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.

Detection of the siRNA transfection rate

After transfection for 6 h, the cells were observed under a fluorescent and bright field microscope to determine the transfection rate. Figure 1a shows the negative transfection control. Figure 1b shows the positive transfection of > 70% compared with the negative control.

Expression of ICOS mRNA and protein levels after ICOS siRNA transfection

To further clarify the influence of transfection on the targeted gene and protein, we determined the expression of ICOS mRNA and protein levels in the different groups. As shown in Fig. 2a, the results of RT-PCR indicate that after ICOS siRNA transfection (EG), the mRNA expression of ICOS in the EG group was significantly reduced compared with the NC and CON groups. Thus, the ICOS siRNA expression carrier effectively transfected cells and successfully reduced the expression of ICOS mRNA.

In addition, we determined the expression of ICOS protein in different groups by Western blot. As shown in Fig. 2b, compared with the NC and CON groups, the expression of ICOS protein in the EG group was significantly reduced, indicating that the expression of ICOS protein in HepG2 cells was successfully reduced through the expression carrier of ICOS siRNA. The expression of ICOS protein was quantitatively analyzed using three independently repeated experiments, and the inhibition efficiency of ICOS protein was calculated after ICOS siRNA transfection to be approximately 65%, as shown in Fig. 2c.

Influence of ICOS knockdown on the proliferation and infiltration of HepG2 cells

The proliferation of cells in different groups was measured by detecting the OD values of HepG2 cells 0, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h after transfection using the MTT experiment method. Then, the cell growth curve was plotted for all groups as time (horizontal axis) versus average absorbance values of A570 (vertical axis) in all groups (Fig. 3a). Statistical methods were used for data analysis. ICOS knockdown significantly suppressed the proliferation of HepG2 cells, and the effect was more pronounced as the time after transfection was prolonged (*P < 0.05).

To detect the influence of ICOS knockdown on the infiltration of HepG2 cells, Transwell invasion assays were performed. As shown in Fig. 3b, compared with the results in the NC and CON groups, the number of invaded chambers in the EG group was significantly reduced (P < 0.05). Therefore, invasion of HepG2 cells was suppressed by reducing the expression of ICOS.

Influence of ICOS knockdown on the expression of AKT and Bcl-2 protein, and phosphorylation of PI3K in HepG2 cells

AKT, also known as protein kinase B, is an important kinase downstream of ICOS. AKT plays an important role in the generation and development of liver cancer cells. Therefore, the expression of AKT was determined in the different groups. As shown in Fig. 4a, compared with the NC and CON groups, the expression of AKT protein in the EG group was significantly reduced (n = 3, P < 0.05), as shown in Fig. 4b.

Bcl-2 is an important biological marker of cell apoptosis. Western blot was carried out and the results are shown in Fig. 4c. Compared with the results of NC and CON groups, the expression of Bcl-2 in the EG group was significantly reduced (n = 3, P < 0.05), as shown in Fig. 4d. ICOS knockdown suppressed the expression of Bcl-2, and thus may suppress HepG2 cell apoptosis.

Because phosphorylation of PI3K protein plays an important role in the ICOS signal channel, Western blot was performed to detect the expression of PI3K phosphorylation. As shown in Fig. 4e, compared with the NC and CON groups, PI3K phosphorylation in the EG group was significantly enhanced, as shown in Fig. 4f.