2.1. Tissue specimen
The study was carried out on 55 patients with HCC diagnosed between 2015 and 2019. Only patients that had undergone hepatectomy without any treatment before surgery, including radiotherapy or chemotherapy, were included in the study. The liver cancer and adjacent tissue specimens were placed in liquid nitrogen immediately upon collection. The study was approved by the Ethics Review Committee of the Second Affiliated Hospital of Nanchang University. The procedure followed the ethical standards of the Human Experiment Responsibility Committee (institution and country) and the 1975 “Helsinki Declaration” (revised in 2008). Informed consent was obtained from all the patients before enrollment.
2.2. Cell culture
The hepatocyte cell line (HL-7702) and four HCC cell lines (SMMC7721, MHCC97H, HCCLM3, and Huh-7) used in this study were purchased from the Shanghai Institute of Cell Biology (Shanghai, China). All cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Solarbio, Beijing, China) supplemented with 10% FBS (Biological Industries, Beit-Haemek, Israel), 100 µg/mL streptomycin, and 100 U/mL penicillin, incubated in an incubator containing 5% carbon dioxide. All the experiments used cells in the logarithmic phase of growth.
2.3. Cell transfection
The SiRNA and plasmids were obtained from Ruibo (Guangzhou), and the HCC cells were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Inc), according to the manufacturer’s instructions. All the transfected cells were incubated in complete medium for at least 24 h prior to transfection and rinsed with phosphate buffered saline (PBS, pH 7.4) before transient transfection.
2.4. RNA extraction and qRT-PCR
Total RNA was isolated from the cells using Trizol reagent (Invitrogen), according to the manufacturer’s instructions. Reverse transcription (RT) and qRT-PCR were performed using PrimeScript RT kit (Dalian, China, Treasure) and SYBR Prime Script RT PCR kit (Dalian, China, Treasure). The sequences of IFITM3, NCAPG, STAT3, CDK1, and GAPDH are provided in the supplementary information. The result was calculated using the 2-ΔΔCt method.
2.5. Western blot (WB)
All the proteins were separated by polyacrylamide sodium dodecyl sulfate gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane (Amersham, USA), and sealed with 5% skim milk at room temperature. The membrane was then treated with reagents containing the rabbit polyclonal antibody IFITM3 (ab109429, 1:1000, Abcam, Cambridge, UK), NCAPG rabbit polyclonal antibody (ab226805, 1:2000, Abcam, Cambridge, UK), the STAT3 polyclonal rabbit antibody (ab68153, 1:1000, Abcam, Cambridge, UK), CDK1 rabbit polyclonal antibody (ab18, 1:10000, Abcam, Cambridge, UK), and GAPDH rabbit polyclonal antibody (ab9485, 1:2500, Abcam, Cambridge, UK). The treated membrane was then incubated at 4°C overnight, after which it was washed thrice with PBST buffer (PBS buffer containing 0.1% Tween-20) for 10 min. Horse peroxidase-labeled anti-rabbit IgG secondary antibody (ab6721, 1:2000, Abcam, Cambridge, UK) was then added to the membrane and incubated for 1 h at room temperature. The membrane was washed thrice with PBST buffer for 10 min. A photometer (GE, USA) was used to detect immune activity.
2.6. Transwell migration analysis
Tests for cell migration and invasion were performed in a Transwell chamber (Corning Inc., Corning, NY, USA) with a polycarbonate membrane. In the Transwell migration analysis, 1×105 cells of both the experimental cell lines MHCC-97H and HCC-LM3 were seeded into the upper chamber of DMEM without serum, while 10% FBS was added to the lower chamber. After incubation for about 18–36 h, the cells were in the upper chamber were wiped off, and those in the lower chamber were stained with 1% crystal violet at 25 °C for 1 min. The stained cells were then observed and counted under an optical microscope (Nikon). The procedure of the Transwell invasion assay was the same as above, except that the upper chamber was coated with 20 µg extracellular matrix gel (Sigma-Aldrich; Merck KGaA).
2.7. Scratch test
NHCC-97H and HCC-LM3 cells were used for the scratch experiment. First, the cells were seeded in a six-well plate and transfected in the six-well plate. While transfecting and changing the medium, the six-well plate was scratched using a 200ul sterile tip, marked as 0 h, and a picture was taken. After 24 h, another picture was taken at the same marked location. The blank distance of the two pictures was compared statistically.
2.8. Co-immunoprecipitation (CO-IP)
The cells were first transfected and then harvested to prepare protein samples, using the following steps: 1. Cells were harvested 24–48 h after transfection by adding an appropriate amount of cell lysis buffer (containing protease inhibitors), lysing them on ice for 30 min, and then centrifuging them12000 rpm for 30 min. 2. A microcentrifuge tube was taken for Western blot analysis, 1 µg of the corresponding antibody was added to the remaining lysate, which was added to the cell lysate, and then incubated at 4 °C overnight. 3. An aliquot of 10 µL of protein A agarose beads was taken, and an appropriate amount of lysis buffer was used. The solution was washed thrice while centrifuging at 3,000 rpm for 3 min each time. 4. An aliquot of 10 µL of protein A agarose beads pretreated to the cell lysate was incubated overnight with the antibody and then incubated at 4 °C for 2–4 h for coupling of the Protein A agarose beads. 5. After the immunoprecipitation reaction, the mixture was centrifuged at 3,000 rpm for 3 min at 4 °C to settle the agarose beads at the bottom of the tube, after which the supernatant was carefully aspirated, and agarose beads were washed 3–4 times with 1 ml of lysis buffer. Finally, 15 µL of 2×SDS loading buffer was added and boiled for 5 min. 6. The proteins were then separated by SDS-PAGE and subjected to Western blot analysis.
2.9. In vivo experiments
For the in vivo invasion and metastasis assays . First, we divided the nude mice into seven groups, and injected HCC-LM3 cells from different treatments into the tail vein. After four weeks, the mice were sacrificed and the lung tissues of the nude mice were collected for HE staining and immunohistochemistry. Afterward,1×107 cells in 100 µL PBS were injected subcutaneously into the sides of male BALB/c-nu/nu mice (6–8 weeks old; n = 6 per group) (Hunan SJA Experimental Laboratory Animal Company) (Hunan, China). After six weeks, the mice were sacrificed (Using cervical dislocation method), and lung tissues were collected for HE staining and immunohistochemistry (IHC). All the animal experiments were approved by the Animal Experiment Ethics Committee of the Second Affiliated Hospital of Nanchang University and were carried out in accordance with the guidance of the British Animal (Scientific Procedures) Act, 1986, and the EU Directive 2010/63/EU.
2.10. HE staining and immunohistochemistry (IHC)
The prepared tissues were fixed with tissue fixative, placed in paraffin blocks, and sectioned using a microtome. The tissue sections were first dewaxed with xylene and then dehydrated with gradient ethanol. The sections were stained with H&E to identify any changes in their morphology and then rehydrated and microwaved in sodium citrate buffer (10 mmol/L, pH 6.0) to recover the antigen. The sections were incubated with 0.3% hydrogen peroxide/PBS for 30 min and then blocked with serum. Subsequently, the tissue was incubated with 1:200 diluted rabbit monoclonal antibody NCAPG (ab226805, Abcam, Cambridge, MA, USA) at 4 °C overnight. It was then washed with PBS every 5 min for three times and then incubated with the secondary antibody at 37 °C for 30 min. Subsequently, the sections were stained with diaminobenzidine (DAB) and hematoxylin dye, after which the excess dye was washed with running water, rehydrated with gradient alcohol, and sealed with neutral resin. Finally, the sections were observed under an inverted microscope, and images were taken.
2.11. Statistical Analysis
Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software Inc.). All the data are expressed as mean ±standard deviation (SD). The two groups were compared using the t-test. Pearson’s χ2 test was used to analyze the relationship between IFITM3 expression and NCAPG. All experiments in the study were repeated thrice. P values of <0.05 or 0.01 were considered statistically significant.