Cell culture and chemicals
Human malignant melanoma cell lines A375 and embryonic kidney cell lines 293T cells were obtained from Cell Resource Center of Peking Union Medical College (IBMS, CAMS/PUMC). Human malignant melanoma cell lines A2058 was kindly provided by Dr. Fang from the Beijing Institute of Genomics. Cells were grown in high-glucose DMEM medium (Gibco) supplemented by 100 U/ml penicillin and 100 μg/ml streptomycin (TransGen Biotech.), and 10% fetal bovine serum (FBS) (Gibco) at 37°C with 5% CO2.
Construction of DHCR24 overexpression and shRNA constructs
The pCDH-DHCR24 plasmid was constructed as follows, DHCR24 was amplified by reverse transcription-PCR using mRNA from A2058 cells, with the forward primer (5’-gcgttctagagctagcgCCgccaccATGGAGCCCGCCGTGTCGCT-3’) and reverse primer (5’-atcAGGCTGATCAGCGGGTTTAAACTCAGTGCCTGGCGGCCTTGCAG-3’) including ~25 bp homology arm with pCDH，the PCR product and pCDH (digested with EcoR1 and BamH1) fragments were then joined through Gibson assembly using NEBuilder HiFi DNA Assembly Master Mix (NEB).
DHCR24 specific shRNA sequences were selected by BLOCK-iTTM RNAi Designer (http://rnaidesigner.thermofisher.com/rnaiexpress/). The shRNA sequences were as follows: shRNA-6 (5’-CCGCGTGTGAAACACTTTGA-3’), shRNA-7 (5’-GCTCTCGCTTATCTTCGATA-3’), and control shRNA (shNC) (5’-GGTACGGTCAGGCAGCTTCT-3’). Short-hairpin sequences were synthesized as oligonucleotides and annealed according to standard protocol. Annealed shRNAs were then subcloned into pLL3.7 shRNA vectors (Addgene).
Establishment of stable melanoma cell lines
The lentivirus was packaged into the helper plasmids (PSPAX2, PMD2.G) by cotransfection of 293T cells. The supernatant of the 293T cells packages lentivirus was harvested. A375 and A2058 cells were transduced with the lentivirus and 8 μg/ml polybrene. Stable transfected cells were incubated at 37°C with 5% CO2 for 48 h, then selected by 1 μg/ml puromycin for one-two weeks and maintained with 0.8 mg/ml puromycin.
Subcutaneous tumor formation
Six- to eight-week-old BALB/c-nu/nu female mice were purchased from Biotechnology Co., Ltd. (Beijing, China) and fed under SPF conditions in Institute of Radiation Medicine, Chinese Academy of Medical Science & Peking Union Medical College (Tianjin, China). All animal experiments were approved by the Institution’s Ethics Committee.
Mice were injected subcutaneously with 1×107 cells (0.1 ml PBS) and weighed twice a week. The volume of tumor was measured after three weeks and calculated by the equation (length ´ width2/2). Tumors were fixed in 4% paraformaldehyde for hematoxylin-eosin (HE) staining.
Real-Time quantitative PCR analyses
Total RNA was prepared from cells using EasyPure® RNA Kit (TransGen Biotech.) and reversely transcribed using TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech.) according to the manufacturer’s protocol. Real-Time quantitative PCR was performed using SYBR SuperMix (TransGen Biotech.) and specific primers for DHCR24 (forward 5’-GTGAAACACTTTGAAGCCAGG-3’, reverse 5’-AGCCATCAAACATCTCCCAG-3’) CYP27A1 (forward 5’-CATGGAGCTATGGAAGGAGC-3’, reverse 5’-TGAAAGCATCCGTATAGAGCG-3’), and GAPDH (forward 5’-AGCCACATCGCTCAGACAC-3’, reverse 5’-TTAAAAGCAGCCCTGGTGAC-3’). All the samples were normalized to the housekeeping gene, GAPDH. Data were calculated using the 2−ΔΔCt method.
Total RNA was obtained from fresh cells using EasyPure® RNA Kit (TransGen Biotech.) following the manufacturers’ protocol. Paired-end read of 100 bp (or 150 bp) were prepared in a BGISEQ-500 platform. The read number of each gene was converted to Fragments-Per-Kilobase per Million mapped fragments (RPKM) and differentially expressed genes were analyzed by DEseq2 package. Heatmap, GSEA enrichment, GO enrichment and KEGG enrichment were obtained via online Dr.Tom (https://biosys.bgi.com/#/main).
Western Blot analysis
Protein lysates from cells were extracted in RIPA buffer, with 1´ protease inhibitors cocktail. Equal number of proteins were resolved by 12% SDS-PAGE gel and transferred onto PVDF membranes. The blots were blocked and incubated with primary antibodies overnight at 4°C. Then membranes were incubated with the secondary antibody at 37°C for 1 h. The blots were probed with anti-DHCR24 (Santa Cruz Biotechnology, sc-398938), anti-CYP27A1(Thermo Fisher Scientific, PA5-27946), anti-Rap1 (Santa Cruz Biotechnology, sc-53434), anti-AKT1 (Santa Cruz Biotechnology, sc-5298) and anti-pAKT1-Thr308/309 (Signalway Antibody, 13311). GAPDH (TransGen, HC301) were used as the internal control. Densitometry analysis was performed using ImageJ software.
Cell growth and cell cycle analysis
Cell proliferation was assesd using TransDetect® Cell Counting Kit (TransGen Biotech.) according to the manufacturer’s instructions. Briefly, cells were plated in a 96-well plate at 3×103 cells/well and the absorbance value (OD) was measured at 450 nm using a microplate reader. Cells were fixed with 70% ethanol at 4 ̊C overnight for cell cycle assay. Cells were added RNase A (100 μg/ml) for 5 min at room temperature, then stained with PI (50 μg/ml, US EVERBRIGHT) for 30 min at room temperature. All samples were detected using a flow cytometer (NovoCyte 2040R, Agilent Technologies) and analyzed using the NovoExpress 1.4.1 software (Agilent Technologies).
Wound healing assay
Cells were seeded in 6-well plates and wounds were generated using a 200-μl micropipette tip in the middle of each well. The migrated distances were measured at 0 and 48 h after scratching in the same wounded region using an inverted microscope. The data were processed using Image J software.
To prepare poly-hydroxyethyl methacrylate (poly-HEMA, Sigma) coated plates, 300 μl of 12 mg/ml poly-HEMA was added to each well of 12-well plates and put on the shaker at 37°C overnight. Cells (5×103 cells/well) were plated in triplicate in poly-HEMA coated 12-well plates using DMEM/F-12 medium (Gibco), supplemented with penicillin (100 U/ml)/streptomycin (100 μg/ml) (TransGen Biotech.), L-glutamine (2 mM, Gibco), B27 supplement (1×, Thermo), bFGF (20 ng/ml, MCE), and hEGF (20 ng/ml, Gibco). Tumorsphere with a diameter greater than 50 μm were counted using an inverted microscope.
Determination of cholesterol metabolites
1×107 cells were collected and added 400 μL methanol: water: chloroform (5:2:2, v/v/v) mixed solution. The samples were homogenized in a microhomogenizer for 3 min, and extracted ultrasonically in an ice-water bath. The supernatant was collected and concentrated with centrifugal concentration dryer. Dried lipids were dissolved with 200 μl methanol: water (4:1, v/v) mixture solution and swirled in ice water bath until mixture is homogenous. Then the sample were filtered with 0.22 μm organic phase pinhole filter for LC-MS analysis.
Liquid chromatography was performed using an UPLC system (Nexera UHPLC LC-30A). The chromatographic column was the Waters UPLC HSS C18 Column (2.1 mm × 100 mm, 1.7 µm). The mobile phase A was water containing 0.1% formic acid, and the mobile phase B was acetonitrile. The temperature of column was 45°C, and the flowing rate of mobile phase was 0.4 ml/min. Gradient conditions were as follows: 0-0.5 min, 40% B, 0.5-1 min, 40%-55% B, 1-8 min, 50%-90% B, 8-9.2 min, 90%-40% B, 9.2-10 min, 40% B. Mass spectrometry analyzed using an AB Sciex Qtrap 5500 system containing an electrospray ionization (ESI) source. The capillary voltage was set to 5500V for positive mode and 4500 V for negative mode. The mass spectra were acquired in a mass range of m/z 100–1000, and collision-activated dissociation (CAD) was medium. Other parameters were as follow: ion source gas 1, 60 psi, ion source gas 2, 50 psi, curtain gas (CUR), 35 psi, and turbo ion spray source temperature, 500 °C.
All standards were purchased from Sigma and prepared at the concentrations of 0.05 ng/ml, 0.13 ng/ml, 0.33 ng/ml, 0.82 ng/ml, 2.05 ng/ml, 5.12 ng/ml, 12.80 ng/ml, 32.00 ng/ml, 80.00 ng/ml, 200.00 ng/ml, 500.00 ng/ml and 1000.00 ng/ml. The content of cholesterol and its metabolites in different cells were obtained according to standards.
Tissue Microarrays (TMAs) and Immunohistochemistry (IHC)
Skin cancer tissue Microarray (K063Me01) was purchased from Xi’an bioaitech Co., Ltd (Xi’an, China). The samples were analyzed by IHC using an anti-DHCR24 antibody according to standard method and microarray instruction. The results were randomly observed 5-10 fields of view, and then taken the average.
Measurement of 27-HC
Human 27-HC ELISA Kit was obtained from Wuhan Fine Biotech Co., Ltd (Wuhan, China). Cells (2x106) were collected and detected for 27-HC content following the manufacturer’s protocol. The OD value was measured by a Microplate Reader (Thermo) at 450 nm. The concentration of cellular 27-HC was obtained according to standards.
The GraphPad Prism v8.0 software was used to perform all statistical analyzes. Student’s t-test was performed to compare two groups, a one-way ANOVA was performed to compare more than two groups. All experiments were repeated at least three times. Data were represented as the mean ± SEM. P value < 0.05 was considered statistically significant.