (ESR) + 0.014 × GH.
The fasting venous blood (10 mL) was collected from each participant. Specifically, 4 mL of the collected blood was placed in an ethylenediamine tetra-acetic acid tube for subsequent extraction of miRNA, and 1.6 mL was kept in a sodium citrate tube for determination of erythrocyte sedimentation rate (ESR) using the Westergren method. Moreover, after the remaining blood was coagulated, the obtained serum was used for determination of C-reactive protein (CRP), rheumatoid factor (RF), and anti-cyclic citrullinated peptide (anti-CCP) antibodies. RF level was measured by immunonephelometry using the BN Prospec system (Siemens Healthcare Diagnostics, Tarrytown, NY, USA), and CRP and anti-CCP levels were measured by enzyme-linked immunosorbent assay .
Isolation of Plasma and PBMCs and Collection of Synovial Fluid
The aforementioned 4 mL venous blood was centrifuged at 3,000 r/min for 5 min to isolate the plasma, and the PBMCs were separated by Ficoll-Paque density gradient centrifugation [12, 18]. The synovial fluid (2 mL) was collected aseptically and kept in an anticoagulative tube containing heparin . The plasma, PBMCs, and synovial fluid were stored at −80°C until usage.
Reverse Transcription Quantitative Polymerase Chain Reaction
Total RNA was extracted from plasma, PBMCs, and synovial fluid by the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) in strict accordance with the provided instructions. The purity and concentration of RNA were determined using a NanoDrop™ 2,000/2,000c spectrophotometer (Thermo Fisher Scientific), and its integrity was tested by agarose gel electrophoresis. Each RNA sample (100 ng in total) was used for reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. cDNA was synthesized by reverse transcription using miScriptII RT kits (Qiagen, Valencia, CA, USA). RT-qPCR was performed using the SYBR Green PCR Mix (Takara, Tokyo, Japan) with the following reaction conditions: at 95°C for 15 min, and then 40 cycles at 95°C for 15 s, at 55°C for 30 s, and at 70°C for 30 s. The relative expression level of miR-124a after normalization to the internal control U6 was computed using the 2−ΔΔCt method . Primer sequences are exhibited in Table 1.
|Gene .||Forward 5’–3’ .||Reverse 5’–3’ .|
|Gene .||Forward 5’–3’ .||Reverse 5’–3’ .|
SPSS 21.0 software (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8.0.1 software (GraphPad Software Inc., San Diego, CA, USA) were used for data analysis and plotting. Variables were checked for consistency with normal distribution by Shapiro-Wilk test. Measurement data were displayed as mean ± standard deviation (SD). The independent sample t test was adopted for comparisons of means between groups. One-way analysis of variance (ANOVA) was used for comparisons of means among multiple groups, and Tukey’s multiple comparisons test was employed for post hoc analysis. Fisher’s exact test was performed for comparative analysis of categorical variables. Pearson coefficient was used to analyze the correlation between miR-124a expression and major clinical indicators. The diagnostic value of miR-124a for RA was evaluated using the area under the curve (AUC) of receiver operating characteristic curve. The sensitivity and specificity were calculated, respectively, and Youden’s index = (sensitivity + specificity −1) was calculated. The value corresponding to the maximum of the Youden’s index was considered the optimal diagnostic cut-off value, and the expression levels of miR-124a in plasma, PBMCs, and synovial fluid < the optimal diagnostic cut-off value were used to assist in the diagnosis of RA. The difference in the AUC was analyzed using MedCalc software. Statistical significance was declared when p < 0.05.
Comparative Analysis of Clinical Baseline Data
A total of 152 participants were included, including 36 healthy people, 36 OA patients, and 80 RA patients. Comparative analysis of the clinical data revealed no significant differences in indicators such as age, gender, height, and weight among the three groups (all p > 0.05), while there were statistically significant differences in CRP, RF, and ESR (all p < 0.05). The OA group and RA group showed no significant difference in age, gender, height, weight, and disease duration (all p > 0.05), but showed significant differences in CRP, RF, and ESR (all p < 0.05); RA patients had a higher anti-CCP level than OA patients and healthy people (all p < 0.01) (Table 2).
|Characteristics .||Control (N = 36) .||OA (N = 36) .||RA (N = 80) .||Pa .||Pb .||Pc .|
|Weight, kg||56.7±8.81||58.4±8.02||59.6 ±9.43||0.395||0.121||0.509|
|Disease duration, years||/||7.03±0.71||6.92±0.58||/||/||0.381|
|Characteristics .||Control (N = 36) .||OA (N = 36) .||RA (N = 80) .||Pa .||Pb .||Pc .|
|Weight, kg||56.7±8.81||58.4±8.02||59.6 ±9.43||0.395||0.121||0.509|
|Disease duration, years||/||7.03±0.71||6.92±0.58||/||/||0.381|
OA, osteoarthritis; RA, rheumatoid arthritis; CRP, C-reactive protein; RF, rheumatoid factor; ESR, erythrocyte sedimentation rate; anti-CCP, anti-cyclic citrullinated peptide; DAS28, disease activity score of 28 joints.
Measurement data were expressed as mean ± SD.
The unpaired t test was used for comparisons between two groups, one-way ANOVA was used for comparisons among multiple groups, and Tukey’s multiple comparisons test was used for post hoc analysis.
Measurement data were expressed as the number of cases, and Fisher’s exact test was used for comparatives between groups.
Pa, OA group compared with control group; Pb, RA group compared with control group; Pc, OA group compared with RA group.
Down-Regulation of miR-124a in Peripheral Blood Plasma, PBMCs, and Synovial Fluid of RA Patients
RT-qPCR results showed that the expression levels of miR-124a in plasma, PBMCs, and synovial fluid of the RA and OA groups were significantly reduced compared to the control group, and the RA group had significantly lower miR-124a level than the OA group (all p < 0.05) (Fig. 1a–c). Taken together, miR-124a was under-expressed in peripheral blood plasma, PBMCs, and synovial fluid of RA patients.
Correlation Analysis of miR-124a in Peripheral Blood Plasma, PBMCs, and Synovial Fluid of RA Patients
The Pearson correlation analysis unveiled that the expression levels of miR-124a in peripheral blood plasma, PBMCs, and synovial fluid of RA patients were positively correlated to some extent. Specifically, miR-124a expression in plasma was positively correlated with miR-124a expression in PBMCs (r = 0.673, p < 0.001) and in synovial fluid (r = 0.595, p < 0.001); in addition, miR-124a expression in PBMCs was positively linked with miR-124a expression in synovial fluid (r = 0.564, p < 0.001) (Fig. 2a–c).
Correlation Analysis of miR-124a in Peripheral Blood Plasma, PBMCs, and Synovial Fluid with Major Clinical Indicators in RA Patients
Existing evidence has shown that RF, ESR, and DAS28 are commonly used activity indicators for monitoring RA in clinical practice [2, 18, 22, 23]. Hence, Pearson analysis was used to further investigate the correlation between miR-124a expression levels in peripheral blood plasma, PBMCs, and synovial fluid with RF, ESR, and DAS28. The results unraveled that miR-124a expression in plasma was negatively correlated with RF, ESR, and DAS28 (r = −0.611, −0.707, and −0.553, respectively) (all p < 0.001) (Fig. 3a–c); miR-124a expression in PBMCs was inversely linked with RF, ESR, and DAS28 (r = −0.639, −0.659, −0.622, respectively) (all p < 0.001) (Fig. 3d–f); and there were different degrees of negative correlations between miR-124a expression in synovial fluid with RF, ESR, and DAS28 (r = −0.535, −0.482, −0.408, respectively) (all p < 0.05) (Fig. 3g–i).
The Expression Levels of miR-124a in Peripheral Blood Plasma, PBMCs, and Synovial Fluid Had High Diagnostic Values for RA Patients
The diagnostic efficacy of miR-124a expression in peripheral blood plasma, PBMCs, and synovial fluid for RA patients was evaluated by receiver operating characteristic curve. The AUC of plasma miR-124a expression for diagnosing RA patients was 0.899, the cut-off value was 0.800, the sensitivity was 68.75%, and the specificity was 94.44%, suggesting that plasma miR-124a level <0.800 could assist the diagnosis of RA (Fig. 4a). The AUC of miR-124 expression in PBMCs for distinguishing RA patients from healthy subjects was 0.937, the cut-off value was 0.805, the sensitivity was 82.50%, and the specificity was 91.67%, indicating that miR-124a level in PBMCs <0.805 had an auxiliary diagnostic value for RA (Fig. 4b). In addition, the AUC of miR-124a level in plasma combined with that in PBMCs for the diagnosis of RA was 0.961 (Fig. 4c), and the MedCalc analysis revealed that the diagnostic value of miR-124a expression in plasma and PBMCs for RA was higher than that of miR-12a level in the independent plasma or PBMCs (p < 0.05) (Fig. 4d). Moreover, the AUC of miR-124a expression in synovial fluid for the diagnosis of RA patients was 0.929, the cut-off value was 0.835, the sensitivity was 80.00%, and the specificity was 88.89%, demonstrating that miR-124a level in synovial fluid <0.835 could aid the diagnosis of RA (Fig. 4e). All in all, miR-124a expression levels in peripheral blood and synovial fluid had high diagnostic values for RA patients.
As an autoimmune disorder, RA is primarily characterized by joint destruction, progressive bone erosion, pannus formation, and chronic inflammation of the synovial joints, and patients typically present joint tenderness and swelling, progressing to serious disability, thereby severely influencing the quality of patients’ physical and mental lives . In recent years, epigenetic mechanisms such as DNA methylation and miRNAs have attempted to explain gene-environment interactions during the development of autoimmune diseases such as RA , providing a new window for understanding the possible pathogenesis of RA. RA is a heterogeneous disease and its specific immunological and genetic/epigenetic background is responsible for the manifestation and course of the disease . Among the epigenetic factors involved in RA, miRNAs impose an imperative role in various biological processes and are considered potential therapeutic targets for autoimmune and inflammatory diseases, including RA . Identification of disease-associated miRNAs will lead us to the post-genomic era, providing real possibilities for manipulating the genetic impact of autoimmune diseases. Therefore, different miRNAs may be good candidates for biomarkers for disease diagnosis, prognosis, treatment, and other clinical applications. Importantly, miR-124a appears to be repressed in RA synovial fibroblasts . This study underlined the diagnostic value of miR-124a expression in peripheral blood and synovial fluid for RA.
Altered miRNA expression is associated with the augmented production of pro-inflammatory cytokines, raised numbers of inflammatory pathways, and other processes that sustain the vicious cycle of autoimmunity, and thus miRNAs may function as potential biomarkers for the diagnosis and prognosis of autoimmune disorders . Additionally, altered expression of various miRNAs has been noted in PBMCs, body fluids, and tissues in RA patients , indicating the involvement of miRNA alterations in RA pathogenesis, diagnosis, and treatment response prediction. miR-124a level is diminished in RA synoviocytes . At present, miR-124a is considered a candidate for the treatment of human RA . Overexpression of miR-124a prominently represses cell proliferation, induces G1-phase cell-cycle arrest, and inhibits the secretion of inflammatory cytokines in fibroblast-like synoviocytes from RA patients . Therefore, we determined the miR-124a expression and noted downregulated miR-124a expression levels in peripheral blood plasma, PBMCs, and synovial fluid of RA patients. Interestingly, the Pearson analysis indicated that miR-124a expression in plasma, PBMCs, and synovial fluid of RA patients had a certain degree of positive correlation. Moreover, the synovial fluid and plasma miRNAs possess the potential to be diagnostic biomarkers for RA . Subsequently, we revealed that miR-124a level in plasma <0.800 and miR-124a level in PBMCs <0.805 both could aid the diagnosis of RA, but the MedCalc analysis indicated that miR-124a level in plasma combined with those in PBMCs had a higher diagnostic value for RA. Besides, miR-124a level in synovial fluid <0.835 could exert a certain auxiliary diagnostic value for RA. It is well known that early detection and treatment of RA are important. After searching the literature, we have found a plethora of miRNAs in several cells or tissues the participate in RA pathophysiology or are consequent to disease processes. miRNAs play an essential role in regulating the transcriptome and development of RA . An existing study has reported the presence of some abnormally expressed circular RNAs in PBMCs of RA patients, and hsa_circ_101328 may be a new and effective biomarker for early diagnosis of RA . However, related research about the diagnostic value of miR-124a for RA is scarce. Some traditional clinical markers for detecting or monitoring RA, such as CRP, ESR, interleukin-6, and anti-citrullinated protein antibodies , have some limitations in diagnosing RA. In addition, RA can be divided into two categories (autoantibody-positive and autoantibody-negative) based on the presence or absence of RF or anti-CCP . However, these traditional markers exhibit low specificity and sensitivity, and there are also false negative results, which may lead to misdiagnosis. Therefore, the diagnosis of RA remains difficult. Growing studies have reported changes in methylation of promoter regions of RA-associated genes or changes in miRNAs in synovial fluid and serum, and some miRNAs are considered markers of disease and are used to predict good response to biological therapy in RA . Evidence suggests that miRNAs have higher application value as potential biomarkers than traditional clinical markers . For the diagnosis of RA, our study found that the sensitivity of plasma miR-124a was 68.75% and the specificity was 94.44%; the sensitivity of miR-124a level in PBMCs was 82.50% and the specificity was 91.67%; and the sensitivity of miR-124a level in synovial fluid was 80.00% and the specificity was 88.89%. It can be found that miR-124a has higher sensitivity (>60%) and specificity (>80%) for the diagnosis of RA than traditional biomarkers, and there are no false negative results, which is the potential advantage of miR-124a as a diagnostic marker for RA. In addition, combining miRNA with traditional diagnostic methods can further improve the accuracy of RA diagnosis. In summary, our findings unraveled that low miR-124a expression in peripheral blood, PBMCs, and synovial fluid could assist in the diagnosis of RA.
RF is believed to be a vital diagnostic and prognostic biomarker for RA, and the anti-CCP is regarded as a RA disease-specific humoral immune response . Some researchers have illustrated the functionality of elevated IgM-RF, CRP, and ESR as validated laboratory parameters for the diagnosis of RA . Accordingly, the comparative analysis of the clinical baseline characteristics revealed that RA patients exhibited increased levels of CRP, RF, ESR, and anti-CCP. In the past several years, DAS28 has been used as a measurement for evaluating disease activity in RA patients . Growing evidence has revealed the correlation between differentially expressed miRNAs with clinical parameters of RA [38‒40]. Likewise, miR-21 and miR-125a which are reduced in RA patients are both negatively correlated with ESR, CRP, and DAS28-ESR scores . Further experiments demonstrated that the miR-124a expression levels in plasma, PBMCs, and synovial fluid were inversely associated with RF, ESR, and DAS28. In addition, miR-21 and miR-125a can mediate RA development by regulating inflammatory responses. For example, miR-21 overexpression alleviates the symptoms of RA by downregulating the Wnt pathway and suppressing the expression of inflammatory factors (IL-6, IL-8) ; miR-125a suppresses inflammatory responses in RA by mediating the Wnt/β-catenin and NF-κB pathways . We thus hypothesized that, similar to these two miRNAs, miR-124a may also target the downstream signaling pathways and suppress inflammatory responses, thereby alleviating the progression of RA. The aforementioned results further documented the strong participation of miR-124a in RA development. This finding is of great significance for understanding the pathogenesis and conducting targeted therapy of RA.
To conclude, as a prospective study, this study for the first time analyzed the expression levels and diagnostic value of miR-124a in peripheral blood plasma, PBMCs, and synovial fluid of RA patients and investigated the correlation between miR-124a and major clinical indicators (such as RF, ESR, and DAS28). The study provided a new reference for the clinical diagnosis of RA, a novel potential biomarker for RA, and exact evidence of the potential role of miR-124a as a useful epigenetic biomarker in assessing RA risk, broadening the practical application value of miR-124a. However, this study failed to comprehensively consider various aspects of patients, such as combined detection of multiple clinical indicator levels and clinical manifestations of patients for diagnosis and treatment. Additionally, the number of included cases was relatively small. In future studies, we shall carry out a larger multicenter study and expand the sample size to increase the credibility of the results. Furthermore, the molecular regulatory mechanism of miR-124a in RA progression is worth exploring. Given that current research on miR-124a is still in its infancy, studies on this new biomarker may advance the use of personalized medicine in RA treatment.
Statement of Ethics
The study protocol was approved by the Ethics Committee of The Third Hospital of Hebei Medical University (approval number: 201800232 TH). All participants voluntarily signed the informed consent. All procedures were strictly implemented according to the Declaration of Helsinki.
Conflict of Interest Statement
All authors declare that there is no conflict of interest in this study.
This work was supported by an experimental and clinical study on reconstruction of the anterior cruciate ligament by a rectangular bone tunnel (182777211).
Xirui Wu is the guarantor of integrity of the entire study; Xirui Wu is responsible for research concepts, study design, and clinical research; Tianhao Wu and Yanlong Zhang are responsible for clinical studies and data analysis; Tianhao Wu is responsible for manuscript editing and data collection; Yanlong Zhang is responsible for statistical analysis; Aqin Peng is responsible for manuscript review; and all authors read and approved the final manuscript.
Data Availability Statement
All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.