Data sources
Two datasets of senescent fibroblast cells including genomic and methylation data (GSE81798 and GSE91071) along with HCC tissue datasets containing methylation data (GSE73003) and expression data (GSE14520) were downloaded from the Gene Expression Omnibus (GEO) database. Data from GPL3921 platform in GSE14520 included 209 HCC patients and another HCC cohort data including 370 HCC patients were obtained from The Cancer Genome Atlas (TCGA) database. And the immunohistochemical results of CDC20 in HCC and normal liver tissues were downloaded from the Human Protein Atlas database (https://www.proteinatlas.org/).
Differentially expressed gene analysis
The differentially expressed gene (DEG) analysis was conducted on two senescent cell datasets. Genes with a fold change (FC) > 1.5 and q-value < 0.05 were selected for further analysis. With methylation data, we removed data located on chromosome X- and Y. At the same time, we identified the differentially methylated genes with P < 0.05. Then, we selected the overlapped DEGs with altered methylation in the lists as SAGs in the senescent cell datasets.
Survival analysis
Log-Rank test was carried out to analyze the difference among groups and the outcomes were presented by Kaplan-Meier curves. We used the median to separate the patients into high- and low- expression or/and methylation groups.
Cell culture and transfection
Human HCC cell lines (SMMC-7721 and Hep G2) were obtained from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured in 37℃ incubator contained 5% CO2 with Dulbecco's Modified Eagle Medium (Hyclone, Logan, UT, USA). CDC20 siRNA: 5’-GGAGCUCAUCUCAGGCCAUtt-3’ (sense) and 5’- AUGGCCUGAGAUGAGCUCCtt-3’ (antisense) and control were obtained from GenePharma (Shanghai, China). The siRNA (100 nM) targeting CDC20 and and control were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
Cell treatment
To establish the senescent cell model, cells were treated with 100 µM hydrogen peroxide (Sigma Aldrich) for 24 h according to the previously described protocol [15, 16]. Then, cells were treated with 5 µM 5-Azacytidine (5-AZA), a DNA methylase inhibitor (MCE MedChem Express, Princeton, NJ, USA) for 48 h [17] and phosphate buffered saline (PBS) was added in the control group.
RNA isolation and qRT-PCR assay
After total RNA was extracted using Trizol agent (Ambion) and quantified by absorbance at 260nm, reverse transcription was performed using the PrimeScript RT reagent Kit. The comparative-Ct method was used to calculate the relative mRNA levels. The primer sequences used were: CDC20: 5’-CTGTCTGAGTGCCGTGGATG-3’ (sense), 5’-CCATGGTTGGGTACTTCCAAATAA -3’ (antisense); β-actin: 5’-ATCGTGCGTGTGACATTAAGGAG-3’ (sense), 5’-AGGAAGGAAGGCTGGAAGAGTG-3’ (antisense). All kits and primers were obtained from Takara and all operations are performed in accordance with the manufacturer’s instructions.
Pyrophosphate sequencing
After total DNA was extracted and quantified, bisulfite treatment was performed. Then, the products were amplified using PCR and sequenced by pyrophosphate sequencing. All kits were purchased from Qiagen and the protocol followed was as per the manufacturer’s instructions. The primer sequences used are given in Table 1.
Western blot
The cells were lysed by RIPA (HAT, Xi’an, China) and the lysate was separated in 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Then, the membranes were incubated in the primary antibody at 4°C overnight (1:1000, proteintech, Wuhan, China) and the secondary antibodies used subsequently along with the room temperature for 2 hours (1:10000, proteintech, Wuhan, China). The visualization was performed using the ECL kit (Millipore, Bedford, MA, USA).
CCK-8 and cell cycle analysis
The cell viability of HCC cell lines was measured using the CCK-8 Kit (Qihai, Shanghai, China), according to the manufacturer’s instructions. After the cells were trypsinized, they were fixed overnight in 70% ethanol at 4°C. After centrifugation, the cells were stained using propidium iodide (PI), incubated at 37°C for 30min and then subjected to flow cytometry (Qihai, Shanghai, China) analysis.
Senescence β-galactosidase (SA β-gal) assay
Cells were stained using the SA β-gal staining kit (Beyotime Inc., Nantong, China), as per the manufacturer’s instructions. The cells are fixed for 15 minutes, incubated with the staining working solution overnight at 37°C and the percentage of SA-β-gal positive cells was calculated by bright-field microscopy (Zeiss 2.0, Germany).
Statistical analysis
All experiments were repeated three times and presented as mean ± SD. The expression levels and beta-values of CDC20 were analyzed using the t-test. P -value less than 0.05 was considered statistically significant. The statistical analysis was conducted using IBM SPSS Statistics software 24.0 (IBM Corp, NY, USA).