Psoriasis is one of the most common chronic cutaneous inflammatory disorders. It impairs patient’s quality of life and social relationships 1. The precise pathogenesis of psoriasis is still not completely understood. Nevertheless, several studies suggest that it is associated with multiple pathogenic mechanisms: genetic, immunological, infectious, and metabolic 2. In humans, almost a thousand microRNAs have been identified, encompassing 1–5% of all genes in the human genome. Increased evidence has shown that microRNA levels are useful biomarkers for diagnosis, prognosis, and possessing therapeutic value in various diseases 3. Several psoriasis-associated microRNAs have already been identified in the skin and have been shown to contribute to its development 4,5. MicroRNAs affect keratinocyte and T-cell function, immune activation, and regulate the strength of inflammatory signals 6. It has been shown that microRNA-369-3p regulates many psoriasis-related genes, including TNF-α, LIMK1, SIRT1, SP3, ADAM10, HES1, and WNT5A7–9. On the basis of these findings, microRNA-369-3p might be involved in the pathogenesis of psoriasis by regulating the expression of these target genes. In addition, IL-22 plays a pathogenic role in psoriasis, where it is responsible for the altered proliferation and differentiation of keratinocytes and induces inflammatory molecules. STAT3-dependent IL-22 signaling and its effects on keratinocytes are negatively regulated by SIRT110. We aimed to study the relationship between microRNA-369-3p, IL-22, and SIRT1 in psoriasis.
Patients and methods
Clinical assessment and patient materials
This case–control study was carried out at the Dermatology, Venereology, and Andrology Department at Assiut University Hospital, Egypt, during the period from March 2014 to June 2015. The study included 25 patients with clinically diagnosed plaque psoriasis and 25 healthy controls. The Local Ethics Committee of the Faculty of Medicine at Assiut University approved the study according to the principles of the Declaration of Helsinki. Patients over 10 years of age with plaque psoriasis were included. All patients (and parents in the case of patients under 18 years of age) have provided informed consent for participation in this study.
Patients with other autoimmune or systemic diseases were excluded. Patients receiving systemic treatment, including immunosuppressive drugs and phototherapy, within a month of the sample collection period and patients who refused to be included in the study protocol were excluded as well. An initial evaluation was performed with regard to patient age, sex, duration, associated symptoms, previous treatment, and other systemic affections. Diagnosis of plaque psoriasis was established clinically. The disease severity of each patient was assessed using the following grading based on Psoriasis Area and Severity Index (PASI) score as follows: mild psoriasis, PASI less than 10; moderate disease, PASI of 10 to less than 20; and severe psoriasis, PASI of at least 20 11.
Serum samples and skin biopsies were taken from each patient and control. Five millilitres of venous blood sample was taken. The resulting serum was divided into small aliquots and preserved at –20°C. Skin biopsies were taken from psoriatic lesion in the patients and from normal skin (usually the thigh) in controls. Skin biopsies (each sample is about 30 mg weight) were preserved immediately in 300 μl of RNA later solution to protect RNA content and then preserved at –80°C.
Total RNA including small RNAs was purified from skin biopsies using miRN easy minikit (Qiagen, Valencia, California, USA) (cat no. 217004) according to manufacturers’ instructions. The RNA content of each sample was measured using nanodrop device to fix the amount of RNA content in the starting material.
Complementary DNA synthesis
Reverse transcription of RNA into complementary deoxyribonucleic acid (cDNA) was performed using miScript II RT Kit (Qiagen) (cat. nos. 218160, 218161) according to manufacturer’s instructions, and to measure the SIRT1 gene expression reverse transcription into cDNA was performed using quantitect reverse transcription kit (Qiagen) (cat. nos. 205310, 205311, 205313, and 205314) according to manufacturer’s instruction.
Real-time PCR (RT-PCR) was performed using 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, California, USA).
A-Detection of microRNA-369-3p expression
PCR amplifications were carried out using miScript SYBR Green PCR Kit (cat. nos. 218073, 218075, 218076; Qiagen), miScript Primer Assays; Hs-miR-369-3p-1 (cat. no. MS00006853; Qiagen) and Hs-SNORD68- 11 (cat. no. MS00033712; Qiagen) as a housekeeping gene. The primer sequence of miR-369 was as follows: AAUAAUACAUGGUUGAUCUUU. The reaction solution was prepared to a volume of 25 µl, which comprised 12.5 µl of QuantiTect SYBR Green PCR Master Mix, 2.5 µl of miScript Universal Primer, 2.5 µl of miScript Primer Assay, 2.5 µl of template cDNA, and 5 µl RNase-free water. The PCR amplification protocol was as follows: 95°C for 15 min, followed by denaturation at 94°C for 15 s, annealing at 55°C for 30 s, and extension at 70°C for 30 s for 40 cycles.
B-Detection of SIRT1 expression
Quantitative Real-Time PCR for detection of SIRT1 gene expression was performed using QuantiTect SYBER Green PCR kit (cat. nos. 204141, 204143, and 204145; Qiagen), miScript Primer Assays; Hs-SIRT1-1-SG (cat. no. QT00051261; Qiagen) and HsGAPDH-1-SG (cat. no. QT00079247; Qiagen) as a house keeping gene. The primer sequence of SIRT1 was as follows: 5′-GCTGGCCTAATAGAGTGGCAA-3′ and 5′-CTCAGCGCCATGGAAAATG-3′. The reaction solution was prepared to a volume of 25 µl, which comprised 12.5 µl of QuantiTect SYBR Green PCR Master Mix, 2.5 µl of Primer A, 2.5 µl of Primer B, 2.5 µl of template cDNA, and 5 µl of RNase-free water. The PCR amplification protocol was as follows: 95°C for 15 min, followed by denaturation at 94°C for 15 s, annealing at 50–60°C for 30 s, and extension at 72°C for 30 s for 40 cycles.
Relative expression level (fold change) for both skin microRNA-369-3p and SIRT1 gene was calculated using the comparative threshold cycle (Ct) method 12. To compare separate data of patients and controls, we used the ΔCt (the difference in threshold cycle between the target and reference genes) of both patients and controls, as this was the last comparable data between them.
Level of IL-22 in serum was measured using Human IL-22 ELISA Kit (cat. no. E-EL-H0106) (Elabsceince Biotechnology Co., Ltd, Hubei, China) according to the manufacturer’s instructions.
Data entry and data analysis were performed using SPSS, version 19 (Statistical Package for Social Science; SPSS Inc., Chicago, Illinois, USA). Data were presented as number, percentage, mean, median, and SD. The χ2-test was used to compare qualitative variables. The Mann–Whitney test was used to compare quantitative variables between two groups. Spearman’s rank correlation was performed to measure correlation between quantitative variables. P-values were considered statistically significant when P less than 0.05.
Characteristics of study participants
This study included 25 patients with psoriasis vulgaris. Their ages ranged from 12 to 70 years, with 64% male and 36% female patients. The study also included 25 age and sex matched controls whose ages ranged from 15 to 60 years, with 68% male and 32% female patients. Disease duration ranged from 2 months to 16 years. The PASI score indicated that 11 patients had mild psoriasis, eight had moderate psoriasis, and six had severe psoriasis.
Skin microRNA-369-3p expression
The mean relative miRNA369-3p expression level (mean fold change) was 4.5-fold higher in patients than in controls (Fig. 1). There was a significant difference between patients and controls, when the mean ΔCt of both groups was compared (P=0.01) (Table 1).
Correlation between relative skin microRNA-369-3p levels and PASI score
A statistically nonsignificant positive correlation was observed between relative skin microRNA-369-3p levels and disease severity as measured using the PASI score (r=0.079, P=0.772).
Skin SIRT1 expression levels
The mean fold change in SIRT1 expression was about 0.6 between patients and controls, indicating nearly similar levels (Fig. 1). There was no significant difference between patients and controls when the means of ΔCt of both groups were compared (P=0.564) (Table 2).
Correlation between skin SIRT1 expression and PASI score
A statistically nonsignificant positive correlation between SIRT1 expression levels and disease severity (r=0.079, P=0.772) was detected.
IL-22 levels were significantly higher in patients with psoriasis (11.62±10.21 pg/ml) than in controls (1.77±1.61 pg/ml) (P=0.000; Table 3). There was a significant positive correlation between IL-22 levels and disease severity as measured using the PASI score (r=0.688, P=0.000) (Fig. 2).
Correlation between microRNA-369-3p, SIRT1 expression, and IL-22
There was a statistically nonsignificant negative correlation between the microRNA-369-3p and SIRT1 expression levels (r=−0.066, P=0.782), and a statistically nonsignificant positive correlation between levels of SIRT1 expression and IL-22 (r=0.179, P=0.425).
We studied the relationship between microRNA-369-3p, one of its predicted target genes SIRT1, and a related cytokine IL-22, which has been demonstrated to be a key cytokine in the development of psoriasis.
It was found that microRNA-369-3p expression in skin samples from patients with psoriasis was significantly higher than that in healthy controls, with a mean fold change of 4.5. This is in agreement with the results of Guo and colleagues, who found that skin microRNA-369-3p levels in patients showed an aberrant 2–3-fold increase in expression relative to that in healthy controls. Therefore, they considered skin microRNA-369-3p as a potential marker that may assist in understanding the pathogenesis of psoriasis 13.
After conducting a correlation analysis, relative skin microRNA-369-3p expression levels were found to correlate positively with PASI scores in patients with psoriasis, although this correlation was not statistically significant. This is in contrast to the results of Guo and colleagues whose data showed a statistically significant positive correlation between microRNA-369-3p levels in skin and PASI scores in patients with psoriasis. This discrepancy might be attributed to a difference in the number and severity of studied groups. In our study, skin specimens were obtained from 25 patients with psoriasis and 25 healthy controls, whereas in the study by Guo et al.13 skin specimens were obtained from 10 patients with psoriasis and 10 healthy controls. In our study, 44% of patients had mild psoriasis, 32% had moderate disease, and 24% had severe psoriasis, whereas in the study conducted by Guo and colleagues, 60% of patients had severe psoriasis, 40% had moderate psoriasis, and mild cases were not included. However, Guo and colleagues showed that there was no significant correlation between serum microRNA-369-3p levels and PASI scores in patients with psoriasis.
It was expected that SIRT1 expression levels would be downregulated in patients with psoriasis because SIRT1 opposes IL-22-induced STAT3 activity by deacetylating STAT3 and reducing STAT3 Tyr705 phosphorylation 10.
In our study, the mean fold change in SIRT1 expression levels was ~0.6 between patients and controls, indicating nearly similar levels of expression. Therefore, there was no significant difference between the patient and control groups.
These results are in agreement with those of Sestito et al.10, whose study showed that patients with psoriasis and healthy donors showed similar levels of SIRT1, which is localized mainly in the cytosol. Immunohistochemistry revealed that basal keratinocytes in lesional skin psoriasis exhibit reduced and less intense SIRT1 staining compared with healthy and nonlesional psoriatic skin, especially at the tips of the dermal papillae close to the CD3 T-cell infiltrate. Sestito et al.10 concluded that the impaired SIRT1 expression was the consequence of the microenviromental cytokine milieu and not an intrinsic defect of the psoriatic epidermis.
In the present study, SIRT1 expression levels in the skin were not significantly correlated with PASI scores. No previous study has shown the correlation between SIRT1 and PASI score to allow for comparison.
In this study, serum levels of IL-22 were significantly higher in patients with psoriasis vulgaris than in controls. Michalak-Stoma et al.14 similarly observed significantly higher levels of IL-22 in patients with psoriasis compared with those in controls. In the study by Boniface and colleagues, IL-22 was detected in the serum of 22 of 33 patients with psoriasis, compared with its detection in only a single serum sample out of 20 normal individuals.
A significant positive correlation was found between IL-22 levels and disease severity as measured using PASI scores. However, in the study by Boniface et al.15, circulating IL-22 concentrations in patients were dispersed and found to correlate with levels of the inflammation marker C-reactive protein (P<0·05), but not with the index of disease severity.
To address our hypothesis, we performed a correlational analysis between microRNA-369-3p, SIRT1 expression, and IL-22, but were unable to detect any significant correlation. To the best of our knowledge, there are no reports about a relationship between these three factors. We expected to observe statistical significance in these correlations, but our study did not prove this hypothesis.
MicroRNA-369-3p may be involved in the pathogenesis of psoriasis. The impaired SIRT1 expression is a consequence of the microenviromental cytokine milieu and is not an intrinsic defect of the psoriatic epidermis 10. Serum levels of IL-22, which correlated with the clinical severity of disease, may represent an objective parameter of successful treatment and may be used as a parameter during follow-up observations.
Further studies are needed to determine whether microRNA-369-3p can affect the expression of other psoriasis-related genes, including LIMK1, SP3, ADAM10, HES1, and WNT5A. Future studies are needed to determine the mechanism of microRNA-369-3p function in psoriasis. This would serve to further the understanding of psoriasis pathogenesis and contribute to the development of new therapeutic strategies.
Conflicts of interest
There are no conflicts of interest.
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