3.1 Cell culture
Five HCC cell lines and human normal liver cell LO2 were employed in this study. All HCC cell lines (Hep-G2, Hep-3B, LM3, Huh7, and MHCC97H) were acquired from the Chinese Academy of Sciences' cell bank, while LO2 was acquired from Southern Medical University's Cancer Research Institute. All cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) from GIBCO, NY, USA, and antibiotics from BIOMYC-3 Antibiotic Solution and Penicillin-Streptomycin Amphotericin B Solution from Biological Industries, Israel, at 37°C and 5% CO2.
3.2 Clinical specimens and immunohistochemistry (IHC)
We purchased a HCC tissue microarrays from Shanghai Outdo Biotech Company, containing 90 samples of HCC and 90 samples of paracancerous tissues. The clinical samples were approved by patients' informed consent and authoritative histopathologists, and passed the review of the ethics committee on the use of the samples(Ctl No.:YB M-05-02). After a series of steps such as dewaxing, hydration, block the activity of endogenous peroxidase and nonspecific antigen,antigen retrieval, incubation with first antibody, staining and sealing. The tissue microarray was observed under Inverted microscope and double-blind scored in accordance with staining intensity(0-Colorless, 1-light yellow,2- light brown, 3-dark brown) and positive area ratio(0: ༜5%, 1:5%-25%, 2:25%-50%, 3:50%-75%, 4༚༞75%) by The Department of Pathology of the Hospital of Integrated traditional Chinese and Western Medicine of Southern Medical University.(Score calculation: staining intensity grade*positive area proportion grade)
3.3 SiRNA,plasmids and transfection conditions
The cells to be transfected were cultured in cell petri dishes for 48–72 hours in advance.SiRNA(Ribo-Bio, Guangzhou, China)and overexpressing plasmid(GeneChem,Shanghai,China) was introduced into cells by Lipofectamine 3000༈Thermo Fisher Scientific Company,USA༉ to obtain cell lines that temporarily silenced TM4SF1 or MYH9(Supplementary Table 1).
3.4 Lentivirus production and infection
Lentivirus human TM4SF1 gene was introduced into lentiviral vector GV367 to construct lentiviral TM4SF1(LV-TM4SF1)(GeneChem,Shanghai,China) to infect hepatoma cells, and stable hepatoma cell lines with loss of TM4SF1 expression was constructed. On this basis, the empty vector lentivirus(LVNC)(GeneChem,Shanghai,China) was used as the control, and the cells were screened with puromycin, and the polyclonal cells with green fluorescence signal were selected for follow-up experiment(Supplementary Table 1).
3.5 Extraction of RNA and QPCR
The total RNA of cells was obtained by using RNA extraction kit(Foregene, Chengdu, China), and cDNA was amplified by reverse transcription kit produced by Takara company. Finally, quantitative polymerase chain reaction of cDNA was performed with the help of Bio-Rad T100 system using primers ordered from Guangzhou IGE Biology Company(Supplementary Table 2), and the changes of genes were detected by calculating the data.
3.6 Western blot analysis and Antibody
The protein of the cell was obtained from the solution system of lytic buffer, protease inhibitor in the ratio of 100:1:1 and phosphatase inhibitor. The pure protein was collected by ultrasound and centrifugation and quantified by BCA protein analysis kit. SDS-polyacrylamide gel electrophoresis was used to separate the protein. It was then moved to a polyvinylidene fluoride membrane and treated at 4°C with the desired primary antibody. The antibodies used in the experiment include TM4SF1, CD44, CD133, OCT4, SOX2, MYH9, NOTCH1, JAGGED1, HES1, β-Tubulin(Supplementary Table 3). After the protein was fully combined with the primary antibody, the chemiluminescence kit(Thermo Fisher Scientific Company,USA) was used to image in the ChemiDoc XRS + molecular imager (Bio-Rad, Hercules, CA, USA)to analyze the expression of the protein.
3.7 Tumour sphere formation
The cells(5×103)were inoculated into six-well ultra-low attachment plates per well (Corning, NY, USA) and cultured with special sphere formation medium(It is composed of 1mL B27, 20uL human EGF of 20ng/mL and 20uL human FGF of 20ng/mL added to 50mL DMEM/F12 culture medium) at 37°C, 5% CO2 for 1–2 weeks. Tumour spheres were observed and photographed with inverted microscope.The proportions of tumour spheres with diameters of 50–100µm, 100–150µm and > 150µm in the total visual field were calculated,and statistical charts were drawn.
3.8 Detection of Side population cells
The cells (1×106) were prepared into a single cell suspension in DMEM containing 2% fetal bovine serum (FBS), The negative control group was treated with verapamil(50umol/L) purchased from Sigma-Aldrich Company. and then incubated with Hoechst 33342 (5µg/ml) purchased from Sigma-Aldrich Company in a 37°C water bath for 90 minutes, shaking gently every 10 minutes.Flow cytometry data were analyzed by FlowJo software (Tree Star Inc.). For analysis..
3.9 Fluorescence-activated cell sorting
For FACS staining, the cells were prepared as a single cell suspension. 30min was incubated with FITCCD133, PE-CD44 antibody and AF647 sheep anti-mouse immunoglobulin antibody (AF647) at 4 ℃, and the stained cells were obtained for LSRFortessa or AriaII (BD) analysis or sorting. Flow cytometry data were analyzed by FlowJo software (Tree Star Inc.).
3.10 Protein Mass Spectrometry
The protein extracted from the tested cells was entrusted to Fitgene Biotechnology company(Guangzhou,China)for protein spectrum analysis, and the genes whose expression degree changed after silencing TM4SF1 were screened. These genes were further screened according to the multiple of gene expression difference and bioinformatics database search, the appropriate genes were determined for further research.
3.11 Coimmunoprecipitation (co-IP)
The total protein of the cells to be tested was extracted for protein value quantification, and 10µg immunoglobulin or specific antibody was added to the 5mg protein and incubated overnight at 4 ℃. Finally, the protein was recovered for Western blot analysis.
3.12 Establishment of Lenvatinib-resistant cell lines
The cells were cultured for a long time by the method of increasing concentration, and the model of Lenvatinib-resistant strain was established. At 37°C and 5% CO2, the cells were grown in DMEM containing 10% fetal bovine serum (FBS). Lenvatinib of 1 mg was administered to the culture media once the cells had stabilized. After 1 week of culture, if the cells could grow stably, they would be changed to a higher concentration of Lenvatinib (increasing 1mg each time), until the cells could grow stably in the culture medium containing 10mg Lenvatinib, indicating that Lenvatinib-resistant cells were induced successfully.
3.13 etermination of CCK-8 cell viability
The cell survival rate and tolerance to Lenvatinib were detected by CCK-8 method. According to previous studies[31],The cells (5×103) were inoculated in a 96-well plate and cultured for 24 hours in 200µ Lenvatinib medium with different concentrations of Lenvatinib at 37°C and 5%CO2. Then after 2 hours of treatment with 10% CCK8, the absorbance was detected at 450nm, and the cell survival rate was calculated.
3.14 Mouse xenografts
All animal experiments meet the requirements of the International guidelines for Animal Care and Conservation and the Animal Research Committee of the academic Medical Center of Southern Medical University. The tumor growth was evaluated by subcutaneous transplantation of xenogeneic oysters in mice.
LM3 with stable expression loss of TM4SF1(LV-TM4SF1) in different concentration gradients was subcutaneously injected into 3-week-old male nude mice weighing 16-25g, and the corresponding concentration of LM3 infected with the empty vector lentivirus(LV-NC) was used as negative control.The growth of the tumor was observed and the size of the tumor was recorded with Vernier caliper every three days. 45 days later, the tumor was taken out, photographed and weighed, and the data of the tumor were collected for statistics and analysis.
The effect of TM4SF1 on drug resistance of human hepatocellular carcinoma cell line Lenvatinib was studied by subcutaneous transplantation in nude mice. Saline containing Lenvatinib was injected into nude mice by intraperitoneal injection in advance. The experimental group of nude mice received subcutaneous injections of various quantities of Lenvatinib-resistant LM3; untreated LM3 served as the negative control. Then, according to the previous method, nude mice were cultured and tumor data were collected for follow-up experiments.
3.15 Primary cell culture
Cut the subcutaneous tumor of nude mice taken from the previous experiment into smaller lumps (about 4mm in diameter), rinse with PBS to remove blood clots and adipose tissue, digest with 0.25% trypsin in a water bath at 37 ℃ for 15–20 minutes, wash again with PBS, then add a small amount of cell culture medium to gently blow and disperse, count the cells and culture them with appropriate concentration. If there is still undigested tissue, you can repeat the above steps to digest it many times.
3.16 Bioinformatics analysis
The study used bioinformatics databases to obtain materials that can support the conclusions of the study. The databases used include: The Cancer Genome Atlas(TCGA), TIMER2.0,Gene Expression Profiling Interactive Analysis(GEPIA). Gene Set Enrichment Analysis(GSEA) technique was used to enrich the KEGG and GO pathways of TM4SF1, and the pathways related to its expression were obtained.
3.17 statistical analysis
Each group of in vitro experiments were repeated three times to ensure the credibility and reproducibility of the study, and the average-standard deviation (x s) was used to express the measured results. SPSS13.0 software was used to analyze the statistical process and data in the study, and t-test was used to compare between groups, the difference was statistically significant(P < 0.05).