Saturday, June 10, 2023

SLC35E1 promotes keratinocyte proliferation in psoriasis by regulating zinc homeostasis – Cell Death & Disease

Human subjects

Psoriatic skin samples were obtained by punch biopsy from patients under local lidocaine anesthesia. Normal adult human skin specimens were acquired from healthy donors who were undergoing plastic surgery. The objectives, procedures, and potential risks were verbally explained to all the participants. Written informed consent (in Chinese) was obtained from all the participants before inclusion in the study.

Animal model

Slc35e1-knockout (KO) mice, generated through the CRISPR-Cas9-mediated deletion of exon 2 to exon5 of the Slc35e1 gene, were obtained from Cyagen (USA) and were maintained under specific pathogen-free conditions. To generate heterozygotes, Slc35e1−/− mice were crossed with C57BL/6J mice obtained from the Animal Experiment Centre of Southern Medical University, GuangZhou, China. The heterozygotes were crossed to generate wild-type (WT) and Slc35e1−/− mice. All animal experiments were performed in compliance with the Southern Medical University Animal Care and Use Committee guidelines. Mice were housed in individually ventilated cages at a maximum density of five mice per cage and kept on 12h-12 h light-dark cycle. Room temperature was maintained at 22 °C ± 1 °C with 30–70% humidity. Mice were fed ad libitum with rodent diet and water. None of the mice were involved in any previous procedures before the study. For the IMQ-induced model of psoriasis, sexual matched mice were mated at 7–9 weeks of age. The mice were administered a daily topical dose of 15 mg of IMQ cream (5%) per ear.

Flow cytometry

Single-cell suspensions of the dorsal skin and ears were generated. The epidermis and dermis of the dorsal skin and ears of WT and Slc35e1−/− mice were separated using 2.5 mg/ml dispase II (Sigma, USA) at 37 °C for 1 h, digested with collagenase type IV at 37 °C for respectively 1 and 2 h to generate single-cell suspensions, and stained with fluorophore-conjugated monoclonal antibodies against CD45 (103108, Biolegend), MCH-II (107622, Biolegend), F4/80 (17-4081-82, eBioscience), γδTCR (118131, Biolegend), Ly6G (127618, Biolegend), CD207 (144204, Biolegend), and CD11b (103208, Biolegend). After washing and resuspending, the cells were assayed with a BD LSRFORTESSA flow cytometer, and the data were analyzed using FlowJo software.

For zinc ion staining, mouse epidermal cells or Normal Human Epidermal Keratinocytes (NHEKs) were incubated with 10 µM FluoZin-3, AM (Invitrogen, USA) for 1 h at 37 °C in an incubator with 5% CO2. Then zinc ions were detected by flow cytometry.


Paraffin-embedded skin specimens were sectioned, stained with hematoxylin and eosin (H&E), and visualized using a light microscope (Nikon, Eclipse 80i, Japan). Epidermal thickness (×100 magnification) was measured using ImageJ software using the following formula: thickness (µm) = area (µm2) / length (µm).

RNA extraction and qPCR

Total RNA was extracted using TRIzol reagent (Yeasen Biotech, cat. 10606ES60) and reverse transcribed into cDNA using the Evo M-MLV RT Kit with gDNA Clean for qPCR II (Accurate Biotechnology cat. AG11711). Real-time quantitative PCR (qPCR) was conducted in a LightCycler 96 (Roche, Basel, Switzerland) using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology cat. AG11701). The relative expression levels of target genes were normalized to those of GAPDH and quantified using the 2−ΔΔCt method. The sequences of the primers used for qPCR are presented in Supplementary Table 1.


Total RNA was extracted from epidermal cells isolated from untreated and IMQ-treated WT and Slc35e1−/− mice (three repetitions per group) using TRIzol Reagent (Invitrogen cat. 15596026). Total RNA was quantified and qualified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), a NanoDrop (Thermo Fisher Scientific Inc.), and 1% agarose gel electrophoresis. Total RNA (1 μg) with a RIN > 6.5 was used for subsequent library preparation. Poly(A) mRNA was isolated using a Poly(A) mRNA Magnetic Isolation Module or a rRNA Removal Kit. mRNA fragmentation and priming were undertaken using First-Strand Synthesis Reaction Buffer and Random Primers. First-strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The double-stranded cDNA was bead-purified and treated with End Prep Enzyme Mix to repair both ends and add a dA-tail in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of adaptor-ligated DNA was then performed using beads and fragments of ~420 bp (with an insert size of ~300 bp) were recovered. Each sample was amplified by 13 cycles of PCR using P5 and P7 primers. Both primers carry sequences that can anneal to the flow cell for bridge PCR, while the P7 primer carries a six-base index allowing for multiplexing. The PCR products were cleaned using beads, validated using a Qsep100 (Bioptic, Taiwan, China), and quantified using a Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).

Libraries with different indices were multiplexed and loaded on an Illumina HiSeq instrument according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was carried out using a 2× 150-bp paired-end configuration while image analysis and base calling were performed by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument. For the psoriasis analysis, two RNA-Seq datasets (accession numbers: GSE54456 and GSE121212) were used, one for normal skin samples and one for psoriasis skin samples.

RNA-Seq analysis

First, reference genome sequences and gene model annotation files of relative species were downloaded from the UCSC, NCBI, and ENSEMBL genome browsers. Then, Hisat2 (v2.0.1) was used to index reference genome sequences. Finally, clean data were aligned to the reference genomes using Hisat2 software (v2.0.1). Transcripts in FASTA format were initially converted from a known gff annotation file and correctly indexed. Then, with this file serving as a reference gene file, gene and isoform expression levels were estimated from the pair-end clean data using HTSeq (v0.6.1). The DESeq2 Bioconductor package, which uses the negative binomial distribution, was used for differential expression analysis. An adjusted p-value (padj) of <0.05 was used to detect differentially expressed genes. GOSeq (v1.34.1) was used to identify enriched Gene Ontology (GO) terms among the differentially expressed genes at a padj of <0.05. TopGO was used to plot DAG. In-house scripts were used for KEGG pathway prediction for the differentially expressed genes.

EdU cell proliferation assay

The thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) has a terminal alkyne group replacing a methyl group at the 5-position of the pyrimidine ring and can be readily incorporated into DNA during synthesis [18]. EdU staining was used for detecting DNA synthesis in proliferating cells in vivo and in vitro. In vivo, EdU was intraperitoneally injected into mice 48 h before euthanasia. In vitro, keratinocytes were incubated with EdU for 2 h at 37 °C with CO2. Skin and keratinocytes were then stained with EdU using an EdU Cell Proliferation Kit (Beyotime,China).


After collection, mouse tissues were immediately embedded in OCT Compound (SAKURA cat. 4583), frozen at −80 °C, and cryosectioned into 7-µm slices using a freezing microtome (Leica, CM1900, Germany). The slices were blocked in 5% BSA for 45 min, incubated overnight with primary antibody at 4 °C, washed with PBS, and stained with fluorochrome-conjugated secondary antibody (mouse anti-rabbit antibody, Life Sciences, cat A11008; 1:500 dilution) for 1 h at room temperature in the dark.

For zinc ion staining, NHEKs were incubated with 10 µM FluoZin-3, AM (Invitrogen) for 1 h at 37 °C in an incubator with 5% CO2.

Western blot

Total protein was extracted from skin using ice-cold RIPA lysis buffer, snap-freezing, and mechanical shearing. Protein supernatants were separated on 10% gradient polyacrylamide gels and then transferred to PVDF membranes (Millipore). After blocking with 5% non-fat dry milk for 1 h at room temperature, the membranes were incubated first with primary antibodies against SLC35E1 (Abcam, ab12150, 1:1000) or GAPDH (Proteintech, 60004-1-Ig, 1:5000) overnight at 4 °C, and then with secondary antibody for 1 h at room temperature. The signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore).

Replicates and statistical analysis

Error bars in this study indicate means ± S.D. for qPCR. Unpaired, two-tailed Student’s t-tests were used for comparisons between two groups of at least three independent biological samples each. P-values < 0.05 were considered significant. All statistical analyzes were performed with GraphPad Prism 8 software.

Source link

Related Articles

Leave a Reply

Stay Connected

- Advertisement -spot_img

Latest Articles

%d bloggers like this: