A total of 20 psoriatic patients who were diagnosed with PV by pathological examination were recruited from outpatient clinics at the Second Xiangya Hospital of Central South University (Changsha, China). Psoriasis disease activity was assessed using psoriasis area and severity index (PASI) scores (22), and blood samples and lesional skins were collected. Non-lesional skin tissues were obtained from 10 of the patients simultaneously. Patient information is presented in Table I. Blood samples were collected from 20 sex- and age-matched healthy controls who were recruited from the medical staff at the Second Xiangya Hospital. Normal skin tissues were obtained from the outpatient operating room at the Department of Dermatology at the Second Xiangya Hospital. The information of all healthy controls is presented in Table II. The present study was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University. Written informed consent was obtained from all subjects.

For determining the severity and extent of psoriasis, PASI scoring was used (22,23). In the four regions of the body, namely the head (h), upper extremities (u), lower extremities (l) and torso (t), the characteristics of the disease, including erythema (E), infiltration (I) and desquamation (D), were evaluated with a score of 1–4, and the involved area (A) of psoriatic lesions was evaluated with a score of 1–6 (Table III). PASI total scores ranges between 0 and 72. Higher scores indicate greater psoriasis severity. The formula used to calculate the total PASI score is as follows: PASI=(E_h+I_h+D_h)xA_hx0.1+(E_u+I_u+D_u)xA_ux0.2+(E_t+I_t+D_t)xA_tx0.3+(E_l+I_l+D_l)xA_lx0.4

Female BALB/c mice (age, 6–8 weeks; 19.0–20.5 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All mice were maintained in specific pathogen-free conditions (20–24°C; relative humidity, 50–55%; 12 h light/dark cycle) with free access to food and water. The IMQ-induced psoriasis-like mouse model was established as previously described (24). The mice were treated with a daily topical dose of 62.5 mg 5% IMQ cream (cat. no. H20030128; Sichuan Med-Shine Pharmaceutical Co., Ltd., Chengdu, China) on their shaved backs for 7 consecutive days. The control mice were treated with the same dose of vehicle cream. All procedures were approved and supervised by the Animal Care and Use Committee of the Second Xiangya Medical School of Central South University.

Peripheral blood mononuclear cells (PBMCs) were separated from the peripheral blood of healthy controls and patients with psoriasis by density gradient centrifugation at 18°C and 600 × g for 30 min (GE Healthcare, Chicago, IL, USA). The cells were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in 5% CO₂, or collected directly for subsequent experiments. HaCaT cells (cat no. BNCC101683; BeNa Culture Collection, Beijing, China), which were stored in liquid nitrogen, were revived and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS at 37°C in 5% CO₂. The medium was refreshed every 2 days and the cells were subcultured when 90% confluence was reached.

Total RNA was extracted from the cells or skin tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and a NanoDrop spectrophotometer (ND-2000; Thermo Fisher Scientific, Inc.) was used for RNA quality control. The mRNA was reverse-transcribed using a PrimeScript^® RT reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China). Each test used 1 µg total RNA and was performed according to the manufacturer's protocol. qPCR was subsequently performed using the SYBR Premix Ex Taq II (Tli RnaseH Plus; Takara Biotechnology Co., Ltd.) using a LightCycler^® 96 (Roche Diagnostics, Basel, Switzerland) thermocycler. The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 45 cycles of 95°C for 5 sec and 60°C for 20 sec, and a final extension (95°C for 1 sec, 65°C for 15 sec and 95°C for 1 sec). The relative expression of the target genes was calculated using the 2^−ΔΔCq method (25) and normalized against the GAPDH internal control. Detailed information on the primers used is summarized in Table IV.

The cells or skin tissues were lysed in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors (Beyotime Institute of Biotechnology, Haimen, China). The proteins were quantified using a Bradford assay (Pierce; Thermo Fisher Scientific, Inc.) and 100 µg protein from each sample was loaded for 8% SDS-PAGE. Following the transfer of proteins onto a polyvinylidene fluoride membrane, the membrane was blocked with 5% skim milk in PBS with 0.1% Tween-20 at room temperature for 1 h. Rabbit anti-WT1 (1:1,000; cat no. ab89901; Abcam, Cambridge, UK) and goat anti-GAPDH (1:2,000; cat no. ab9483; Abcam) primary antibodies were incubated with the membrane at 4°C overnight. Horseradish peroxidase (HRP) goat anti-rabbit immunoglobulin (Ig)G (H+L) (1:5,000; cat no. AS014; ABclonal Biotech Co., Ltd., Woburn, MA, USA) and HRP donkey anti-goat IgG (H+L) (1:5,000; cat no. A00178; GenScript, Piscataway, NJ, USA) secondary antibodies were incubated at room temperature for 2 h. The data was analyzed using a GE-ImageQuant LAS 4000 mini (GE Healthcare). The quantification of WT1 was normalized against GAPDH by densitometric analysis with Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). The images were cropped for presentation.

Skin tissues were fixed in formalin at room temperature overnight and embedded in paraffin. The 6-µm-thick sections were stained with hematoxylin for 10 min, and then stained with eosin for 2 min at room temperature. For immunohistochemistry, the sections were stained with rabbit anti-WT1 (1:200; cat no. ab89901; Abcam) or rabbit anti-Ki67 polyclonal antibodies (1:100; cat no. ab15580; Abcam) at 4°C overnight, according to the manufacturer's protocol. Image analysis was performed using a DMI 4000B microscope (magnification, ×100) and Leica Qwin Std analysis software version 3 (both Leica Microsystems GmbH, Wetzlar, Germany).

A total of 12 h prior to transfection, the HaCaT cells were seeded in a 6-well cell culture plate at 6×10³ cells/well. The cells were transfected with 20 nM WT1 siRNA, 5 µg WT1 plasmid or their corresponding controls using Lipofectamine^® 2000 (Invitrogen, Thermo Fisher Scientific, Inc., USA) for 6 h in Opti-minimum essential medium (Opti-MEM; Gibco; Thermo Fisher Scientific, Inc.). The Opti-MEM was removed and replaced with DMEM supplemented with 10% FBS for cell culture. The WT1 expression plasmid and the empty control plasmid were purchased from Vigene Biosciences, Inc. (Rockville, MD, USA). The WT1 and negative control siRNAs (sequences unavailable) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The WT1 siRNA consisted of a mixture of two oligos: 5′-AAATATCTCTTATTGCAGCCTGGGT-3′ and 5′-TTTCACACCTGTATGTCTCCTTTGG-3′.

HaCaT cells were seeded in 96-well plates in triplicate and transfected with WT1 siRNA or the WT1 overexpression plasmid as described above. The cells were cultured in 100 µl DMEM with 10% FBS for 24, 48, 72, 96 or 120 h. The CCK-8 kit (Beyotime Institute of Biotechnology) was used to evaluate cell proliferation. A total of 10 µl CCK-8 solution was added to each well and the cells were incubated for 3 h at 37°C in 5% CO₂. The cell viability was detected at 450 nm using an EnSpire Multimode Plate Reader (PerkinElmer, Inc., Waltham, MA, USA).

HaCaT cells were seeded in 24-well plates in triplicate and transfected with WT1 siRNA or the WT1 overexpression plasmid as described above. After 24 or 48 h, the level of cellular apoptosis was detected using a Fluorescein Isothiocyanate Annexin V Apoptosis Detection kit II (BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol. The data were acquired using a flow cytometer (BD Canto II; BD Biosciences) and analyzed using FlowJo software version 10 (FlowJo LLC, Ashland, OR, USA).

HaCaT cells were stimulated with 0, 10, 20 or 50 ng/ml IL-17A, IFN-γ or IL-22 (all PeproTech, Inc., Rocky Hill, NJ, USA). The cells were collected after 24 h for RT-qPCR analysis.

Data are presented as the mean ± standard error of the mean of at least three experiments. Statistical analysis was performed using GraphPad Prism 6.0 software (GraphPad Software, Inc., La Jolla, CA, USA) and P<0.05 was considered to indicate a statistically significant difference. The data were assessed for normality of distribution and a similar variance between groups. A two-tailed unpaired Student's t-test was used for comparisons between two groups and one-way analysis of variance with the corresponding post-hoc test (Bonferroni or Dunnett's) were used for the comparison of multiple groups. When the data were not normally distributed or there were not equal variances between two groups, a two-tailed Mann-Whitney U-test was used for statistical analysis. Correlation analysis was performed using a Spearman's r test.

To examine the role of WT1 in the pathogenesis of psoriasis, the mRNA expression level of WT1 was initially determined in 10 non-lesional skins and 20 lesional skins taken from patients with psoriasis and skin samples taken from 20 normal human controls using RT-qPCR. The results demonstrated that the mRNA expression of WT1 was significantly increased in non-lesional skins and lesional skins from patients with PV, compared with the normal controls (Fig. 1A). Similarly, the protein expression of WT1 in non-lesional skins and lesional skins from patients with PV was also increased compared with the normal controls (Fig. 1B and C). The results suggested that WT1 mRNA and protein expression levels in lesional skins were slightly increased compared with those in non-lesional skins from patients with psoriasis, although there were no statistical differences (Fig. 1A-C). Notably, the WT1 mRNA expression level in the psoriatic skin lesions was positively correlated with the PASI scores of the patients (Fig. 1D). In addition, immunohistochemistry with anti-WT1 antibodies was used to detect the expression and location of WT1 in the skin tissues. It was confirmed that the expression of WT1 was primarily enhanced in the epidermis of the psoriatic skin lesions (Fig. 1E). However, no significant differences were observed in the mRNA and protein expression levels of WT1 in the PBMCs of patients with PV compared with the normal controls (Fig. 1F-H).

Furthermore, the present study detected the WT1 expression in an IMQ-induced psoriasis-like mouse model, which closely resembles the human psoriasis phenotype, according to a previously published study (24). IMQ cream was applied to the shaved backs of BALB/c mice for 7 consecutive days to establish this model. As expected, the IMQ-treated mice developed typical psoriasis-like lesions with evident clinical and histological alterations (Fig. 2A and B). The results demonstrated that the expression of Ki67, a marker exclusively associated with cell proliferation, was significantly increased in the IMQ-treated mice compared with the controls, which indicated that the excessive proliferation of keratinocytes was induced by the IMQ (Fig. 2C). Consistent with the results of the human samples, the mice exposed to IMQ expressed significantly higher levels of WT1 mRNA and protein in their non-lesional skins and skin lesions compared with the vehicle-exposed mice (Fig. 2D-G).

Abnormal proliferation and apoptosis of keratinocytes is a pathological hallmark of psoriasis (26). To examine the role of increased WT1 in the pathogenesis of PV, HaCaT cells were transfected with a WT1 overexpression plasmid or a control plasmid. The results of the western blot analysis demonstrated that the WT1 protein was successfully overexpressed in the WT1 expression plasmid-transfected cells (Fig. 3A). A CCK-8 assay was performed to detect the proliferation of HaCaT cells. The results demonstrated that WT1 overexpression promoted the proliferation of HaCaT cells, particularly on days 4 and 5 following transfection (Fig. 3B). Apoptosis analysis was subsequently performed. The results of the flow cytometry revealed that the proportion of Annexin-positive and propidium iodide-negative cells, which represent early apoptotic cells, was significantly decreased in the WT1 plasmid transfection group compared with the control group (Fig. 3C and D).

To further investigate the functional mechanism of WT1 in the pathogenesis of psoriasis, WT1 expression was knocked down in HaCaT cells using siRNA. The western blot analysis demonstrated that WT1 expression was significantly inhibited in the HaCaT cells transfected with WT1 siRNA compared with the negative control (Fig. 4A). The transfected HaCaT cells were harvested at different time points for the CCK-8 assay. The results revealed that the proliferation ability of HaCaT cells was significantly decreased in the WT1 siRNA-transfected group compared with the control group (Fig. 4B). In addition, the effect of WT1 knockdown on the apoptosis of HaCaT cells was detected by flow cytometry, and it was observed that the proportion of early apoptotic HaCaT cells was clearly increased in the WT1 siRNA-transfected group compared with the control siRNA group (Fig. 4C and D).

It has been previously demonstrated that epidermal keratinocytes are responsive to immune cell-derived cytokines, including IFNs, IL-17 and IL-22, which are increased in the skin lesions of psoriasis (27). To investigate the possible mechanisms associated with WT1 upregulation, HaCaT cells were stimulated with IL-17A, IFN-γ and IL-22. The results of RT-qPCR demonstrated that cytokines at higher concentrations, including IFN-γ, IL-17A and IL-22, promoted WT1 mRNA expression (Fig. 5). These results indicated that the overexpression of WT1 may be due to the stimulation of proinflammatory factors in the local skin lesions of patients with psoriasis.