Patients and tissue samples collection
A total of 7 psoriatic samples were collected from patients with vulgaris psoriasis from Qilu Hospital of Shandong University. The patient did not receive systemic and local treatment within 3 months. All of them had typical psoriasis vulgaris clinical characteristics. 10 normal tissues were collected from healthy volunteers. Healthy subjects had no family history of psoriasis or any other autoimmune diseases. This study was approved by the Ethics Committee of Shandong University, Qilu Hospital (Jinan, China) and written informed consent was obtained from all patients.
Affymetrix GeneChip Human Gene 2.0 ST Array (Invitrogen) was used for circRNAs expression profiling. Cells were cryopulverized and homogenized using the Biopulverizer™ (Biospec) and Mini-Bead-Beater16 (Biospec), respectively. The homogenized samples were separated. RNA was precipitated, washed with 75% ethanol, and dissolved in RNase-free water. Quick Amp Labeling Kit (Agilent p/n 5190-0442) was used to label the reaction. Purified the labeled/amplified RNA and then measured the labeled cRNA quality. Hybridization was performed using Agilent Gene Expression Hybridization Kit (Agilent p/n 5188-5242). The results were detected by Agilent microarray scanner (Agilent p/n G2565BA).
Cell isolation and culture
The skin specimens of young children’s foreskins were got from Qilu Hospital. The specimens were digested with dispase II (Sigma, lot#BCBR9297V) at 4°C overnight to dissect the epidermis from the dermis. Then, the epidermal specimens were digested with a 0.25% trypsin-0.01% EDTA mixture (37°C, 10-15 min) to obtain single cell suspensions. Cells were grown and maintained in keratinocyte medium (ScienCell, CA, USA). The purified NHEKs were obtained after 2–3 passages, and the third passage of NHEKs was used for the subsequent experiments. Human immortalized keratinocyte (HaCaT) cells were purchased from Procell Life Science& Technology Co., Ltd, and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) containing 10% Fetal Bovine Serum (FBS) (Sangon Biotech, China), 100 μg/ml streptomycin and 100 U/ml penicillin. Human keratinocyte cell line hTert (Ker-CT)(ATCC® CRL4048™) from American Type Culture Collection (ATCC) was cultured in KGM-Gold™ with BulletKit™ (Lonza 00192060). All cells were incubated in a humidified chamber at 37℃ with 5% CO2.
RNA fluorescence in situ hybridization (FISH)
Cy3-labeled probes were designed and synthesized by RiboBio (RiboBio Biotechnology). RNA FISH was conducted using the Fluorescent in situ Hybridization Kit (RiboBio Biotechnology). Briefly, tissue paraffin sections and cell samples were fixed with 4% paraformaldehyde and digested by proteinase K. Blocking by the prehybridizing solution and then hybridized overnight by the Cy3-labeled SPRR2C probe at 4°C. Images were acquired by the A1RþMP Confocal Laser Microscope System (Nikon).
RNA extraction, qRT-PCR, nuclear-cytoplasmic fractionation, RNase R treatment, and nucleic acid electrophoresis assays
Total RNA was extracted from tissues and cells with Trizol reagent (Invitrogen, Carlsbad, CA, USA) and was reverse transcribed to cDNA using a Transcriptase Kit (Takara, Otsu, Japan). qRT-PCR was carried out using the TB Green PCR Master Mix (Takara) in CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad). GAPDH and U6 were used as internal controls respectively. The primers used are listed in Supplementary Table 1. The relative gene expression was calculated with 2 –ΔΔCT method. RNAs from the nucleus and cytoplasm of HaCaT and Ker-CT cells were separated by the Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek Corporation, St. Catharines, Ontario, Canada) following the manufacturer's instructions. RNase R treatment was executed at 37 °C with 4 U/µg of RNase R (Geneseed Biotech Co., Ltd., Guangzhou, China), for 30 min. The cDNA and genomic DNA (gDNA) of circOAS3 and GAPDH from HaCaT and Ker-CT cells were amplified by divergent primers and convergent primers, respectively. PCR products were detected with 2% agarose gel electrophoresis at 90 V for 40 min. The bands were observed by UV irradiation. All the experiments were repeated three times.
Construction of psoriatic model in vitro
When cell confluence reached about 60-70%, cells were starved in serum-free DMEM for 12 h. Then M5 (interleukin‐17A [IL‐17A], tumor necrosis factor‐α [TNF‐α], IL‐1α, IL‐22 and Oncostatin‐M, 10 ng/mL of final concentration; PeproTech), a cocktail of cytokines, was used to induce a psoriatic-inflammation-like condition in NHEK, HaCaT and Ker-CT cells in serum-free DMEM for another 24 h.
For gene silencing, small interfering RNA (siRNA) (GenePharma, Shanghai, China) of circOAS3 or Hsc70 was transferred into cells using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, USA). For gene overexpression, cells were transfected with the eukaryotic expression vector pcDNA3.1 (GenePharma) and (Supplementary Fig. S1) by using Jet PRIME transfection reagent (Polyplus, USA). Cells were collected for further treatment or analysis at different time points after transfection. These transfection methods were performed according to the manufacturers’ instructions.
Total proteins of cells were prepared using RIPA lysis buffer (Beyotime, Beijing, China) following the manufacturer’s protocols. Equal amounts of protein were loaded on an SDS-PAGE and then transferred electrophoretically to PVDF membrane (Millipore, USA). After blocking with TBST (5% milk), the membranes were incubated overnight with primary antibody (1:1000) at 4 °C. After washing and incubation, the membranes were incubated with secondary antibody (1:2000) in TBST. Protein expression levels were detected by ECL Plus (Millipore, Billerica, MA, USA) with a Bio-Imaging System. The following primary antibodies were used in this study: p-JNK1/2, JNK1/2, p-STAT3, STAT3 (all from Cell Signaling Technology, Boston, USA), Hsc70, IL-6, cyclinD1, Histone3, and GAPDH (all from Abcam, Cambridge, UK). p-NF-κB，NF-κB (all from Santa Cruz Biotechnology, Texas, USA).
CCK-8 assay and EdU assay
Cell counting kit‐8 (CCK‐8) assay was measured using Cell Counting Kit-8 (CCK8) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) at 24, 48, and 72 h after being transfected as described above. CCK-8 solution was added and incubated for b1h at 37 °C in the dark. The Optical density (OD) value was measured at 450 nm wavelength by microplate reader (BioTek, U.S.A.). For the EdU assay, 1×105 cells were inoculated to 24-well plates using an EdU cell proliferation kit (Ribobio, Guangzhou, China). The percentage of EdU-positive cells was counted in four random fields per well.
Flow cytometry analysis of cell apoptosis and cell cycle
Cellular apoptosis was detected using the Annexin-V-FITC Apoptosis Kit (Solarbio, Beijing, China) following the manufacturer’s instructions. The cell cycle was detected using a DNA staining cell cycle kit (Solarbio).
Enzyme-linked immunosorbent assay (ELISA)
After 24 h of transfection as mentioned above, cells were stimulated by M5, and culture supernatants were collected after 24 h. The secretions of IL-6 were measured by using specific ELISA kits (Elabscience, China). The absorbance at a wavelength of 450 nm was detected with microplate reader (BioTek, U.S.A.).
RNA pull-down assay and mass spectrometry
The interaction between circOAS3 and RNA-binding protein was detected by Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocols. Biotin-labeled probes targeting the junction site of circOAS3 were synthesized by RiboBio (Guangzhou, China) and a control probe was used as a control. Linear circOAS3 was transcribed with Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Thermo Fisher Scientific, USA), circularized using T4 RNA ligase I and digested by RNase R. The cells were lysed and incubated with a biotin-labeled circOAS3 probe. Afterward, cell lysates were subjected to streptavidin agarose magnetic beads at normal temperatures. Finally, interacting proteins were identified by mass spectrometry and Western blot. The sequences of the probe were shown in Supplemental Table 2.
RIP was executed by the Magna RIP kit (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. HaCaT and Ker-CT cells were lysed with RNA lysis buffer, then cell lysates were incubated with the RIP buffer containing magnetic beads conjugated to anti-Hsc70 (Abcam, #ab51052) or negative control IgG antibody (Millipore, Billerica, MA, USA) for 4 h at 4°C. After washing three times with washing buffer, western blot and qRT-PCR were implemented to detect enriched circOAS3
All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software). Data from at least three independent experiments were presented as the mean +/- standard deviation (SD). Comparisons were performed using Student’s t-test, and p < 0.05 was considered statistically significant.