Cell lines and drug treatment
HepG2215 and HepG2 cells were purchased from the American Type Culture Collection. HepG2215 and HepG2 cells were identified by Shanghai FuHeng BioLogy Co., Ltd. (www.fudancell.com) and Shanghai Biowing applied biotechnology Co., Ltd.(http://www.biowing.com.cn/), respectively, and cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C and in an atmosphere with 5% CO2 and 100% humidity. Cisplatin was purchased from Jinan Qilu Pharmaceutical Factory, Co., Ltd. (Jinan, China). HepG2 and HepG2215 cells were treated with cisplatin.
Lentivirus production and infection
The plasmids that encode the lentiviruses expressing shRNA molecules were obtained from the RNAi Consortium shRNA Library. The shRNA target 21-mer sequences were: shControl, CCTAAGGTTAAGTCGCCCTCG; human shYAP1
#3CCGGGACCAATAGCTCAGATCCTTTCTCGAGAAAGGATCTGAGCTATTGGTCTTTTTG. HepG2215 cells were placed in a 24-well tissue culture dish (2 ×105 cells per well) and infected with 80 μL shRNA lentivirus supernatant and 4 μg/ mL polyene. The infected cells were placed in RPMI medium containing 10% fetal bovine serum and 3 μg/ mL purinomycin, and detected 72 h after infection. Western blot was used to detect shRNA knockout efficiency.
Human Protein Atlas database
Human Protein Atlas(HPA)(https://www.proteinatlas.org/) provided each protein in 64 cell lines, 48 kinds of normal human tissues, and 20 kinds of cancer tissues. We collected 365 patients from HPA database and plotted survival time curves.
Survival analysis of PD-1 and PD-L1
Kaplan-Meier Plotter (KM) (https://kmplot.com/analysis/) is a clinical database of survival analysis, Log rank p-value and hazard ratio (HR), and 95% confidence intervals to assess the relationship between PD-1 and PD-L1 and overall survival.
Animal experiment
All animals were fed in the SPF with constant temperature (22-24 °C) and a dark–light cycle of 12 h/12 h, and housed in plastic cages. The protocol was approved by the Ethics Committee for Animal Experiment of Hebei University of Chinese Medicine (Permit number: YXLL2018002).
Immunofluorescence assay
All fluorescence images were observed under a confocal instrument. The primary antibodies were rabbit anti-CD4(GB11064, Seville Biological) and rabbit anti-CD8(GB13429, Seville Biological). The secondary antibody was FITC goat anti-rabbit IgG-HRP (GB22303, Seville Biological). Nucleus was blue by labeling with DAPI. Positive cells were green or red according to the fluorescent labels used. The image was captured using a fluorescence microscope (Olympus BX51, Japan).
Flow cytometry analysis
Spleens of mice were removed and placed in 5 mL PBS+1%FBS+2 mM EDTA solution. The spleen was ground and filtered to obtain spleen cells; Lyse the erythrocytes, count the cells, adjust to 1x106 cells/ml; Adding the antibody according to the antibody instruction, stained and tested on the machine. Finally, the results were analyzed.
Hematoxylin and eosin (H&E) staining
All organs were removed quickly, and the liver tissues were fixed with 4% formaldehyde for 24-48 h and then embedded with paraffin. The section thickness was 5 µm. The sliced sections were stained with haematoxylin and eosin (H&E), and change of histopathological was obtained using a microscope (Leica DM2500, Germany).
Immunohistochemistry (IHC)
Immunohistochemical staining was performed according to the instructions of Nakasi Jinqiao Kit. Finally, sections were determined by microscopic observation of the brown peroxidase in liver tissue at a 200× and 400× magnification. The results of immunohistochemistry were examined by 2 senior histopathologists. The cytomembrane/cytoplasm stained with light yellow or tan were regarded as positive cells. The optical density was measured by an Image-Pro Plus v6.0 software (Media Cybemetic, USA).
Measurement of inflammatory cytokines
IL-10 (ml002285, mlbio), IL-4 (ml002149, mlbio), IL-7 (ml002212, mlbio), IL-2
(ml002295, mlbio), IL-1β (ml063132, mlbio), CCL-2 (ml037533, mlbio), TGF-β (ml057830, mlbio) and IFN-γ (ml063095, mlbio) were determined using ELISA kits according to the manufacturer’s instructions.
Western blot analysis
HepG2215 and HepG2215 shYAP1 cells were seeded in 6-well plates (3×105 cells/well) and treated as described above. HepG2215 and HepG2215 shYAP1 cells were treated with DDP for 24 h. After a brief clean up at PBS, the cells were lysed directly in the SDS sample buffer (50 mM Tris–HCl pH 6.8, 1% SDS, 10% glycerol, 5% β-mercaptoethan, 0.01% bromophenol blue). The primary antibodies were rabbit YAP1 (D8H1X) XP monoclonal antibody (CST,14074s, diluted by 1:1000), rabbit PD-1 polyclonal antibody (Proteintech, 18106-1-AP, diluted by 1:2000), mice PD-L1 monoclonal antibody (Abcam, ab269674, diluted by 1:2000). rabbit GAPDH monoclonal antibody(Abways, AB0037,diluted by 1:5000).The secondary antibody was goat anti-rabbit IgG-HRP (Absin, abs20002, diluted by 1:10,000). The bands were detected by the ECL (enhanced chemiluminescence) detection system (Vilber, Fusion FX5 Spectra, France). The band intensity was measured by an Image-Pro Plus v6.0 software (Media Cybemetic, USA).
Ultrasonic testing
At the beginning and end of the experiment, ultrasound was used to measure the number of liver tumors in the mice using an imaging system (Vevo 2100, Visual Sonics Inc., Toronto, Canada) with an MS250 ultrasound transducer. Soon, the mice were anesthetized and their abdomens shaved. M-mode recording of short and long axis views of planed abdominal wall was performed.
DEN/TCPOBOP‑induced HCC model in C57BL/6 mice
Yap1LKO mice have been successfully constructed by Guangzhou Cyagen Biosciences (Guangzhou, China). The mice were identified by our group. The modeling method was introduced as previously described[11]. Yap1flox/flox were used as the control. 3-week-old male C57BL/6 mice were injected intraperitoneally with 25 mg/kg bodyweight N-nitrosodiethylamine (DEN). Then at the age of 4 weeks, the mice were injected with a dose of 3 mg/kg body weight TCPOBOP. The mice were divided into four groups after the model was successfully built. For the DDP group, the DDP dissolved in saline. The control mice were injected with saline. The Ethics Committee approved all experimental animal protocols for the Animal Experiment of Hebei University of Chinese Medicine (Permit number: YXLL2018002).
Statistical analysis
All statistical tests were performed by SPSS23.0 statistics software (SPSS, Chicago, IL). All the in vitro experiments were repeated for at least three times. Data were presented as Mean±SD. When more than two groups were enrolled, the means were compared between each two groups with one-way ANOVA. Differences were considered statistically significant when P<0.05.