Cell line and reagents
The related reagents are described below. DMEM cell culture medium was purchased from Gibco Company (Waltham, Massachusetts, USA). Trypsin was purchased from Sigma Company (St. Louis, Missouri, USA). Lipidosome LIPOFECTAMINE 2000, Opti-MEM low-serum medium, rabbit-anti-human polyclonal antibody, rabbit-anti-rat polyclonal antibody marked with HRP, and siRNA for the negative control group were purchased from Invitrogen Company (Waltham, Massachusetts, USA). Protein lysis buffer (RIPA) was purchased from Novogen Company (Mauguerand, France). A protein quantitative reagent (BCA kit) was purchased from Pierce Company (Waltham, Massachusetts, USA). MTT and BCA staining kits were purchased from Ribo Bio. Co., Ltd. (Beijing, China). The HCC cell line, HepG2, was provided by the Jiangsu Key Laboratory of Medical Molecular Technology (Jiangsu, China).
HepG2 cell cultures
HepG2 cells were removed from liquid nitrogen, and quickly thawed in a water bath at 37 °C. After centrifugation, the cells were collected, and placed in DMEM culture medium containing 10% fetal calf serum, then cultured at 37 °C in a 5% CO2 incubator. The culture medium was replaced periodically. After the tumor cells grew to approximately 80% of the bottle wall, they were digested and transferred by 0.25% trypsin, then cultured in a fresh culture bottle.
Design and synthesis of siRNA
The ICOS gene sequences were obtained from the Genebank database, and the corresponding siRNA sequences for the ICOS gene were designed using siRNA design software. The sequences were as follows: positive-sense strand, 5′-GGAACUUGCCAUCAAGAUCTT-3′; and negative-sense strand, 5′-AAUGUCGAUAGGAACUUGCTT-3′. The sequences for siRNA in the negative control group were as follows: positive-sense strand, 5′-CCAACUUTCCAUCAACAUCTT-3′; and negative-sense strand, 5′-GGUCUAGAUACCTTCUUGGAA-3′.
Grouping and transfection
The experiment was divided into the following three groups: ICOS siRNA transfection group (Group A); negative control (NC) group (transfection with an irrelevant siRNA sequence; Group B); and blank control (CON) group (Group C). HepG2 cells (1 × 105 cells/mL) were cultured in a 6-well plate in a 5% CO2 incubator for siRNA transfection. The cells were washed 2 times in serum-free DMEM, then added to serum- and antibiotic-free DMEM culture medium (0.8 mL/well), and finally cultured at 37 °C in a 5% CO2 incubator for 1 h. Then, 10 μL of siRNA solution and 175 μL of serum-free DMEM were added and mixed uniformly so that the siRNA transfection solution was acquired. After 6 h of cell and siRNA transfection solution co-culturing, the transfection rate was observed under a microscope in bright and fluorescent field.
Detection of the ICOS mRNA expression by RT-PCR
To detect the level of ICOS mRNA in the transfected cells, HepG2 cells were collected and the total RNA was extracted according to the instructions in the kit provided by Alphainnotech Company (city, state, USA). The ratio of the absorption value at 260–280 nm was measured on a micro-spectrophotometer to evaluate the purity of RNA; the ratio was 1.8 ~ 2.1. Primer 5.0 was adopted for primer design (Ribo Bio. Co. Ltd.). The primers for ICOS detection were as follows: forward primer, 5′- CGT CAC GAC CTA CGA TA-3′; and reverse primer, 5′- GCC CCG CGC CGA GGC AG-3′. β-actin was used as the internal reference; the primers were as follows: forward primer, 5′- GGT GTG ATGGTG GGT ATG GGT-3′; and reverse primer, 5′- CTG GGT CATCTT TTC ACG GTC-3′. Amplification was performed using a Takara reverse transcription and amplification kit (Tokyo, Japan). The relative expression of mRNA in all samples were calculated according to 2−ΔΔCt method.
Detection of ICOS, AKT, Bcl-2, and PI3K protein expression by western blot
Cell lysis buffer (Wuhan Zhongzhi Biotechnologies Co., Ltd., Wuhan, China) was used to split the HepG2 cells into different groups for total protein extraction from cells. Phosphatase inhibitors (Sigma Company) were added into the corresponding cell lysis buffers to detect the level of protein phosphorylation. SDS-PAGE electrophoresis was performed after the extracted total protein in different groups was denatured. After electrophoresis, the protein was transferred to prepared nitrocellulose membranes, and the nitrocellulose membranes were sealed with 5% skim milk powder for 2 h. After washing, the first antibody was added and incubated overnight at 4 °C. On the next day, the membranes were washed once in PBS and twice in TBST. Then, the second antibody labeled with horseradish peroxidase was added and incubated at room temperature for 2 h. After the membrane was washed, ECL chemiluminescence was used for the signaling exposure with X-ray film, then fixed and scanned. β-actin was used as the internal reference. The dilution ratios of antibodies for anti-ICOS, -AKT, -Bcl-2, and -β-actin or the corresponding phosphorylation antibodies were 1:1000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and the second goat anti-rat or goat anti-rabbit antibodies labeled with HRP were diluted to 1:5000.
Detection of HepG2 cell proliferation by the MTT colorimetric method
HepG2 cells (1 × 105 cells/mL) were incubated in 96-well plates. After siRNA transfection for 6 h, 20 μL of MTT was added to each well periodically and incubated at 37 °C for 4 h. The liquid supernatant in the well was removed, then 150 µL of DMSO was added to dissolve the crystal. An enzyme-linked immune detector (Alphainnotech Company) was used to detect the absorbance value for each well at 570 nm.
Transwell invasion experiments
For the Transwell assay, Matrigel was thawed at 4 °C overnight, then diluted (5–1 mg/mL) in cold serum-free DMEM cell culture medium. Diluted Matrigel (100 μL) was placed in the upper chamber (polycarbonate membrane [diameter, 8 μm]) of a 24-well plate. The Matrigel was incubated in the Transwell chamber at 37 °C for at least 4 h to gel. HepG2 cells were harvested and suspended in serum-free DMEM medium at a density of 10 × 6 cells/mL. The gelled Matrigel was gently washed with warmed serum-free DMEM culture medium. The cell suspension (100 μL) was placed onto the Matrigel and 700 μL of DMEM culture medium containing 10% fetal calf serum was added to the lower Transwell chamber, and incubated at 37 °C for 18 h. The Transwell chambers were removed from the 24-well plates, twice-washed in pre-cooled PBS, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet for 5–10 min. After staining, the cell suspensions were photographed under a microscope (Olympus Company, Japan), and the number of cells passing through the Transwell membranes in different groups was counted. The experiments were repeated 3 times to acquire the average.
Statistics processing
Quantitative data are expressed as the mean ± standard deviation (\(\overline{x} \pm s\)), and SPSS 23.0 software was used for statistical analysis. The measurement data between two groups were compared with a t-test, and the measurement data among several groups were compared with a one-way analysis of variance (ANOVA). Fisher’s LSD test was used to calculate statistical significance. A P < 0.05 indicates a statistically significant difference.
