Patients and materials

To perform the GWAS, white blood cell samples from 191 GC patients were obtained as the GC group between April 2018 and May 2019 at the First Affiliated Hospital of Soochow University (Suzhou, China). Samples from 288 volunteers without cancer were collected as the healthy control (HC) group. Table S1 shows the clinicopathological features of the 191 GC patients and baseline information of the healthy controls. The GC patients were staged based on TNM pathological staging criteria (8th edition) established by the American Joint Committee on Cancer (AJCC). We also conducted a questionnaire survey on all the enrolled gastric cancer patients and healthy control volunteers. The contents of the questionnaire including basic information (Gender, Age) and life behaviour (frequency of smoking, frequency of drinking, white meat preference, and pickled food preference). The study protocol was approved by the Institutional Review Board of the First Affiliated Hospital of Soochow University (ethical official number: (2018) No. 016) and was conducted in accordance with the principles of the Declaration of Helsinki. All patients and volunteers were well informed, and written consent was obtained from the study subjects or the legal surrogates of the patients before enrolment.

DNA extraction and quality control

Ten millilitres of peripheral venous whole blood from all GC and HC participants who had not consumed any food or water was collected into EDTA anticoagulant tubes. After gentle mixing and centrifugation at 4 °C and 1900 × g for 10 min, the upper plasma layer was discarded, and the buffy coat layer was retained as leukocytes. Fifty microlitres of white blood cells was placed into a 1.5 ml centrifuge tube, 500 μl of red blood cell lysate was added, and the tube was vortexed to mix for 10 s and centrifuged at 20,000 × g for 1 min. The waste solution was discarded, and 200 μl of nuclear lysis buffer, 20 μl of proteinase K (20 mg/ml), and 20 μl of SDS (20%) were added and mixed well. After incubation at 60 °C for 20 min, 30 μl of magnetic beads (Beckman XP Beads) was added and mixed by vortexing. Then, 400 μl of absolute ethanol was added and mixed thoroughly. The tube was immediately put on the magnetic frame for 2 min until the liquid was clear, the supernatant was carefully discarded, 1 ml of 80% ethanol was added and mixed for 30 s, the tube was put into the magnetic frame for adsorption for 2 min, and the waste solution was discarded. Then, the tube was dried at room temperature for 5–10 min, and 100 μl of Buffer TE was added for elution, incubated at 56 °C for 5 min, and centrifuged at 20,000 × g for 1 min. The tube was put on a magnetic stand for 2 min, and the supernatant was carefully aspirated. After the gDNA was extracted, quality control was carried out as follows: A Nanodrop spectrophotometer was used to determine the purity of the DNA (OD260/280 ratio of 1.8–2.0); agarose gel electrophoresis was performed to analyse the degree of DNA degradation and confirm that there was no obvious degradation, band aggregation without dispersion, or contamination with RNA or proteins; a Qubit fluorometer was then used to accurately quantify the DNA concentration, and DNA samples with an OD value between 1.8 and 2.0 and a total content of more than 100 ng were eligible for use in gene chip detection.

GWAS genotyping and analysis

GWAS genotyping and analysis were performed by 1Gene, Inc. (Hangzhou, China). First, > 100 ng of leukocyte DNA was quantitatively pipetted into a deep-well plate according to the instructions of the Affymetrix Axiom 2.0 assay. After random amplification, the DNA was fragmented; after thorough mixing, alcohol precipitation was performed; and after drying, the DNA was resuspended in buffer. A small amount was taken for quality control by gel electrophoresis. The fragmented DNA that passed the quality control tests was transferred to a hybridization chip for the high-temperature denaturation and hybridization reactions. After the hybridization reaction was completed, the chip was loaded into an Affymetrix Titan gene chip scanner. The gene chip was customized by Affymetrix. Genotyping data were obtained after staining and signal scanning.

Next, 200 HC and 150 GC samples were randomly selected as the training set, and the remaining 88 HC and 41 GC samples were used as the validation set for the subsequent GWAS. All samples were strictly quality controlled. The detailed quality control and filtration process is shown in Table S2. Briefly, all initial data were filtered according to the following sequence: ① Polymorphic SNP loci; ② Sample data with inconsistent sex information; ③ SNPs on nonautosomal sex chromosomes; ④ SNPs with a genotyping success rate of less than 95%; ⑤ SNPs with a minimum allele frequency of less than 0.05; ⑥ Samples with a genotyping success rate of less than 95% in the samples; ⑦ Samples with a nondesired ancestry; and ⑧ SNPs that were not in Hardy–Weinberg equilibrium. A total of 256,830 SNPs were finally accrued after filtering. Then, the additive model, dominant model and recessive model were performed on the training set. Based on the preliminary analysis results, we finally selected the additive model and applied it to the validation set data to validate the top 5 significant loci in the training set. The SNPs with a P value < 0.05 in the validation set were considered significant.

In the training set, we performed all 3 models—the additive model, dominant model and recessive model—to identify the appropriate model. For these 3 models, the numbers of SNPs that we found after data filtering and analysis were 12, 16 and 5, respectively (Table S2). The P value threshold was set according to the quantile–quantile plot (Figure S1). Considering the features of GC and the initial results of the GWAS, we finally chose the additive model for further study.

Differential expression and functional enrichment assay of the TCGA dataset and GEO dataset

RNA-seq data with the corresponding clinical data for 343 GC tissues and 30 adjacent tissues were downloaded from the TCGA database (https://cancergenome.nih.gov/). The DSE66229 dataset was downloaded from the NCBI GEO database (https://www.ncbi.nlm.nih.gov/geo/) and was used for quantification and differential expression analysis of genes with the edgeR package [18] and limma package [19] in R software (4.1.2). Genes with a FC of ≥ 1.5 or ≤ 0.67 and a false discovery rate (FDR) of < 0.05 were considered to be significantly dysregulated between GC tissues and adjacent tissues. The clusterProfiler package [20] in R software was used to perform functional enrichment analysis with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. All KEGG pathways were filtered with a significance threshold, namely, an adjusted P value of < 0.05.

Protein structure prediction analysis

To study the effects of mutations on protein stability, the structure of DHCR7 was predicted with I-TASSER [21]. The effect of the single mutation L68P (rs104886038) on protein stability was predicted using DynaMut2 [22]. More importantly, we also investigated the effects of the combined L68P mutation with other mutations on protein stability. First, fifty-six mutation sites in DHCR7 were obtained from the UniProt database (https://www.uniprot.org/uniprot/Q9UBM7), and the mutations that were detected in our above SNP microarray were retained for further analysis. Then, the potential co-mutations with L68P were identified as the mutation sites that have a shortest path length smaller than the average shortest path length between L68P (rs104886038) and the remaining residues in the amino acid network. Our previously developed tool ANCA [23] was used to construct the amino acid network of DHCR7 and calculate the shortest path length between residues.

Cell culture

The cell lines GES-1, AGS, MKN-45, MKN-28 and HGC-27 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). HGC-27 cells were cultured in DMEM (EallBio, Beijing, China, #03.1006C), and the other cells were cultured in RPMI-1640 medium (EallBio, Beijing, China, #03.4007C). The above culture media were supplemented with 10% foetal bovine serum (FBS; EallBio, #03. U16001DC) and 1% penicillin–streptomycin (NCM Biotech, Suzhou, China, #C100C5). All cells were cultured in a humidified incubator with 5% CO2 at 37 °C.

Cell transfection and lentiviral infection

Two commercial nonoverlapping DHCR7 siRNAs (DHCR7-siRNA-1 and DHCR7-siRNA-2) and the control siRNA were purchased from RiboBio (Guangzhou, China). Target sequence of siRNA were provided in Table S3. The expression plasmids carrying wild-type (WT) or mutant (MT) human DHCR7 cDNA and the corresponding control plasmids were purchased from Realgene Biotechnology (Nanjing, China). Cells were transfected with siRNAs or expression plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.

Lentiviruses carrying DHCR7 cDNA or DHCR7 short hairpin RNA (shRNA) containing the sequence of DHCR7-siRNA-1 were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Figure S2A and Figure S2B provided the maps of the shuttle plasmids carrying DHCR7 shRNA or cDNA, respectively. Briefly, 3 packaging plasmids (Figure S2C) and the shuttle plasmid carrying shRNA or cDNA were co-transfected in 293 T cells. After 72 h, the supernatant was collected, centrifuged at 4000 rpm for 4 min at 4 °C. Then, the supernatant was filtered with a 0.45 μm filter, and the filtrate was ultracentrifuged at 4° C, 20,000 rpm for 2 h. The obtained virus concentrate was subjected to titer detection and transfection. An empty backbone vector was used as the control. AGS, MKN-28 and HGC-27 cells in exponential growth phase were grown to 30% confluence and infected with lentiviral particles (MOI: 40). After 72 h, GFP-expressing cells were counted under a fluorescence microscope. Western blotting was used to further confirm the transfection or infection efficiency.

Western blot analysis

Cells were harvested and lysed in SDS Lysis Buffer (Beyotime, #P0013G) containing protease inhibitors and phosphatase inhibitors (Beyotime, #P1045). Protein concentrations were measured with an Enhanced BCA Protein Assay Kit (Beyotime, #P0010S) according to the manufacturer’s instructions. Total protein (30 μg) was separated by 10% SDS–PAGE (NCM Biotech, Suzhou, China, #P2012) and transferred onto 0.45-µm PVDF membranes (GE Healthcare Life Science, Germany). The membranes were blocked with 5% BSA (Fcmacs, Nanjing, China, #FMS-WB021) for 1.5 h and then incubated with the indicated primary antibodies at 4 °C overnight. The next day, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the membranes were visualized with ECL reagents (NCM Biotech, Suzhou, China, #10,100) using a ChemiDocTM MP Imaging System (Bio-Rad). All antibodies and their dilution used for western blot analysis were listed in Table S4.

CCK8 assay

A Cell Counting Kit-8 (NCM Biotech, Suzhou, China, #C6005) was used to evaluate cell proliferation. GC cells from different experimental groups were plated in 96-well plates at a density of 3000 cells per well. Then, 10 μl of CCK8 reagent was added to each well for staining at 37 °C for 2 h. The absorbance at 450 nm was measured.

EdU incorporation assay

Cells were plated into 48-well plates and cultured overnight. Then, EdU working solution (10 μM, Beyotime, #C0075) was added to the cells. After incubation for 2 h at 37 °C, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Then, the cells were stained with Click Additive Solution according to the manufacturer’s instructions. Nuclei were stained with Hoechst 33,342 (Beyotime, #C1025). Cell counting was subsequently performed under a fluorescence microscope.

Colony formation assay

GC cells were plated in 12-well plates at a density of 800 cells per well. The cells were cultured at 37 °C for 14 days. After removing the cell culture supernatant, the cells were fixed using 4% paraformaldehyde. Then, the cells were stained with Crystal Violet Staining Solution (Beyotime, #C0121). Images were acquired using an inverted microscope.

Transwell migration and invasion assays

For the migration assay, 4 × 104 GC cells in 300 μl of serum-free medium were plated in the upper compartments of a 24-well plate containing an 8-μm pore size membrane (Corning, #353097), and 500 μl of medium containing 20% serum was added to the bottom compartments. For the invasion assay, Matrigel (diluted 1:10; Corning, #356234) was used to coat the membrane in the upper compartment. After culture at 37 °C for 48 h, the cells attached to the lower surface of the membrane in the upper compartment were fixed with 4% paraformaldehyde and stained with Crystal Violet Staining Solution. Images were acquired using an inverted microscope.

Cholesterol measurement

The cholesterol levels in cells or tissues were measured using a total cholesterol assay kit (Jiancheng, Nanjing, China, #A111-1–1) following the manufacturer’s instructions. Briefly, 1 × 107 cells were digested and resuspended in 100 μl of PBS containing 1% Triton X-100. After lysis on ice for 45 min, the cells were centrifuged at 4 °C and 4000 rpm for 10 min. The supernatant was collected for cholesterol measurement. To measure the tissue cholesterol, the tumor tissue was mixed 1:9 by weight and volume with PBS solution containing 1% Triton X-100 and was homogenized. After centrifuging at 4000 rpm, 4 °C for 10 min, the supernatant to taken for the determination of cholesterol. To measure the cholesterol level, 2.5 μl of cell supernatant was added to 250 μl of working solution per well in a 96-well plate. In addition, ddH2O and the calibrator were added to the blank and calibrator wells, respectively. After incubation at 37 °C for 10 min, the absorbance at 510 nm was measured. Protein concentrations were calculated using the following equation: \(Cholesterol\;concentration=\left(\left(_-_\right)\right)/\left(\left(_-_\right)\right)\times calibrator\;concentration\;\div\;protein\;concentration\;in\;sample\). 

Filipin III staining

GC cells were plated in 48-well plates at a density of 2 × 104 cells per well and cultured at 37 °C overnight. After fixation using 4% paraformaldehyde, the cells were stained with Filipin III (Sigma, St. Louis, USA, #SAE0088) at room temperature (RT) for 2 h. The stained cells were imaged using an inverted microscope. All images were processed using ImageJ software [24]. Briefly, individual cells were segmented on the images according to the GFP signal. First, an intensity threshold was applied to determine the cell contours. A watershed algorithm (find maxima) was used to divide the image into discrete areas. Then, the thresholded and segmented images were merged to generate a mask showing the contours of individual cells. A threshold was applied to the filipin staining image to decrease the background signal and to focus the analysis on bright filipin puncta corresponding to cholesterol accumulation. The segmentation mask was applied to this thresholded image, and the filipin signal intensity was measured in each cell.

Apoptosis assay

The apoptosis rate was determined by a PE Annexin V Apoptosis Detection Kit I (BD Biosciences, #559763). Cells were collected and washed with cold PBS. Then, the cells were resuspended in 1 × Binding Buffer at a concentration of 1 × 106 cells/ml. Then, 5 µl of Annexin V-PE and 5 µl of 7-AAD were added to the cell suspension. After incubation for 15 min at RT in the dark, apoptosis was analysed by flow cytometry. Annexin-V + /7-AAD- cells and Annexin-V + /7-AAD + cells were considered apoptotic cells.

Xenograft tumor model

The animal experiments were approved by the Institutional Animal Care and Use Committee of Soochow University (Suzhou, China). Five-week-old female BALB/c nude mice and NSG mice were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). BALB/c nude mice were randomly assigned to the OE-DHCR7 group or the OE-NC group (n = 5 per group). To establish the xenograft tumor model, 1 × 107 DHCR7-overexpressing MKN-28 cells or empty vector MKN-28 cells were injected subcutaneously into the right flanks of the mice in the OE-DHCR7 group or the OE-NC group, respectively. The xenograft tumors were measured every 3 days by using digital callipers, and tumor volumes were calculated. After 2 weeks, all mice were sacrificed, and tumor masses were analysed. In addition, the xenograft tumor tissues were used for cholesterol detection and immunohistochemistry (IHC).

In rescue assays with inhibitors targeting cholesterol biosynthesis pathway and DHCR7, NSG mice were randomly assigned to the OE-DHCR7 group and tamoxifen (TAM) treatment (OE-DHCR7-TAM) group (n = 5 per group). 1 × 107 DHCR7-overexpressing MKN-28 cells were injected subcutaneously into the right flanks of the mice. The xenograft tumors were measured every 3 days by using digital callipers, and tumor volumes were calculated. After 2 weeks, mice in OE-DHCR7-TAM were administrated TAM (10 mg/kg, i.g.) once a day, for 7 consecutive days. At the same time, mice in OE-DHCR7 were were administrated an equal dose of solvent (corn oil). All mice were sacrificed on the day 28 after cell injection, and tumor masses were analyzed as above.

Murine lung metastasis model

Five-week-old female NOD/SCID mice and NSG mice were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). NOD/SCID mice were randomly assigned to the OE-DHCR7 group or the OE-NC group (n = 4 per group). Mice were injected with DHCR7-overexpressing MKN-28 cells or empty vector MKN-28 cells (3 × 106 cells/200 μl PBS per mouse) via the tail vein. 6 weeks after injection, mice were sacrificed and the lungs were excised and fixed with 4% paraformaldehyde, and stained with hematoxylin and eosin (HE). Lung metastatic nodules were counted in a blinded manner by two experienced pathologists. In rescue assays with inhibitors targeting cholesterol biosynthesis pathway and DHCR7, NSG mice were randomly assigned to the OE-DHCR7 group and the OE-DHCR7-TAM group (n = 5 per group). All mice were injected with DHCR7-overexpressing MKN-28 cells (3 × 106 cells/200 μl PBS per mouse) via the tail vein. After 2 weeks, mice in OE-DHCR7-TAM were administrated TAM (10 mg/kg, i.g.), once a day, for 7 consecutive days. At the same time, mice in OE-DHCR7 were administrated an equal dose of solvent (corn oil). 6 weeks after injection, all mice were sacrificed and the lung tissues were obtained for metastatic nodules caculation and HE staining.

Statistics

All statistical analyses and graphing in this paper were performed with GraphPad Prism (9.3.0) and R software (4.1.2). For normally distributed data, Student’s t test was used, while for nonnormally distributed data, the Wilcoxon rank-sum test was used. Regression analysis was based on a logistic regression model. A P value < 0.05 was considered statistically significant.