Cell lines and reagents
Human HCC Cell lines SK-Hep1, HepG2 and Huh-7 and human immortalized liver Cell lines THLE-2 and THLE-5 were purchased from the National Collection of Authenticated Cell Culyures. HCC cell lines were grown in RPMI-1640 (Hyclone, Salt Lake City, Utah, USA) containing 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials, Hangzhou, China). Human immortalized liver cells were cultured in RPMI-1640 containing 15% fetal bovine serum. Stored in 5%CO2, 37℃ constant temperature incubator for culture.
The main reagents used in this study are as follows: IL-6protein was purchased from Sino Biological LNC, It is soluble in MP Biomedicals (CA, USA) at 100mg/mL. Tocilizumab was purchased from MedChemExpress, it goes into dimethyl sulfoxide. Sorafenib purchased from MedChem Express (Monmouth Junction, NJ, USA), dissolved in dimethyl sulfoxide. IL-6R antibody was purchased from Thermofisher. Cleaved Caspase-7, Caspase-7, Cleaved Caspase-3, Caspase-3, phospho-P70S6K, P70S6K, MMP9, MMP2, phospho-JAK2, JAK2, Phospho-Stat3, STAT3, β -actin and Secondary horseradish peroxidase (HRP) Conjugated goat anti-rabbit antibodies are purchased from Cell Signal Technology (Danvers, MA, USA).
Western Blot
The cells were placed in the culture dish for about 3 days, and the cells were basically covered at the bottom of the cell culture dish, when there were about 1.0*107 cells. The cells were placed in protein phosphatase and loading buffer mixed at a ratio of 50:1. The cells were first fully lysed on a shaker for about half an hour, then centrifuged in a low-temperature centrifuge for 30min. Finally, 5× Loading buffer was added and mixed, and then the cells were placed in a water bath at 99 ° C and fully heated for about half an hour. Cool naturally at room temperature, centrifuge and store in the refrigerator at minus 20 degrees. Remove when in use. Whole cell lysates of different treatment groups were prepared, and the extracted total protein was dissolved in 10% SDS-PAGE and transferred to PVDF membrane (EMD Millipore). The membrane was blocked in 5% skim milk at 37°C for 1h. After blocking, the membrane was incubated with specific antibody at 4°C overnight, and the secondary antibody (diluted 1:2000) was incubated at room temperature for 1h. Bands were visualized and exposed with the ECL Luminescence Kit (EMD Millipore), and ImageJ 1.44P software (National Institutes of Health, National Institutes of Health) was used for immunoblot quantification. The gray values of different imprinted signals were compared with the control group to analyze the expression of target gene protein.
Immunocytochemistry
The cells with a concentration of about 2.0×106 cells/mL were planted in 24-well plates, and the cells were observed under a microscope when the cell density reached about 80% after 24h growth in the incubator. Wash with PBS twice and fix in 4% paraformaldehyde for 15 min, then seal with H2O2 for 15 min. Incubate with closed serum at room temperature for 20min. 200 µL primary antibody (IL-6R, 1:100, PA5-102425) was added to each well and incubated overnight in a refrigerator at 4°C. Add the second antibody working solution and incubate for 30min at room temperature. The streptomycin working solution was added with horseradish peroxidase and incubated at room temperature for 30 min. DAB chromogenic solution (Beyotime Biotechnology, Shanghai,China) was used for dark staining for 15min, and hematoxylin (Beyotime Biotechnology, Shanghai,China) was used for nuclear staining for 10min. Finally, it was dehydrated with 95% alcohol and sealed with neutral resin for two seconds before being photographed under a microscope. H2O2, serum, secondary antibody and horseradish labeled streptomycin working solution were all products of SP kit (SP-9000, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.).
Edu Method To Detect Cell Viability
About 1.0×105 cells were planted in 24-well plates, and the experiment was carried out when the cells stuck to the wall and the growth basically covered the bottom of the petri dish for about two days. First replace the culture medium, add 0.5 microliter EdU reagent and then put it into the incubator for incubation for about 3 hours. Then take it out and wash it with PBS washing solution for 3 times for about 3 to 5 minutes each time. Apply 4% paraformaldehyde for about 15 minutes. The percentage of EdU positive cells was observed under a fluorescence microscope to measure cell proliferation.
Jc-1 Measures Cell Apoptosis
1.0×106 cell suspension was prepared and planted on a 24-well plate and incubated in an incubator. The cells were adherent for 24h and then treated with 1µM sorafenib. 48h later, the slipper was removed for experiment. Fix with 4% paraformaldehyde for 15min. It was then stained with JC-1 reagent for about 30 minutes. The nuclei were stained with DAPI for 10 minutes. The apoptosis level was determined by observing the intensity of red-green fluorescence under fluorescence microscope.
Transwell
After the cells were starved in serum-free medium for 24h, about 105~106 200µ L cell suspensions were taken and planted in Transwell cells in the middle and upper chambers of 24-well plates. 750µL medium containing 15% serum was added to the lower chamber of Transwell and placed in an incubator for 24h before absorption of the medium. Remove the chamber and transfer it to PBS for cleaning twice. Methanol was fixed at room temperature for 15 min, 0.1% crystal violet was dyed at room temperature for 15 min. Finally, the film was sealed with gum and photographed with fluorescence microscope. The number of cells is a measure of how invasive a cell is.
Scratch Healing Experiment
The cells were measured with a ruler and marked with a "horizontal line" on the back of the petri dish before inoculation. The cells were diluted to a certain extent and then planted in a petri dish and cultured in an incubator until they were spread to the bottom of the dish and then removed, scratch the line perpendicular to the ground with the 10 microliter point of the ruler. The scar widths of 0h, 24h and 48h were photographed by microscope and cell mobility was calculated.
Immunofluorescence
About 2.0×105 cells were planted on a 24-well plate. After incubation for 24h in a constant temperature incubator of 37℃ and 5%CO2, fixation with 4% paraformaldehyde for 20min. Dye with hematoxylin for 15 minutes. Eosin was stained for 5min. Finally, the film was sealed with neutral gum and photographed under a positive fluorescence microscope.
Clone Formation Experiment
About 1,000 cells are planted evenly in a six-well plate, Culture in 37℃ constant temperature incubator for 10 days. When the clone group in the orifice are visible to the naked eye. The cells were fixed with 4% paraformaldehyde for 15min at room temperature. After fixation, it was stained with crystal violet for 15min and photographed.
Xenotransplantation Of Nude Mice
All experiments were performed in accordance with the Animal Research: Reporting of In Vivo Experiments guidelines and in accordance with the principles and procedures approved by the Animal Experiment Ethics Committee of Anhui University of Science and Technology (Anhui Province, People's Republic of China). Nude mice (female, 6 weeks old) were provided by Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. SK-Hep1, SK-Hep1IL − 6, SK-Hep1STAT3− and SK-Hep1IL − 6+STAT3− were implanted into the subcutaneous ribs of nude mice, respectively. The body weight and tumor volume of the mice were recorded three days later, and the mice were sacrificed on day 30.
Statistical analysis
Experimental data were obtained through at least three independent experiments. Data are expressed as mean ± standard deviation. Statistical differences between the two groups were analyzed using the T-test. Analysis of variance (ANOVA) was used to compare differences between multiple groups. Tukey test to compare the mean values of multiple experimental groups. P < 0.05 is considered a statistically significant difference. GraphPad 8 statistical software was used for all analyses.
