Clinical tissue samples
Five adult cases of psoriatic leision (PS) tissue and the same amount of psoriasis lesions-adjacent normal skin tissue (PN) and healthy control-derived normal skin tissue (NN) were collected from Shanghai Tenth People’s Hospital, none of which had underwent medical treatment. The psoriasis severity was evaluated and graded by the Psoriasis Area Severity Index (PASI). Clinical tissue samples were stored at liquid nitrogen immediately after sampling [13].
Mice
All animal experiments were performed on ICR background mice and approved under the guidelines of the Animal Experimental Ethics Committee of Hubei University of Chinese Medicine. Mice were obtained from Vital River Laboratories (Beijing, China) and were maintained under SPF conditions. The IMQ-induced psoriasis mouse model was induced in 8–12 weeks of age mice. The mice were applied to a daily topical dose of 62.5 mg IMQ cream (5%) (MedShine, #120503; China) on the shaved dorsal skin or 25 mg on ears for 6 consecutive days. As negative controls, wild-type (WT) mice were treated with the same dose of Vaseline cream. Erythema, scales, and thickness were scored independently on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; and 4, very marked.
Cell culture and transfection
Human keratinocyte HaCaT cells were seeded into a six-well plate (5×105 cells/well) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, USA) containing 10% fetal bovine medium with 5% CO2 in a 37°C incubator. miR-214-3p mimics or inhibitors and their negative controls were designed and synthesized by RiboBio Inc. (Ribobio, China). The sequences of miR-214-3p mimic: sense strand: 5′ CGACGUGGUCGGACGGACGACU 3′ and antisense strand: 5′ AGUCGUCCGUCCGACCACGUCG 3′; miR-214-3p inhibitor is single-strand: 5′ AGUCGUCCGUCCGACCACGUCG 3′. When cells grew to approximately 70% confluent, miR-214-3p mimics or its inhibitors or negative controls was transfected at a final concentration of 100 nM using Lipofectamine 3000 Transfection Reagent (Invitrogen, USA) according to the manufacturer's instructions. The cells were then harvested for the extraction of RNA or protein.
RNA extraction and quantitative real-time PCR (qPCR)
Total RNA was extracted using Trizol reagent (TaKaRa, Japan) and chloroform. The concentration of total RNA was measured using Nanodrop 2000/2000c (Thermo Fisher Scientific). To separate the epidermis from the dermis and following epidermis RNA extraction, an overnight incubation of the dorsal skin at 4°C in dispase II (2.5 U/mL, BD, USA) was performed. Complementary DNA was synthesized by the cDNA RT kit (Applied Biosystems, USA). TaqMan probes were used to quantify the expression of miR-214-3p and specific RT primers were used to quantify the expression of other genes. The qRT-PCR was performed using All-in-One™ miRNA qRT-PCR Detection Kits (GeneCopoeia, Inc., USA). TaqMan probes were obtained from Guangzhou RiboBio company. The other primer sequences were listed as follows: FOXM1, forward: 5′- CGTCGGCCACTGATTCTCAAA -3′ and reverse: 5′- GGCAGGGGATCTCTTAGGTTC -3′; Foxm1, forward: 5′- CTGATTCTCAAAAGACGGAGGC -3′ and reverse: 5′- TTGATAATCTTGATTCCGGCTGG -3′; NEK2, forward: 5’- TGCTTCGTGAACTGAAACATCCG -3’ and reverse: 5’- CCAGAGTCAACTGAGTCATCACT-3’; KIF20A, forward: 5’- TTGAGGGTTAGGCCCTTGTTA-3’ and reverse: 5’- GTCCTTGGGTGCTTGTAGAAC -3’; CENP-A, forward: 5’- CTCCCATCAACACAGTCGGC-3’ and reverse: 5’- GAAGTCCACACCACGAGTGA-3’; CENP-F, forward: 5’- ACCTTCACAACGTGTTAGACAG -3’ and reverse: 5’- CTGAGGCTCTCATATTCGGCA-3’; CCNB1, forward: 5’- AACTTTCGCCTGAGCCTATTTT-3’ and reverse: 5’- TTGGTCTGACTGCTTGCTCTT-3’; CCND1, forward: 5’- CAATGACCCCGCACGATTTC -3’ and reverse: 5’- CATGGAGGGCGGATTGGAA-3’; U6, forward: 5’-ATTGGAACGATACAGAGAAGATT-3’ and reverse: 5’-AGGAACGCTTCACGAATTTG-3’; GAPDH, forward: 5′-AACTTTGGGATTGTGGAAGG-3′ and reverse: 5′-ACACATTGGGGGTAGGAACA -3′. U6 and GAPDH were used as endogenous controls. Relative gene expression levels were calculated using the 2−ΔΔCt method.
Cell proliferation, apoptosis, and cell cycle assay
Cell Counting Kit-8 (CCK-8) (Beyotime, China) was used to measure cell proliferation. In brief, the HaCaT cells were seeded in 96-well plates at a density of 5×103 cells/well. After transfecting miR-214-3p mimics or inhibitors or controls for 24, 48, and 72 h, CCK-8 solution (10 μL) was added to each well, and then the plates were incubated for 1 h at 37°C in the incubator. The absorbance was measured at a wavelength of 450 nm using a Microplate Reader (Bio-Rad, USA). For cell cycle assay, transfections were done in 6-well plates (4×105 cells/well). Following transfecting with miR-214-3p mimics or inhibitors or controls for 48 h, the cells were collected, washed in ice-cold phosphate-buffered saline (PBS), and fixed with ice-cold 75% ethanol at -20°C overnight. Then, the cells were washed with ice-cold PBS, centrifuged, and incubated with 0.1% RNase A solution for 30 min at 37°C. Subsequently, cells were incubated in 0.4 mL propidium iodide (PI) at 4°C for 30 min in the dark and the cell cycle distribution was analyzed using FACSAriaTM III Cells (BD, USA). The results were analyzed using Flowjo software. For apoptosis analysis assay, cells were seeded in 12-well plates (2×105 cells per well). After transfected with miR-214-3p mimics or inhibitors or controls for 24 h, cells were stained with Annexin V-FITC/PI Apoptosis Detection kit (Beyotime, China) in the dark for 15 min according to the manufacturer’s instructions. The percentage of early and late apoptotic cells were acquired and analyzed using Flowjo software.
Western blot
HaCaT cells were lysed in RIPA buffer containing proteases and phosphatase inhibitors. The total lysate was denatured for 10 min at 95°C in a metal bath. The concentration of protein was quantified using BCA kit (Beyotime, China) and an equal amount of protein was separated on SDS-PAGE gels. Separated protein bands were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% skimmed milk for 1 h at room temperature, incubated with appropriate primary antibodies FOXM1 (1: 1,000; Cell Signaling Technology, 5436S), NEK2 (1: 1,000; Santa Cruz Biotechnology, sc-55601), KIF20A (1: 1,000; Santa Cruz Biotechnology, A300-879A), CENP-A (1: 1,000; Abcam, ab13939), CENP-F(1: 500; Novus Biologicals, NB500-101), Cyclin B1 (1: 1,000; Santa Cruz Biotechnology, sc-245), Cyclin D1(1: 1,000; Cell Signaling Technology, 2978S), GAPDH (1: 5,000; Cell Signaling Technology, 2118) at 4°C overnight. After three times washing with TBST, the membranes were subsequently incubated with HRP-conjugated secondary antibody (1: 10,000) at room temperature for 1 h. Protein bands were visualized using ECL substrates on Amersham Imager 600 (General Electric Company, USA) and analyzed by Quantity One software (Bio-Rad, USA). GAPDH was used as an endogenous control to obtain the optical density ratio of the detected proteins.
RNA pull-down
Biotinylated antisense-miR-214-3p and miR-214-3p were transcribed in vitro and labeled with Biotin RNA Labeling Mix (Roche, USA). 500 μg whole-cell lysates from HaCaT cells were incubated with 1 μg of labeled RNA for 30 min at 25°C. Then biotin-labeled RNA-protein complex was captured by streptavidin agarose beads (Bimake, USA). Eluted RNA-bound protein was detected by western blotting.
H&E staining
The mouse dorsal and ear skin treated with IMQ or Vaseline cream was fixed with 4% paraformaldehyde at 4°C overnight, followed by dehydration in 30% sucrose and embedded in OCT (SAKURA, USA). Frozen sections (5 μm) were stained with hematoxylin and eosin (H&E, Sigma-Aldrich, 51275, 318906) according to standard procedures.
Luciferase reporter assay
The DNA fragments of FOXM1 3′-UTR were amplified and cloned into pmirGLO Dual-Luciferase vector (Promega, USA) to generate the WT FOXM1 3′-UTR luciferase vector. The mutated FOXM1 3′-UTR luciferase vector was generated by site-directed mutated PCR. HaCaT cells were seeded in a 24-well plate (5×104 cells/well) and then co-transfected with miR-214-3p mimics (50 pmol) and luciferase reporter plasmid (100 ng) and hRluc-neo plasmid (500 ng) using Lipofectamine 3000 (Invitrogen, USA). After 24 h of transfection, the firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, USA). The ratio of firefly luciferase to Renilla luciferase was calculated for each well.
RNA-ChIP
Immunoprecipitation of RNA binding protein–RNA complexes (RNA-ChIP) was performed using EZMagna RIP Kit (Millipore, USA) according to the protocol provided by the kit. In brief, epidermal cells were lysed by RIP lysis buffer after treatment with IMQ or Vaseline cream. Then the lysate was incubated with magnetic beads and anti-Argonaute 2 (AGO2, 5 μg) or control IgG antibody (Abclonal, AC005, 5 μg) with rotation for 6 h at 4°C. The immunoprecipitate was washed and incubated with Proteinase K and then subjected to qRT-PCR.
In vivo administration of miR-214-3p mimics
To overexpress miR-214-3p in the skin, 7 ug miR-214-3p mimics or scrambled controls with the transfecting agent (mirVana; Life Technologies, USA) were injected intradermally into the shaved mouse dorsal skin on days 1, 2, and 5 during the application of IMQ. Mice were sacrificed and analyzed on day 6.
Statistical analysis
All experiments were repeated at least three times. Data were presented as mean ± SEM. The differences between two groups were assessed using the two-tailed Student t-test. Multiple comparisons were analyzed by One-way ANOVA with post hoc Tukey test. GraphPad Prism 8.0 was used for the statistical analysis. p < 0.05 was considered statistically significant.