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Abstract
Background: Psoriasis is a chronic immune-mediated inflammatory disease affecting the skin and/or joints. MicroRNAs (miRNA) are single-stranded non-coding RNA molecules that bind to messenger RNA (mRNA) and regulate gene expression. Studies on peripheral blood mononuclear cells (PBMCs) in psoriatic patients showed an upregulation of miRNA 223. Aims and Objectives: a) To estimate and compare the expression of miRNA 223 in cases of chronic plaque psoriasis and controls. b) To correlate the expression of miRNA 223 with the severity of chronic plaque psoriasis. Materials and Methods: This study included 80 subjects (40 with psoriasis and 40 with age- and sex-matched healthy controls) attending the dermatology OPD of a tertiary care hospital from January 2018 to June 2019. A detailed history, determination of Psoriasis Area Severity Index (PASI) score and estimation of miRNA 223 by quantitative real-time polymerase chain reaction (qRT-PCR), was done in all subjects. Results: The expression of miRNA 223 (ΔCt) was higher in cases than in controls. The observed mean ΔCt was higher in severe (12.90 ± 0.46) than in mild (9.81 ± 1.70) and moderate (10.58 ± 1.26) psoriasis. The difference in expression of miRNA with varying severity of psoriasis was significant. The mean difference of ΔCt between mild to severe was (3.09) (P ≤ 0.001) and moderate to severe was (2.31) (P = 0.013). Among cases, the expression of miRNA 223 was higher in those exhibiting Koebner's phenomenon compared to those without Koebner's phenomenon (P = 0.0424). Conclusion: Expression of miRNA 223 was higher in psoriatic patients than in controls and the expression increased with the severity and activity of the disease suggesting the upregulation of miRNA 223 with the progression and activity of the disease.
Keywords: Koebner's phenomenon, MicroRNA, miRNA 223, psoriasis
Introduction 
Psoriasis is an immune-mediated proliferative inflammatory disorder predominantly involving the skin, joints and nails.[1] Although the pathogenesis of psoriasis is not yet understood completely, it is thought that the disease is initiated and maintained by a chronic inflammatory cycle that occurs with the interaction of keratinocytes and immune cells.[2] Also, environmental, genetic, infectious, metabolic and psychological factors play a critical role.[3] Immunologically interleukin (IL) -17 produced by T helper (Th) 17 cells contributes to the development of psoriasis by inducing the production of proinflammatory cytokines.[4]
MicroRNAs (miRNAs, miR) are small 21–25 nucleotide RNA molecules that bind to the 3'-untranslated region (UTR) of messenger RNA (mRNA) and are involved in regulating gene expression by mRNA degradation and inhibiting translation.[5],[6] These miRNA molecules regulate innate and adaptive immune responses and modify the expression of inflammatory mediators.[7] Peripheral blood mononuclear cells (PBMCs) from patients with psoriasis have shown an upregulation of miR 223 along with miR 193b and miR 143.[8]
These miRNAs may serve as specific disease biomarkers. Therefore, this study was undertaken to estimate and compare the expression of miRNA 223 in cases of chronic plaque psoriasis (CPP) and controls and to correlate the expression of miRNA 223 with the severity of CPP.
Materials and Methods This hospital-based case-control study was performed on 80 subjects among which 40 were cases of Type 2 CPP and 40 were age and sex-matched healthy controls. Our cases and controls were from the southern states of India, Karnataka and Kerala which mainly constitute population of a dravidian origin. The study was conducted from January 2018 to June 2019 at a tertiary care hospital. Ethical clearance from Institutional Ethics Committee was obtained before the start of the study.
Our case group comprised patients of CPP aged 18 years and above of either gender who consented to participate in the study. All cases included in the study were not on systemic or topical medication for the previous three months and two weeks, respectively. The control group comprised age- and gender-matched healthy individuals who did not have CPP. The exclusion criteria for the cases and controls were subjects with a family history of psoriasis, known autoimmune disease, malignancy, long-standing kidney and/or liver disease, and pregnant and lactating women.
A detailed history was taken, and a complete physical examination was done including the extent of lesion, local examination of lesions, presence of Koebner's phenomenon (KP), body surface area (BSA) involvement, determination of Psoriasis Area and Severity Index score (PASI). PASI was interpreted as mild <5, moderate 5–10 and severe >10.
Five ml of peripheral venous blood was taken in an EDTA tube, and total RNA was isolated from the PBMC using the mirVana miRNA isolation kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's protocol. The concentration and purity of total RNA were measured with an ultraviolet Eppendorf BioSpectrometer. Quantification of miRNA was done using TaqMan MicroRNA Assays in a two-step RT-PCR.
In the reverse transcription (RT) step: complementary DNA (cDNA) was reverse transcribed from total RNA sample using specific miRNA primer from the TaqMan miRNA Assay and reagents from TaqMan miRNA RT Kit.
In the PCR step: PCR amplification of cDNA was done using TaqMan miRNA Assay together with the TaqMan Universal PCR Master Mix.
The data for gene expression was analysed by viewing the amplification plot and setting baseline and thresholds. The comparative CT method was used, and delta Ct (ΔCt) was obtained which represented the expression of miRNA 223 after normalization by endogenous control Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Normalization is required to correct the difference in the amount of cDNA loaded into the PCR reaction.
The data collected was analysed using BDSS Corp. Released in 2020. coGuide Statistics software, Version 1.0, India: BDSS Corp. Quantitative variables were presented as mean and standard deviation, and for categorical variables, frequency and proportion were used. Categorical outcomes were compared between study groups using the Chi-square test. For normally distributed quantitative parameters, the mean values were compared between study groups using an independent sample t-test (2 groups) and one-way ANOVA (more than two groups). For non-normally distributed quantitative parameters, median and interquartile range (IQR) were compared between study groups using Mann–Whitney U test (two groups) and Kruskal–Wallis test (more than two groups). The association between quantitative explanatory and outcome variables was assessed by calculating Spearman's rank correlation coefficient represented by rs. P value < 0.05 was considered statistically significant.
Results In our study of 80 participants, 54 (67.5%) were males and 26 (32.5%) were females with a male-to-female ratio of 2.08:1. The mean age of cases was 43.6 ± 14.78 years and controls was 43.55 ± 15.13 years. The youngest patient was 18 years of age, and the oldest was 72 years of age. The majority of the patients (45%) were below 40 years [Table 1].
The duration of psoriasis ranged from 2 months to 588 months. In the psoriatic group, there was an equal distribution of disease duration for ≤5 years and >5 years being 20 (50.0%). The mean duration of psoriasis was 122.35 ± 134.38 months.
The PASI score in our study ranged from 0.6 to 19.8. In our study, 25 (62.50%), 11 (27.50%) and 4 (10.00%) cases were categorized as having mild (<5 PASI), moderate (5-10 PASI) and severe (>10 PASI) psoriasis, respectively [Table 2]. The mean and median PASI scores observed in the current study were 4.98 ± 4.50 and 3.25, respectively.
Table 2: Distribution of cases based on Psoriasis Area and Severity Index (PASI)There was a significant association between the duration of psoriasis and disease severity which was assessed by using the PASI score [P = 0.043].
ΔCt was analysed in 80 participants and the mean value of ΔCt for the cases (10.49 IQR 9.63 to 11.53) was higher compared to the controls (10.43 IQR 8.5 to 11.79) but the difference was not statistically significant (P = 0.397).
Among the 40 subjects of psoriasis, mean ΔCt was higher in severe (12.90 ± 0.46) psoriasis compared to mild (9.81 ± 1.70) and moderate (10.58 ± 1.26) psoriasis [Table 3].
Table 3: Comparison of PASI score with Delta CT (n=40) [One-way analysis of variance]The overall P value in defining the difference of ΔCt across the severity of psoriasis reported significance, and hence post hoc test was done to see the pairwise comparisons. The difference between mild to severe (3.09) (P ≤ 0.001) and moderate to severe (2.31) (P = 0.013) psoriasis was statistically significant.
The activity of the disease was determined by the presence of the Koebner phenomenon which was observed in 6 (15%) cases of psoriasis. Mean ΔCt was found to be higher in cases showing the Koebner phenomenon compared to cases without the Koebner phenomenon. The difference was statistically significant (P = 0.042) [Table 4].
Discussion MicroRNAs predominantly reside in the cells, their recognition in the cell-free environment has provided a diagnostic significance and identified them as biomarkers for disease.[9]
MiRNA is involved in the pathogenesis of psoriasis by inducing both proliferation of keratinocytes and activation of T cells and release of IL.[8] Various miRNAs have been studied in the skin tissue of patients with psoriasis with miRNA 223 being upregulated in dermal inflammatory infiltrate and epidermis of psoriatic lesions.[10] MiRNA 223 is observed to be upregulated in rheumatoid arthritis,[11] inflammatory bowel disease,[12] influenza infection, Epstein–Barr virus-positive B-cell lymphoma, osteosarcoma, metastatic gastric cancer cell, and ovarian cancer.[13]
In this study, we observed that ΔCt was higher in cases with severe psoriasis compared to moderate and mild psoriasis, the expression of miRNA 223 increased with the progression of the disease. It was also observed that ΔCt was statistically significantly higher in cases showing Koebner's phenomenon which indicates the activity of the disease. This suggests that miRNA 223 expression might have a role in disease progression and activity of the disease.
Lovendorf MB et al.[14] studied the expression of miRNA 223 in different blood compartments (whole blood, plasma, PBMC) in patients of psoriasis and healthy controls. They studied the expression of miRNA 223 in PBMC of 76 patients with moderate to severe psoriasis which showed a significant upregulation in comparison with the control. Their study further showed that miRNA 223 expression significantly correlated with PASI score, leukocyte, neutrophil, eosinophil and monocyte counts.
García-Rodríguez et al.[7] studied 11 patients with psoriasis who were treatment-naive or in the washout phase of treatment and 11 healthy controls. The relative expression of miRNA was calculated using 2-ΔΔCT method, and the values were shown as median. They observed that the relative expression of miRNA223 in PBMC was significantly increased in cases compared to controls [0.9 (0.56-1)], and it remained elevated following treatment with methotrexate or topical therapy. However, no correlation was observed with the PASI score. The small sample size was the limitation of this study.
Pivarcsi A et al.[15] analysed the miRNA 223 expression in serum before and after treatment with etanercept and found that the expression of miRNA 223 was abundant in patients with psoriasis and expressed preferentially by leukocytes. There was no correlation with PASI score in untreated psoriatic. They also studied the expression level in the serum of miRNA 223 in untreated CPP patients and healthy controls and found no significant difference between the two groups.
In our study, there was an upregulation of miRNA 223 in CPP patients compared to controls but the difference was not statistically significant. These variations in results could be attributed to racial and geographical variation, the methodology employed for isolation of miRNA, variation in the endogenous control used and different blood compartments studied.
Løvendorf et al.[10] in their study of psoriatic skin tissue samples observed that miRNA 223 has a role in increasing the IL-17 which is involved in the pathogenesis of psoriasis.
A single miRNA can target more than one mRNA and deregulate various genes.[16] Synthesized miRNAs are known to negatively alter gene expression by binding to the 3' UTR mRNA.[17]
One of the predicted targets of miRNA 223 is the Roquin gene, and this gene is known to decrease the level of IL-17A. Hence, when miRNA 223 acts on its target Roquin gene it negatively modulates its expression and leads to an increase in IL-17A.[18]
The role of miRNA 223 has also been identified in inflammation where it is involved in the differentiation of granulocytes and chemotaxis of inflammatory cells.[13]
This indicates that miRNA 223 has a critical role both in the pathogenesis of psoriasis and in the progression of inflammatory process.
In our study, there was an upregulation of miRNA 223 expression in PBMC of CPP with the progression of the disease and activity of the disease. Early identification of rising expression of miRNA 223 can help in identifying the progression towards severe psoriasis, and hence, early intervention can be undertaken to prevent the same.
It can also help in identifying the effectiveness of treatment by determining the pre- and post-levels of miRNA 223 following treatment but we did not investigate if the expression remained upregulated or if it down-regulated following the treatment of the disease.
Limitations
The limitation of this study was small sample size, hospital-based study, and the results cannot be extrapolated to the general population and no follow-up of the patients in the study.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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