RAS‐association domain family 1A regulates the abnormal cell proliferation in psoriasis via inhibition of Yes‐associated protein

Abstract Psoriasis is a chronic, inflammatory skin disease with a high incidence and recurrence; however, its exact pathogenesis and aetiology remain unclear. This study aimed to analyse the effect of the upstream negative regulator RAS‐association domain family 1A (RASSF1A) on Yes‐associated protein (YAP) in psoriasis. Skin lesions of 22 patients with psoriasis and 19 healthy controls were used. Human epidermal keratinocytes stimulated by M5 (IL‐1α, IL‐17, IL‐22, TNF‐α and oncostatin M) were used to establish a psoriatic cell model. BALB/c mice treated with topical imiquimod were used to establish a psoriatic mouse model. As the methylation level of RASSF1A increased, its expression in psoriatic patients and mice model decreased. Addition of the methylation inhibitor 5‐Aza‐CdR or RASSF1A‐overexpressing lentivirus vector increased RASSF1A and reduced YAP expression; meanwhile improved skin lesions, reduced cell proliferation, induced cell cycle arrest in the G0/G1 phase, increased apoptosis, reduced inflammatory cytokines and activities of ERK, STAT3 and NF‐κB signalling pathways. The results indicated that RASSF1A could play a role in the treatment of psoriasis by inhibiting YAP expression. Based on these findings, targeted drugs that can inhibit the methylation or increase the expression of RASSF1A may be useful for treating psoriasis.

psoriatic lesions and is involved in the pathogenesis of psoriasis through the regulation of KC proliferation and apoptosis. 6 RASassociation domain family 1A (RASSF1A), the most common negative regulator of RAS, is expressed in most normal tissues but is down-regulated or absent in some cell lines and tissues of tumours. 7 The core of the kinase cascade of the Hippo signalling pathway includes Mst1/2, Sav1, Lats1/2, Mob1, YAP and TAZ. The signalling transduction process of Hippo pathway is composed of a series of kinase cascade phosphorylation reactions and YAP is its downstream effector, 8 which regulates cell proliferation and apoptosis, and regulates the growth, development, and size of tissues and organs.
When phosphorylated by upstream kinases at serine 127, pS127-YAP is inactivated and retained in the cytoplasm, preventing it from functioning as a transcription factor. 8,9 However, the relationship between RASSF1A and the Hippo-YAP signalling pathway remains unclear. Studies have demonstrated that RASSF1A is rarely mutated, and its low or loss of expression is mainly due to hypermethylation of the CpG island in the promoter region. 10 As a negative upstream regulator of Hippo-YAP signalling pathway, [11][12][13]   Hospital of Xi'an Jiaotong University and informed consents were obtained. All the psoriatic specimens were confirmed by pathological examinations. Additionally, 10 psoriasis samples and 10 healthy control samples from the collected samples were frozen in liquid nitrogen for subsequent detection of protein and methylation levels.

| Histology and immunohistochemistry
Tissue samples were paraffin-embedded and sectioned. Hematoxylin and eosin (HE) and immunohistochemical stainings were performed following a standard staining procedure. 14 The thickness of the skin lesions was measured using Image Pro-Plus 6.0 software (Media Cybernetics). Based on the results of the HE staining examined under microscope, the average thickness was determined from mean values in three microscopic fields. The results of immunohistochemistry analysis were classified as negative (−), weakly positive (+), moderately positive (++) and strongly positive (+++) after doubleblind examination by two pathologists. were recognized as the psoriatic cell models. 15 In the present study, we used 10 ng/mL M5 (PeproTech) to stimulate KCs for 48 hours.

| Establishing the psoriatic cell model
The stimulated KCs were divided into three groups, which were then treated with different concentrations of 5-Aza-CdR (5, 10, 20 μmol/L; MedChemExpress), a widely used methylation inhibitor.

| Lentivirus infection
An RASSF1A overexpression vector (the CMV promoter is shown in Figure S1A) was constructed by Shenyang Wanleibio Technology.

| Establishing the psoriatic mouse model
Currently, using topical imiquimod on the backs of mice for seven consecutive days is universally acknowledged as the method to establish a psoriatic animal model. 16 Twenty-five 6-to 8-week-old female BALB/c mice (18-20 g) were routinely raised in specific pathogen-free conditions. All animal experiments were approved by the ethics committee of the Second Affiliated Hospital of Xi'an Jiaotong University. The mice were randomly divided into five groups: control, psoriasis and three 5-Aza-CdR groups (treated with a different concentration of 5-Aza-CdR). In the psoriasis groups, 62.5 mg of imiquimod cream (Hubei Keyi Pharmaceutical Co., Ltd.) was applied daily to the back of the mice. In the control group, an equal amount of Vaseline (Nanchang Baiyun Pharmaceutical Co., Ltd.) was applied daily. In the 5-Aza-CdR group, different concentrations (5, 10 and 20 μmol/L) of 5-Aza-CdR were applied besides imiquimod application. At the end of 7 days, mice were anaesthetized with phenobarbital (Shenyang Wanleibio Co., Ltd.) and euthanized.

| Quantitative real-time PCR (qRT-PCR)
TRIzol (Invitrogen) was used to extract total RNA from cells or skin tissues, and cDNA was obtained by reverse transcription. The sequences of the primers synthesized by Shanghai Biotechnology Co., Ltd. are shown in Supplemental Table S1. The standard curves for each group were obtained through qRT-PCR. After standardization to glyceraldehyde-3-phosphate dehydrogenase levels, relative YAP expression was calculated using the 2 −ΔΔCT method. The experiment was repeated three times, and three technical replicates were included for each data point.

| Western blotting
Total protein from cells or tissues was extracted and quantified.

| Detection of RASSF1A gene promoter methylation
Tissue DNA was extracted according to the manufacturer's instructions (Bioteke). Methylation of the RASSF1A promoter was detected through methylation-specific PCR (MSP). DNA samples were treated using an EZ DNA methylation kit (ZYMO Research). Then, PCR assay and agarose gel analysis were performed. Primer sequences of methylated RASSF1A (M-RASSF1A) and unmethylated RASSF1A (U-RASSF1A) are shown in Table S2.

| Cell proliferation assay
Cells were inoculated into a 96-well plate at the concentration of 5 × 10 3 cells/200 μL culture medium per well. MTT (20 μL) was added to each well 24, 48 and 72 hours later. After 4 hours of incubation in the dark, 150 μL of dimethyl sulphoxide was added to each well, and the plate was shaken for 10 minutes. The absorbance value of each well at 570 nm was measured. Five technical replicates were evaluated for each group. Experiments were repeated three times, and five technical replicates were performed for each data point.

| Cell apoptosis
Cell apoptosis was measured according to the manufacturer's instructions (Jiangsu Kaiji Biotechnology Co., Ltd.). Approximately, 1-5 × 10 5 cells were suspended in 500 μL binding buffer, stained with 5 μL Annexin V-FITC and 5 μL PI staining solution, and incubated in the dark for 5-15 minutes at room temperature. The cell apoptosis was measured by flow cytometry. Three independent experiments were performed.

| ELISA
The concentrations of IL-1 β, IL-6, IL-17 and IL-23 from supernatant of culture medium or mouse serum samples were determined through standard ELISA using an ELISA kit according to the manufacturer's instructions (Lianke Biotech). Optical density (OD) values at 450 nm were then obtained using a spectrophotometer to construct the standard curve. Experiments were repeated three times, and three technical replicates were included for each data point.

| Statistical analysis
All data are presented as the mean ± standard error of the mean. SPSS 19.0 software (SPSS, Inc.) was used for statistical analysis.
Pearson chi-square and Mann-Whitney U tests were used to analyse the immunohistochemistry results. Unpaired Student's t test was used for comparisons between two groups. One-way analysis of variance was used for experiments performed with 3 or more groups and the LSD test was used as a post hoc for multiple comparisons. P < .05 was considered statistically significant.

| Expression and methylation of RASSF1A in psoriasis
The immunohistochemistry results showed that the expression rate of RASSF1A protein was 78.95% (15/19) in normal skin tissues and 50.00% (11/22) in psoriatic lesions (Table 1, Figure 1A). The qRT-PCR and Western blot results showed that RASSF1A mRNA and protein expression in normal skin were higher than that in psoriatic lesions ( Figure 1B,C). MSP assays showed that the methylation level of RASSF1A increased gradually in normal skin tissue, normal tissue beside psoriatic lesions and psoriatic tissues, indicating that the low expression of RASSF1A may be due to promoter methylation ( Figure 1D).

| Validation of RASSF1A overexpression efficiency and its effect on YAP expression
To confirm the effect of RASSF1A overexpression on YAP expression, we infected cells with a RASSF1A-overexpressing lentivirus.
After 48 hours of lentivirus infection, RASSF1A mRNA and protein were overexpressed in the group infected with the RASSF1A overexpression lentivirus compared to their expression in the control group (Ctrl) and the group infected with empty virus vector (BLK).

| Effect of 5-Aza-CdR on RASSF1A and YAP expression in psoriatic cells
The MSP results showed that different concentrations of 5-Aza-CdR dose-dependently reduced the RASSF1A methylation level in psoriatic cell model induced by M5 ( Figure 1F). Western blotting showed that 5-Aza-CdR dose-dependently increased RASSF1A expression in psoriatic cells; the level did not return to pre-M5 levels.

| Effect of RASSF1A overexpression and 5-Aza-CdR on cell proliferation
The results of MTT showed that cell proliferation increased at all time points after adding M5, which is consistent with the characteristic of abnormal proliferation in psoriasis. 5-Aza-CdR dosedependently inhibited cell proliferation, but the level did not return to pre-M5 levels. The cell proliferation ability also increased following incubation with M5. After RASSF1A overexpression, the proliferation of cells decreased significantly, but did not return to pre-M5 levels ( Figure 2A).

| Effect of RASSF1A overexpression and 5-Aza-CdR on cell cycle
Results showed that the proportion of cells in the G0/G1 phase decreased and that of cells in the S phase increased after adding M5, indicating that the proportion of actively replicating cells was increased. This is consistent with the abnormal psoriasis cell proliferation model (Figure 2A). Adding 5-Aza-CdR increased the G0/G1 phase ratio and decreased the S phase ratio, which became more obvious with increasing 5-Aza-CdR concentrations but did not

| Effect of RASSF1A overexpression and 5-Aza-CdR on cell apoptosis
The results showed that the KC apoptosis rate decreased when M5 was added and increased when 5-Aza-CdR or RASSF1A was added, but did not return to pre-M5 levels ( Figure 2D; Figure S1E). Further, Western blot analysis showed that expression of the apoptosisrelated proteins cleaved-caspase-3 (c-caspase-3), BAX, p53 and p21 was decreased, whereas that of Bcl-2 and p73 was increased.

| Effect of 5-Aza-CdR on YAP and RASSF1A expression in psoriatic mouse model
Immunohistochemistry analysis confirmed that RASSF1A expression in the Pso was lower than that in the Ctrl in mice ( Figure 4A) and similar to that in human samples ( Figure 1A). Addition of 5-Aza-CdR increased the positive rate of RASSF1A expression, and the trend was more obvious with increasing 5-Aza-CdR concentrations.
Semiquantitative analysis showed that the intensity of RASSF1A expression in the epidermis of the Ctrl group significantly differed from that in the Pso group (P = .032). Moreover, the expression rate of YAP in the Pso group was higher than that in the Ctrl group. The positive rate of YAP expression decreased after adding 5-Aza-CdR, and the trend was more obvious with increasing 5-Aza-CdR concentrations ( Figure 4B). Semiquantitative analysis of the expression intensity showed that RASSF1A expression in the Ctrl group was significantly higher than that in the Pso group (P = .009), and there was a significant difference between the Pso group and the 10 μmol/L 5-Aza-CdR group (P = .031) or 20 μnol/L 5-Aza-CdR group (P = .013) ( Table 3). MSP confirmed that 5-Aza-CdR reduced RASSF1A methylation ( Figure 4C), and Western blotting confirmed that 5-Aza-CdR induced RASSF1A expression while reducing YAP expression and driving pS127-YAP, p-LATS1/2 and p-MST1/2 in a dose-dependent manner ( Figure 4D).

| Effects of RASSF1A overexpression and 5-Aza-CdR on cytokine expression
ELISA results showed that the concentrations of IL-1β, IL-6, IL-17 and IL-23 in the supernatant of the psoriatic model of cells or mouse serum were increased. RASSF1A overexpression or addition of 5-Aza-CdR to the cells and topical 5-Aza-CdR administration on the back of mice reduced cytokine expression to different degrees but the levels did not return to the Ctrl level ( Figure 5A,B). This suggests that RASSF1A reduces the expression of these cytokines at different degrees to reduce the inflammatory response in psoriasis.

| Effect of RASSF1A overexpression and 5-Aza-CdR on the expression of various signalling pathways
To explore which signalling pathways are involved in the effect of and STAT3 levels were basically unchanged, indicating increased activities of these signalling pathways. However, RASSF1A overexpression or addition of the methylation inhibitor 5-Aza-CdR reduced the expression of p-ERK, p-STAT3, NF-κB P65 (n) and increased that of NF-κB P65 (p); changes in the expression of p-AKT were less obvious ( Figure 5C).

| D ISCUSS I ON
Our previous study showed that YAP expression increased in pso- In this study, we found that the expression of RASSF1A decreased, whereas its methylation increased in psoriasis lesions compared to that in normal skin tissues. Thus, we predicted that a relationship exists between the expression of RASSF1A and YAP.
We assumed that loss of RASSF1A expression is mainly caused

DATA AVA I L A B I L I T Y S TAT E M E N T
No data have been shared during the current study.