Background Research on miRNAs as regulators of immune system has fast emerged. miRNA expression profile is believed to be reflective of specific physiologic and pathologic processes and can serve as potential biomarkers of disease activity.
Objectives In the present study, we proposed to test the hypothesis that, “patients with ankylosing spondylitis (AS) have an altered miRNA expression profile compared to control subjects.” We also proposed to test whether the expression profile of selected miRNAs identified in the study correlates with disease activity as measured by BASDAI and ASDAS and markers of inflammation:ESR and CRP.
Methods Fifty-one patients ≥18 years of age with AS based on modified New York classification criteria and 23 healthy controls were prospectively recruited. Subjects with malignancy in last 5 years, RA, SLE, evidence of HIV or chronic hepatitis B or C infection were excluded. Patients and controls were screened, consented and peripheral blood samples (5 ml) were obtained. ESR and CRP were measured using routine laboratory methods. Validated Questionnaires to assess disease, and functional activity in AS were administered to patients. Sixty-eight circulating miRNAs in the plasma of all the study subjects were profiled by Firefly Multiplex Circulating miRNA Assay (Abcam, Cambridge, MA) using the Immunology Panel. Forty microliters of plasma samples were first digested using supplied enzyme mix and then the assay was performed following the instructions provided by manufacturer. The assay beads were scanned on BD Accuri C6 flow cytometer. Data were analyzed using Firefly Analyis Workbench (Abcam). Descriptive analyses included continuous variables (the mean ± SD) and the categorical variables percentage. Linear regression analysis was performed to assess the association between BASDAI, BASMI, ASDAS, Rapid 3, ESR, CRP and differentially dysregulated miRNAs in AS patients.
Results There were 34 males/51 with mean age of 49.8± 12.2; 30.6% African-Americans, 47.0% were HLA-B27 positive, 29.4% were negative and 23% indeterminate, and 45% on anti-TNF alpha inhibitors. Mean (±SD) values were: BASDAI score 5.23 (±2.5); BASFI score 4.7 (±2.8); PGA, 5.1 (±2.4); ASDAS-CRP score 3.4 (±1.13); RAPID 3 score 13.76 (±7.21); ESR, 22.4 mm/hr (±22.0) and CRP levels, 1.3 mg/dl (±1.9). Six microRNAs (miR-181b-5p, miR-181a-5p, let-7i-5p, miR-34a-5p, miR-30b-5p and miR-146a-5p) were found to be differentially expressed in AS patients compared to healthy controls (p<0.05). miR-181b-5p also showed a significant linear relationship with BASDAI score (p=0.0083) and ASDAS (p=0.0547). Some of the predicted targets of miR-181b-5p according to miRTarBase have been shown to inhibit TIMP-3 which is a tissue inhibitor of matrix metalloproteinase and TMED 7 which is involved in TLR-4 signaling. MicroRNA-181b has also been shown to serve as a potent regulator of downstream NF-κB signaling
Conclusions Patients with AS as compared to controls, have dysregulated expression of selected miRNAs in the plasma; and levels of miR-181b-5p were significantly higher in patients with active disease and showed significant correlation with BASDAI and ASDAS scores. miR-181b-5p is predicted to target genes regulating inflammatory processes and may be proposed as a potential biomarker for disease activity in AS.
Acknowledgement The study is funded by National Institute of Allergy and Musculoskeletal diseases
Disclosure of Interest None declared
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