Cell lines and cell culture
The human keratinocyte cell line HaCaT, murine keratinocyte cell line JB6 Cl 41-5a, human squamous cell carcinoma cell line A-431 and HEK293T were maintained in Dulbecco’s modified Eagle’s medium (DMEM, 01-052-1A, VivaCell, Shanghai, China) with 10% fetal bovine serum (04-001-1A, VivaCell, Shanghai, China) and 1% antibiotics (10,000 μg/ml streptomycin and 10,000 units/ml penicillin, 03-031-1B, Biological Industries, Israel) at 37 °C and 5% CO2.
Antibodies and reagents
Chemical reagents, including Tris base, glycine, NaCl, and SDS for molecular biology and buffer preparation, were purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies used were mouse monoclonal anti-CD147 antibody (1:1000, ab666, Abcam, MA, USA), rabbit monoclonal anti-phospho-RSK2 (Ser227) antibody (1:1000, 3556 s, Cell Signaling Technology, MA, USA), rabbit monoclonal anti-RSK2 antibody (1:1000, 5528 s, Cell Signaling Technology, MA, USA), mouse monoclonal anti-c-fos antibody (1:1000, 66590, proteintech, IL, USA), mouse monoclonal anti-CD33 antibody (abcam, ab11032, MA, USA) and mouse monoclonal anti-GAPDH antibody (1:3000, 60,004-1-Ig, Proteintech, IL, USA).
Transgenic mouse model
Xenograft tumor models were constructed by Shanghai Model Organisms (Shanghai, China). Briefly, for CD147 epidermal overexpressing (EpiCD147-OE) mice, CD147 genomic DNA was inserted into pCAG-pcDNA3.1 (pCAG-CD147), followed by digestion with pvul. The 5.3-kb fragment was isolated and purified before microinjection into fertilized one-cell stage mouse oocytes. Transgenic mice were generated from C57BL/6 J embryos by standard microinjection procedures. The transgenic line EpiCD147-OE was established by breeding heterozygous mice with C57BL/6 J mice. Littermates were used as controls. For CD147 epidermal knockout transgenic mice (KRT14 Cre+/CD147fl/fl, EpiCD147-KO), a donor vector including a 4.5-kb 5′ arm of homology, 2.0-kb loxP and a 4.6-kb 3′ arm of homology was constructed by in-fusion cloning. Cas9 mRNA, gRNA and donor vector were microinjected into fertilized C57BL/6 J oocytes to obtain CD147fl/+ mice. Chimeras were then mated to KRT14 Cre+ mice to generate KRT14 Cre+/CD147fl/+ or KRT14 Cre−/CD147fl/+. KRT14 Cre+/CD147fl/+ and CD147fl/+ mice were mated to generate KRT14 Cre+/CD147fl/fl and KRT14 Cre−/CD147fl/lf, respectively. To identify transgenic animals, DNA was extracted from the tail biopsies of 3-week-old mice and analyzed by PCR with probes and primers (P1 and P2 for EpiCD147-OE, P3 and P4 for EpiCD147-KO) designed to discriminate between human and murine genomic CD147 DNA, as shown below.
P1: 5’CGATCACGAGACTAGCCTC’3.
P2: 5’GTCATCTGCGTCCACTATGT’3.
P3: 5’AGCGATGGATTTCCGTCTCTGG’3.
P4: 5’AGCTTGCATGATCTCCGGTATTGAA’3.
DMBA/TPA skin carcinogenesis model
The backs of the mice were shaved 2 days prior to the start of the treatment, and then the mice received 250 μg/mL DMBA in acetone (200 μl/mouse) for 2 weeks, followed by 50 μg/mL TPA in acetone (50 μl/mouse) every 3 days for 24 weeks. The appearance of papillomas > 2 mm in diameter was recorded every week from the first appearance at 8 weeks. Tumors were measured by calipers, and tumor volume was calculated using the following formula: length × width × height × 0.52. After 24 weeks, the mice were sacrificed, and the tumor tissues were harvested and fixed in 10% buffered formalin, embedded in paraffin, sectioned at a thickness of 5 μm, and stained with H&E or subjected to immunohistochemical analysis.
Flow cytometry
The skin lesions of mice were digested with collagenase IV (2 mg/mL) and DNAse I (50 U/ml) for 2 hours. Then, the cell suspensions were blocked and stained with reagents from the Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies) to distinguish between live and dead cells. Then cells were labeled with indicated antibodies, including CD45 (103116, Biolegend), Gr-1 (108428, Biolegend), CD11b (101208, Biolegend) for 30 min on ice. Samples were then analyzed on flow cytometer.
Protein preparation and Immunoblotting
Cells were lysed in RIPA buffer, and protein concentrations were determined by a BCA Protein Assay Kit (Santa Cruz, CA, USA). Membrane protein was purified by Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Scientific™, MA, USA). A total of 30-50 μg of protein was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). Immunoreactions were detected by an imaging system (Bio-Rad, USA).
Electrophoretic mobility shift assay
Nuclear protein was extracted by NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (78835, Thermo Scientific™, MA, USA) under the guidance of the manufacturer’s instructions. Three microgram of nuclear protein was subjected to AP-1 kit (AP-1 IRDye 700, 829-07925, LI-COR Biosciences, USA) according to the manufacturer’s instructions.
Cell proliferation assays
Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) assay (Bimake, USA) according to the manufacturer’s instructions. Cells were seeded into 96-well plates at 3000 cells/well and cultured for 24, 48, 72, or 96 h. Then, 10 μL of CCK-8 solution was added to each well, and the 96-well plate was incubated for 2 h at 37 °C and 5% CO2. The fluorescence of each plate was measured using a spectrophotometer at an emission wavelength of 450 nm (Beckman, USA). Six replicates per sample were analyzed.
Plasmid and Lentiviral vector construction
Lentivirus plasmids including pLKO.1, pSPAX2, pMD2G and CD147 shRNAs were purchased from Thermo Scientific, MA, USA. The pLVX-CD147-puro (CD147-overexpressing lentivirus plasmid) was constructed in our lab. For the transfection experiments, cells were transfected with different plasmids using TurboFect Transfection Reagent (Thermo Scientific, MA, USA). The reagent and DNA were diluted in DMEM and incubated for 20 min. The mixture was added to cells growing in the plates for 36 to 48 h to facilitate transfection. To establish stable CD147 knockdown and overexpression cells, pLKO.1-shCD147 or pLKO.1-shMock and pLVX-CD147-Puro or pLVX-IRES-Puro plasmids were cotransfected with packaging plasmids (pSPAX2 and PMD2G) into 293 T cells. The supernatant fractions containing lentiviral particles were collected separately at 48 and 72 h, and JB6, HaCaT and A431 cells were infected with lentiviral particles in medium supplemented with 10 μg/mL polybrene. At 16 h after infection, the medium was replaced with fresh medium containing a suitable concentration of puromycin. The appropriate experiments were performed with these cells until all control cells (uninfected) were dead (usually 36-48 h) in the puromycin-containing medium.
Transwell invasion assay
For the invasion assay, a Transwell experiment was performed with 8 μm pore chambers inserted into 24-well plates (Corning, NY, USA). Matrigel (BD Biosciences, NJ) was diluted (1:7) in serum-free DMEM and was then added to each chamber and allowed to solidify completely. Transfected cells were obtained, resuspended in serum-free medium at a concentration of 4 × 104/100 μL and seeded in the upper chambers, while 550 μL of DMEM containing 30% FBS was placed into the bottom chamber as a chemotactic factor. After 24 or 48 h, cells were fixed in 4% paraformaldehyde for 15 min at room temperature. Nonmotile or noninvaded cells on the top surface of the filter were removed, while motile or invaded cells on the bottom surface were stained with crystal violet. ImageJ software was used to quantify the invaded and migrated cells. Three fields per well were counted with an inverted microscope system (Ti-S, Nikon, Tokyo, Japan).
Scratch assay
Cells in complete medium were seeded in a 6-well plate at a density of 1 × 105 cells/well, and a straight line was scratched on the cell monolayer with a 200 μL pipette tip. Then, cells were washed with PBS three times to remove debris and then replenished with fresh medium.
Immunohistochemistry
Formalin-fixed, paraffin-embedded tumor sections were baked at 65 °C, deparaffinized in turpentine, rehydrated through a series of graded alcohols, and immersed in hydrogen peroxide to block endogenous peroxidase activity. Antigen retrieval was performed by heat treatment in a pressure cooker in citrate buffer (pH 6.0) for 3 min. Primary antibodies are as follows: CD147 (1:400, ab666, Abcam, MA, USA) and PCNA (1:300, YM6090, ImmunoWay Biotechnology Company, TX, USA). Sections for immunohistochemistry staining were blocked for nonspecific binding by incubation in normal goat serum at room temperature. Subsequently, slides were incubated with a primary antibody at 4 °C overnight. The next day, sections were incubated with a biotin-conjugated secondary antibody for 20 min and then with peroxidase-conjugated streptavidin for an additional 30 min. Next, 3,3′-diaminobenzidine tetrahydrochloride was used to visualize the reaction, and slides were then counterstained with hematoxylin.
RNA-sequencing (RNA-Seq)
The cDNA library construction, library purification and transcriptome sequencing were implemented according to the Wuhan Huada Sequencing Company’s instructions (www.genomics.org.cn, BGI, Shenzhen, China).
Quantitative real-time PCR analysis
Total RNA was extracted from skin lesions of transgenic mice and cells with Trizol reagent. Total RNA (3 μg) was used as the template for the reverse transcription reaction (SuperScript III First-Strand Synthesis System for Reverse Transcription PCR, Invitrogen). All PCR primers used in this study are as follows:
CXCL1
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Forward
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5’TGCGCTGCCAGTGCTTGC’
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Reverse
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5’TTCCGCCCATTCTTGAGTGTGG’
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CXCL2
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Forward
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5’CACTGGTCCTGCTGCTGCTG’
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Reverse
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5’GGCGTCACACTCAAGCTCTGG’
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CXCL10
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Forward
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5’TGCCTCATCCTGCTGGGTCTG’
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Reverse
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5’CATTCTCACTGGCCCGTCATCG’
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CCR1
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Forward
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5’AAGGTCAAAGCCGTGCGTCTG’
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Reverse
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5’GGCCAGGTCCAGTTGCTTACTC’
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CCL2
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Forward
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5’ACGCCCCACTCACCTGCTG’
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Reverse
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5’CCTGCTGCTGGTGATCCTCTTG’
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CCL3
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Forward
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5’CCACCACTGCCCTTGCTGTTC’
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Reverse
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5’GCGTGGAATCTTCCGGCTGTAG’
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CCL4
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Forward
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5’CTTGCTCGTGGCTGCCTTCTG’
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Reverse
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5’AGCTGCCGGGAGGTGTAAGAG’
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IL-1β
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Forward
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5’GCAGCAGCACATCAACAAGAGC’
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Reverse
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5’AGGTCCACGGGAAAGACACAGG’
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MMP8
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Forward
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5’CACGTCTGGAGTGTAGCATCGC’
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Reverse
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5’TGGTTGAAAGGCATGGGCAAGG’
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GAPDH
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Forward
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5’GAGTGGGTGTCGCTGTTGAAGTC’
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Reverse
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5’GGCAAATTCAACGGCACAGTCAAG’
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The qRT-PCR assays and data collection were performed on a 7500 real-time PCR system (Applied Biosystems, USA). The data were analyzed by using the 2-△△CT values.
ELISA assays
ELISA was used to detect the levels of CXCL1 and CXCL2. ELISA kit (ml037774, ml058180, Mlbio, Shanghai, China) were used. All operations were performed in strict accordance with the manufacturers instructions.
Luciferase reporter gene assays
pGL3 is the luciferase reporter vector (E1751, Promega, USA). pENTER is the vector with kanamycin selection and CMV promoter and C-terminal FLAG and His tags (pENTER, P100001, Vigene, USA). pRLTK is renilla luciferase control reporter vectors (P100001, Promega, USA). HEK293T cells were transfected with CD147, PGL3-AP-1 and pRLTK. 293 T cells were transfected with CD147 or pEnter, pGL3-AP-1 or pGL3-ctrl and pRLTK pasmids. After 24 h of transfection, the firefly and renilla luciferase activity in the cell lysates was analyzed with a dual luciferase assay kit (Promega, Madison, WI) following the protocol. For each transfection, the luciferase activity of four replicates was averaged.
Chromatin Immunoprecipitation
HaCaT cells (10^7) infected with sh-Mock and sh-CD147 were collected and Chromatin Immunoprecipitation were performed according to the protocol provided by EZ CHIP KIT (Millipore, 17-371RF, MA, USA). Soluble lysates were rotated with 5 μl antibody (c-Jun, Cell Signaling Technology, 9165 s, MA, USA) overnight at 4 °C with protease inhibitors. CXCL1 promoter regions were amplified by PCR (50 cycles) using the following primer pairs:
CXCL1-Primer-1
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Forward
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5’AGAGTTCATGTTATACAG’
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Reverse
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5’TAACATCAGTGCTGTCTG’
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CXCL1-Primer-2
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Forward
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5’GAAGGGTGCTTGCACACC’
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Reverse
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5’GGGGTTCAGGTTTGATC’
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CXCL1-Primer-3
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Forward
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5’TCTCTAGAACTTCTGGAG’
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Reverse
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5’GCAGACATGCATTCGGATG’
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CXCL1-Primer-4
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Forward
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5’CAAACATTTATAAGGCAC’
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Reverse
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5’GAACACAGGCAAGGCTGC’
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CXCL1-Primer-5
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Forward
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5’GACTGGAGTACATATACTATC’
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Reverse
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5’TCGGTTACTCAGGGGTAC’
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Statistical analysis methods
Statistical results are presented as the means ± S.D.s and were analyzed by Student’s t-test or one-way ANOVA to evaluate the statistical differences. A p-value of < 0.05 was considered statistically significant.