Cell line and specimens
The immortalized normal liver cell line THLE-3 and HCC cell lines Hep3B, HepG2, Huh1, Huh7, SK-Hep1, SUN-182 and SUN-475 were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China), All the cell lines were cultured using DMEM adding 10% fetal bovine (FBS; HyClone, USA), 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich, USA).
All the HCC tissues and adjacent tissues were obtained from the HCC patients who were histopathologically and clinically diagnosed in the First Affiliated Hospital of Gannan Medical University were frozen and stored in liquid nitrogen until further use. Prior patient consent and approval were obtained from the Institutional Research Ethics Committee. The patients were selected based on the following criteria: pathological diagnosis of HCC, primary resection without pre-operative anticancer treatment. 8 patients were included in the present research. 5 patients were female and 3 patients were male. Mean age of first diagnosis were 59 years (range 52–69 years). All patients were completely resected, and we collected the resected tissues. Non-cancerous tissues were defined > 2 cm distance from the margin of the resection.
Establishment of stable cell lines
The plasmids of SSR2 overexpression, SSR2 downregulation and corresponding control were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The process of screening stable cell lines was performed according to previously published methods [19]. Briefly, plasmids were transfected into 293T cells to produce lentiviral particles. After 48 hours, the particles were collected and used to infect SNU-182 and Huh1 cells overnight. Then, transfection medium was then replaced using normal culture media. The stable cells were screened by treating using 5 µg/ml puromycin for 7 days.
Western blotting assay
Total protein was obtained by radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China). And nuclear proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime; China). Extracted proteins were subjected to western blotting according to previously described methods [20]. Briefly, equal amounts of protein (25 µg/well) were separated using 10.5% polyacrylamide gels and subsequently transferred onto PVDF membranes. Then, the membranes were probed with primary antibodies, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Cat: ab288151 or ab6728; 1:500; Abcam; USA). And the luminescence of western blotting bands was using Ultra-sensitive ECL Chemiluminescence Kit (Beyotime; China) GAPDH was used as the loading control of total protein. The primary antibodies are as follows: anti-SSR2: (Cat: AT31G6; 1:400; GeneTex; USA); anti-p-YAP (Ser127): (Cat: 13008; 1:1000; Cell signaling Technology; USA); anti-YAP (Cat: ab205270; 1:1000; Abcam; USA); anti-GAPDH (Cat: ab8245; 1:2000; Abcam; USA); EF-1α (Cat: sc-21758; 1:1000; Santa Cruz Biotechnology, Inc.; USA). And the bands of western blotting were analyzed using iBright CL750 imaging system (ThermoFisher Scientific; USA).
RT-PCR analysis
The total RNA was extracted using TRIzol (Invitrogen, USA). And total cDNA were synthesized using Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany) according to the manufacturer’s directions. qPCR was performed on a 7500 Fast Real time PCR system (Applied Biosystems, USA) using the SYBR Green PCR Kit (Invitrogen, USA). The primers used were as follows: SSR2, forward, 5’- CACTGCTGAACAGATACGCCGT-3’, reverse, 5’- GCCAAAGTCTTCTGGAGGGAAG-3’; CCNE1, forward, 5’- TGTGTCCTGGATGTTGACTGCC-3’, reverse, 5’- CTCTATGTCGCACCACTGATACC-3’; CTGF, forward, 5’- CTTGCGAAGCTGACCTGGAAGA-3’, reverse, 5’- CCGTCGGTACATACTCCACAGA-3’; CCN1, forward, 5’- GGAAAAGGCAGCTCACTGAAGC-3’, reverse, 5’- GGAGATACCAGTTCCACAGGTC-3’; GAPDH, forward, 5’- GTCTCCTCTGACTTCAACAGCG-3’, reverse, 5’- ACCACCCTGTTGCTGTAGCCAA-3’.
Colony formation assay
Colony formation assay were performed according previously described [21]. Briefly, 0.5 × 103 cells were seeded into 6-well plate and maintained in an incubator at 37°C for 12 h. Vehicle or 15 µg/mL DDP were added into the well. 10 days later, the cells were fixed using 4% paraformaldehyde, and then stained with 0.1% crystal violet. The stained colonies were counted under a light microscope.
Flow cytometer assay
The apoptosis rate of HCC cells was detected by flow cytometer assay according previously described [22]. 0.5 × 105 cells were seeded into 10 cm plates, and maintained in an incubator at 37°C for 12 h. Vehicle or 15 µg/mL DDP were added into the well. 48 h later, the cells were collected, and washed twice using PBS, and re-suspended using binding buffer. Then, we treated cells using FITC Annexin V and propidium iodide for 15 min at room temperature away from light. At last, we examined the cells using an EPICS XL flow cytometer (Beckman-Coulter, USA).
TUNEL assay
Mouse xenograft tissues were minced and digested with collagenase and DNAses (Life Technologies, USA) at 37°C for 30 min, and then filtered with 40-micron cell strainers (BD, USA). Then, the 0.5 × 103 cells were fixed by 4% paraformaldehyde, and subsequently permeabilized using 0.1% sodium citrate and 0.1% Triton X. DNA fragmentation was probed using TdT-mediated dUTP nick end labeling (TUNEL) kit (Roche Applied Science, Switzerland) following the manufacturer. Fluorescent images were captured using an EVOS fluorescent microscope (Advanced Microscopy Group, USA).
Anchorage independent growth assay
Anchorage independent growth assay was performed according to the previous method [23]. Briefly, the completed medium adding 1% agar was put into the cell plate. Subsequently, the mixture containing 0.5 × 103 cells, 0.3% agar and 2 mL complete medium were added into the upper of the plate. And the plate was maintained in 37°C incubator for 10 days. We finally counted the colonies with diameter larger than 0.1 mm.
In vivo animal model
In this study, 4-week-old male BALB/c nude mice were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China), and these mice were maintained in aseptic conditions. 4 days later, 1 × 106 cells were suspended in serum-free DMEM/Matrigel (1:1). Anesthesia was carried out with isoflurane (Piramal Critical Care, PA, USA) through anesthesia machine (RWD, Guangdong, China). 3% induction concentration is used. The mouse is put into the induction box after isoflurane is filled with induction box for about 1 min, and then the induction box is closed, and the mouse is completely anesthetized (This process takes about 2–3 min). The induction box can be gently shaken to check whether the mouse is fully anesthetized. The mouse’s body is overturned into a side position, and doesn’t restore lying position, which means that the mouse is fully anesthetized. Subsequently, the mouse is removed from the induction box. The concentration of isoflurane in induction box maintains 1.5%, and the head of the mouse is fixed in the anesthesia mask. The experiments can be performed. Under anesthesia, a 1 cm transverse incision in the upper abdomen of mice was made. The complex was injected into the left hepatic lobe of the mice by a 28-gauge needle. The liver was returned to the peritoneal cavity and the abdominal wall was closed. 3 mg/kg DDP were intraperitoneally injected into mice every 4 days. After 40 days, all mice were sacrificed. All experiment procedures were approved by the Institutional Animal Care and Use Committee.
Statistical analysis
Statistical analyses were performed using the SPSS version 19.0 statistical software package. The data are present as the mean ± standard deviation. Student’s paired t-test was used to analyze the statistical difference between paired tissues, and comparisons among more than two groups were analyzed using variance (ANOVA) followed by Dunnett’s test. P < 0.05 was considered as statistically significance.