Study outline
To clearly demonstrate the specific process of this study, we have summarized the process as shown in the figure (Fig. 1).
Clinical samples
A total of 106 GC patients were enrolled in this study from 2016 to 2019. The detailed patient characteristics were showed in Table (1). None of the enrolled patients received preoperative radiotherapy, chemotherapy, or immunotherapy before. This study was approved by the Institutional Review Board of Huai’an NO.2 Hospital and Institute (HEYLL2011915). The methods were carried out in accordance with the Declaration of Helsinki. At recruitment, written informed consent was obtained from each subject.
Immunohistochemistry (IHC) analysis
The samples were incubated with DCAF1 (1:500 dilution, Proteintech, Wuhan, China, 11612-1-AP). The intensity of IHC staining and the proportion were evaluated by two senior pathologists independently. The intensity of IHC staining was graded as follows: “0” (no staining), “1+” (weakly positive), “2+” (moderately positive), and “3+” (strongly positive). The proportion of staining was graded as follows: 0 (negative), 1 (< 33%), 2 (33–66%), and 3 (> 66%). Staining scores were calculated as follows: score (maximum of 9) = staining intensity × staining proportion 23. The median score was chosen as the cut-off value to separate the DCAF1 low expression group and DCAF1 high expression groups.
Cell culture
Human GC cell lines (MGC803, HGC27) were were donated by the Tumor Central Laboratory of Drum Tower Hospital. MGC803 and HGC27 were cultured in RPMI-1640 (Gibco, USA) with 10% fetal bovine serum (Gibco, USA), 100U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA) at 37℃ in a humidified incubator containing 5% CO2 and periodically tested Mycoplasma negative using MycoAlert Mycoplasma Detection Kit (Lonza). The cell lines were sub-cultured every 2–3 days following digestion at room temperature with 0.5 ml trypsin/EDTA per well (Sigma-Aldrich Ltd, UK).
Plasmids and lentivirus transfection
The plasmid constructs
The shRNA hairpins targeting human DCAF1 (sh-DCAF1) or the control shRNA (sh-NC) were cloned into the pLV3ltr-Puro-U6 vector (Krystal Biotech) (Appendix Table S1). The resultant plasmids were designated sh-DCAF1 or sh-NC. All the plasmids were synthesized by Genewiz and sequenced to confirm the orientation and integrity.
Lentiviral transduction
Recombinant lentiviral particles were produced by OBiO Technology. GC cells were infected with viral supernatant containing 8 mg/mL polybrene. Stably DCAF1-knockdown (KD) cells were selected using 2 mg/mL puromycin. In these cells, DCAF1 expressions were tested by Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot assays.
CCK-8 assay
The cell viability was detected by Cell Counting Kit-8 (CCK-8) (Biosharp, Hefei, China) according to the manufacturer’s instructions. Briefly, 5 × 103 cells/well were seeded onto 96-well plates in quintuplicate and cultured until entirely adherent. At 12, 24, 36 and 48 hours after seeding, 10 µL CCK-8 solution was added to each well and incubated for 2 hours. The cell viability was determined by measuring OD value at 450 nm.
Clone formation assay
A total of 200 MGC803 or HGC27 cells per well were seeded in 6 cm plates. When colonies were visible after 14 days, cells were washed with PBS, fixed with the fixation fluid (methanol: acetic acid ¼ 3:1) and dyed with crystal violet. The colony number in each plate was imaged and then counted.
Wound healing and transwell assay
For wound healing assays, a wound was scratched by a 10 µL pipette tip when the cell layer of MGC803 or HGC27 reached about 90% confluence. Cells were continued cultured at 37°C with 5%CO2, and the average extent of wound closure was quantified. In transwell assays, transfected MGC803 or HGC27 cells were added to upper transwell chambers pore (8 µm, Corning). A medium containing 10% FBS (650 µL) was added to the lower wells. After 24 hours, cells migrated to the lower wells through pores were stained with 0.2% crystal violet solution and counted.
Western blot
Total proteins were extracted from the cells or clinical samples using RIPA lysis buffer containing 1‰ PMSF. Protein specimens were separated with SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, ISEQ00010). The PVDF membranes were incubated with various antibodies (Appendix Table S2). After incubation with corresponding secondary antibody for 2 hours, the protein bands were detected with ECL Western Blot Substrate (Thermo Scientific, 34075).
qRT-PCR
Total RNA was isolated from culture cells or tissue specimens with TRIzol reagent (Invitrogen, 94402). To remove genomic DNA, each RNA sample was treated with DNase I (RNase-free) (Thermo Fisher,18068015). Each RNA sample was then reverse transcribed into cDNAs using PrimeScriptTM RT Master Mix (TaKaRa, RR036A). The relative expression of DCAF1 was calculated by using the 2-ΔΔCt method. Indicated primers were listed in Supplementary Table S3. Each sample was examined at least in triplicate. qRT-PCR product specificity was confirmed by a melting-curve analysis.
RNA-seq
To gain insight into how DCAF1 regulate gene expression in GC cells, we performed RNA-seq of MGC803 cells transfected or transduced with different expression constructs. Total RNA was isolated from cultured cells using TRIzol. RNA-seq of MGC803 cells transfected with sh-DCAF1 was performed using Illumina NovaSeq 6000 sequencing platform (Illumina, USA). Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to whole transcriptome using tophat2 or Bowtie2 with default parameters. Reads count of samples were calculated and converted to FPKM (fragments per kilobase of exon model per million reads mapped). KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses of differentially expressed genes (|log2(FC)|>1, P < 0.05) from RNA-seq were performed.
Statistical analysis
Statistical analysis was performed using SPSS 21.0 (Chi-cago, USA) and GraphPad Prism 8.0 software (San Diego, CA, USA). All data were presented as the mean ± standard deviations (SD), the comparisons between two groups were analyzed by two-tailed Student’s t-test. Pearson’s chi-squared test was used for correlation analysis. Survival curves were calculated by the Kaplan-Meier method and compared with the log-rank test. A Cox regression model was used to analyze independent risk factors for survival. A P-value < 0.05 was considered to reflect statistical significance.
TCGA analysis
RNA-sequencing expression (level 3) profiles and corresponding clinical information for DCAF1 were downloaded from the TCGA dataset (https://portal.gdc.com). R software GSVA package was used to analyze, choosing parameter as method = 'ssgsea'. The correlation between genes and pathway scores was analyzed by Spearman correlation. All the analysis methods and R packages were implemented by R version 4.0.3. p value < 0.05 was considered statistically significant.