2.1 Patient samples
Skin lesions of 22 psoriasis patients (12 males and 10 females) were collected from the Department of Dermatology tissue bank at the Second Affiliated Hospital of Xi'an Jiaotong University from September 2016 to August 2019, and 19 normal skin tissues (10 males and 9 females) from cosmetic surgeries were selected as controls. The study was approved by the ethics committee of the Second Affiliated Hospital of Xi’an Jiaotong University. Specimens were obtained with informed consent, and confirmed by pathomorphological examination. Tissue samples were paraffin-embedded and sectioned for immunohistochemical staining. The results were classified as negative (-), weakly positive (+), moderately positive (++), and strongly positive (+++) after double blind examination by 2 pathologists. Additionally, 10 psoriasis samples and 10 healthy controls were frozen in liquid nitrogen for subsequent protein and methylation level detection.
2.2 Establishing the psoriatic cell model
Human epidermal KCs (ScienCell) were routinely cultured in KC medium (ScienCell). We used 10 ng/mL M5 (mixture of IL-1 α, IL-17, IL-22, TNF-α and oncostatin M, Peprotech) to stimulate the cells for 48 h using the scheme for establishing a psoriatic cell model12. 5-Aza-CdR is a widely used methylation inhibitor. Three groups with different concentrations (5, 10, 20 µmol/L) of 5-Aza-CdR (MedChemExpress) were set-up when M5 was added.
2.3 Lentivirus infection
RASSF1A overexpression vector was constructed by Shenyang Wanleibio Technology. Cells were adjusted in the logarithmic growth period and infected with lentivirus according to the manufacturer’s instructions. RASSF1A and YAP expression was detected 48 h after infection.
2.4 Establishing the psoriatic mouse model
Twenty-five 6-8-week-old female BALB/c mice (18‒20 g) were selected and fed under specific pathogen-free conditions. Experiments were approved by the ethics committee of the Second Affiliated Hospital of Xi’an Jiaotong University. After 1 week of adaptive feeding, the mice were randomly divided into 5 groups: control, psoriasis, and three 5-Aza-CdR groups with different concentrations. In the psoriasis group, 62.5 mg of imiquimod cream (Hubei Keyi Pharmaceutical Co., Ltd.) was applied daily to the back13. In the control group, equal amount of vaseline (Nanchang Baiyun Pharmaceutical Co., Ltd.) was applied daily. In the 5-Aza-CdR group, different concentrations (5, 10, 20 µmol/L) of 5-Aza-CdR were prepared with dimethyl sulfoxide (DMSO) and applied with imiquimod. Seven days later, the mice were anesthesia with phenobarbital (Shenyang Wanleibio Co., Ltd) and then sacrificed, and skin lesions taken.
2.5 Quantitative real-time PCR (qRT-PCR)
Trizol (Invitrogen) was used to extract the total RNA from cells in each experimental group, and cDNA was obtained by reverse transcription. PCR primers were synthesized by Shanghai Biotechnology Co., Ltd. The primer sequence is shown in Supplemental Table S1. The standard curve for each group was obtained by qRT-PCR, and the CT value was computer-analyzed. After standardization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), relative expression was calculated with the 2-ΔΔCT method.
2.6 Western blot
Total protein was extracted and quantified. Twenty-five micrograms of protein was separated by 10% g polyacrylamide gel electrophoresis (PAGE), and then transferred to a nitrocellulose membrane. Skim milk 5% was used to block non-specific antigen binding, and then primary antibodies were added (RASSF1A, Abcam, ab23950; YAP, Cell Signaling Technology, #4912; Cyclin A, Santa Cruz, sc-596; Cyclin B1, Proteintech, 55004-1-AP; Cyclin D1, Santa Cruz, sc-246; Cyclin E, Santa Cruz, sc-25303; CDK1, Wanleibio, WL02373; cleaved-caspase-3, Wanleibio, WL02348; Bcl-2, Wanleibio, WL01556; BAX, Proteintech, 50599-2-lg; p53, Santa Cruz, sc-126; p21, Wanleibio, WL0362; AKT, Wanleibio, WL0003b; p-AKT, Wanleibio, WLP001a; ERK, Wanleibio, WL01864; p-ERK, Wanleibio, WLP1512; STAT3, Wanleibio, WL03207; p-STAT3, Wanleibio, WLP2412; NF-κB P65, Wanleibio, WL01980; β-actin, Santa Cruz, sc-47778; Histone H3, Wanleibio) and incubated at 4 ℃ overnight. Next, secondary antibodies were added and incubated at room temperature for 1 h, and finally exposed on X-ray film.
2.7 Detection of RASSF1A gene promoter methylation
Tissue DNA was extracted according to the manufacturer’s instructions (bioteke). Methylation of RASSF1A promoter was detected by methylation-specific PCR (MSP). Flowing treatment of DNA samples with EZ DNA methylation kit (ZYMO), PCR assay and agarose gel analysis were performed. Primer sequences of methylated RASSF1A (M-RASSF1A) and unmethylated RASSF1A (U-RASSF1A) primers are shown in Supplemental Table S2.
2.8 Cell proliferation assay
MTT assay was used to assess cell proliferation. Cells were taken in the logarithmic growth period and inoculated on a 96-well plate with 5 × 103 cells/ 200 µL culture medium per well. After 24, 48, and 72 h of culture, 20 µL of MTT was added into each well. After 4 h of incubation in the dark, 150 µL DMSO was added into each well and shaken for 10 min to fully dissolve the crystals. The 490 nm absorbance value of each well was measured by a microplate reader. Five technical replicates were set-up for each group.
2.9 Cell cycle
Cells were digested according to the cell cycle kit manufacturer’s instructions (Jiangsu Kaiji Biotechnology Co., Ltd.), washed twice with precooled phosphate buffered saline (PBS), and fixed overnight with 75% ethanol at -20 ℃. Then, 100 µg/mL RNaseA and 50 µg/mL propidium iodide (PI) staining solution were added, and cells were incubated at 37 ℃ in the dark for 30 min. Cell cycle was measured by flow cytometry (FACSCalibur, BD Biosciences).
2.10 Cell apoptosis
Cells were digested according to the cell apoptosis kit manufacturer’s instructions (Jiangsu Kaiji Biotechnology Co., Ltd.) and washed twice with precooled PBS. Cells (1‒5 × 105) suspended in 500 µL binding buffer were collected, stained with 5 µL Annexin V-FITC and 5 µL PI staining solution, and then incubated at room temperature in the dark for 5‒15 min. Cell apoptosis was measured by flow cytometry.
2.11 ELISA
Using the ELISA Kit’s instructions (Lianke Biotech), supernatant or mouse serum samples were detected by standard ELISA. Optical density (OD) 450 nm values were then obtained using a spectrophotometer to draw the standard curve. The concentrations of IL-1 β, IL-6, IL-17, and IL-23 were then measured.
2.12 Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). SPSS 19.0 software was used for statistical analysis. Pearson chi-square and Mann-Whitney U tests were used to analyze the immunohistochemistry results. The Student t-test was used for comparisons between 2 groups. One-way analysis of variance (ANOVA) was used for experiments with more than 2 groups and least significant difference (LSD) test was used as a post-hoc for multiple comparisons. P < 0.05 was considered statistically significant.