Experimental materials
FaDu cells of human hypopharyngeal squamous cell carcinoma (Shanghai Jikai Kein, China); BALB/c nude mice (Beijing Spafu, China).
Experimental methods
The stable PVT1 knockout FaDu cell line was constructed
After the cells reached the logarithmic growth phase, a suspension was prepared at a density of 5 × 104 cells/ml. A 2 ml aliquot of this suspension was seeded into each well of a six-well plate and incubated at 37 °C in a 5% CO2 environment. Once the cells reached 80–90% confluence, the medium was replaced with 1 ml of fresh serum-free medium containing 40 µl of HitransGP and an appropriate amount of sh-PVT1 lentivirus suspension (GenePharma, Shanghai). The cells were then incubated under the same conditions. After 16 h, the medium was replaced with fresh normal medium, and the cells were further cultured. Following 72 h of infection, the medium was replaced with fresh medium containing 1 µg/ml puromycin, and the cells were incubated for an additional 48 h to select for stable PVT1 knockdown in FaDu cells. Green fluorescence expression was observed using a fluorescent inverted microscope, and the success of PVT1 knockdown was confirmed via qRT-PCR.
CCK-8 cell proliferation assay
After the cells grew to the logarithmic phase, the cell suspension at a density of 8 × 104/ml was prepared, and 200ul of the cell suspension was evenly seeded in 96-well plates. Each group was set up with six multiple Wells and cultured in an incubator at 37℃ with 5% CO2. At 4 h, 24 h, 48 h, and 72 h of culture, 10 µl CCK-8 solution was added to the cells, respectively, and the cells were incubated with 5% CO2 and 37℃ for 2 h. The 96-well plate was shaken and mixed on the excellent shaker. The absorbance value at 450 nm was measured using a microplate reader.
Clone formation assay
After the cells were grown to logarithmic phase and made into cell suspension, 800 cells/well were seeded uniformly in six-well plates containing 2 ml medium and cultured in an incubator at 37℃ with 5% CO2. When cell mass could be seen after 2–3 weeks of cell culture, the culture was terminated. The supernatant was discarded, washed 3 times with PBS buffer salt solution, and stained with crystal violet staining solution for 15 min, and then the crystal violet staining solution on the surface was cleared, the colony formation area was scanned, and the number of cell clones was counted.
Cell scratch assay
Draw lines on the back of the 6-well plate so that the same field of view can be located when taking photos; After the cells grew to a logarithmic phase, the cell suspension at a density of 5 × 105/ml was prepared, and 2 ml cell suspension was seeded in a six-well plate and cultured in an incubator at 37℃ with 5% CO2. When the cell density increased to more than 90% confluence, the cells were immediately replaced with serum-free medium, and the wound was scratched with 10 µl of the gun tip perpendicular to the marker line. Pictures were taken at 0 h, 6 h, 12 h, 24 h, and 48 h to observe cell migration.
Transwell assay
The cells in the logarithmic growth phase were prepared with serum-free medium for cell suspension, and 200 µl cell suspension (1 × 105 cells) was uniformly seeded in the upper chamber of the Transwell chamber of a 24-well plate. Then 800 µl medium containing 10% fetal bovine serum was added to the lower chamber of a 24-well plate and cultured in an incubator at 5% CO2 and 37℃ for 24 h. The cells in the upper chamber were gently wiped off with a cotton swab, washed with PBS buffer salt solution, and stained with crystal violet staining solution for 15 min to clean the surface crystal violet staining solution. Photos were taken and counted in six randomly selected fields of view for microscopic observation.
Matrigel was coated in the upper Transwell chamber, diluted with a serum-free medium at a ratio of 1:4, and then 40 µl diluted Matrigel was evenly spread on the bottom membrane of the upper chamber and incubated in the incubator at 37℃ for 4 h. The remaining experimental procedures were the same as the cell migration experiment.
Real-time quantitative PCR (qRT-PCR)
The primer sequence of qRT-PCR is shown in Table 1.
ELISA
Human TGF-β1 ELISA Kit (BOSTER, Wuhan, China) was used to detect the expression level of TGF-β1 in cells according to the instructions. The absorbance (OD value) at 450 nm was determined by enzymoleter, and the standard curve was drawn by ELISA Calc software to calculate the sample concentration.
Tumor formation experiment in nude mice
The cells in the logarithmic growth phase were resuspended in PBS buffer salt solution at a cell density of 1 × 107 cells /ml. Four-week-old female nude mice were selected and acclimatized for 1 week, with 6 mice in each group. After the nude mice were anesthetized by ether inhalation, 100 µl cell suspension was thoroughly mixed and injected subcutaneously into the middle and posterior part of the right axilla of nude mice with a syringe. After the injection, the injection point was pressed for 60 s with a cotton swab. After the tumor was visible to the naked eye, the length and width were measured every 3 days, and the volume V= (length × width2) ×0.5 was calculated. After 6 weeks, all the mice were sacrificed, the tumors were removed, and then the net weight of the tumors was measured.
TGF-β 3’‑UTR reporter vector construction, transfection, and dual luciferase assay
The TGF-β 3’UTR gene sequence was synthesized, and SacI/HindIII restriction enzyme sites were added at both ends of the sequence according to the polyclonal sites of the pMIR-REPORT plasmid. TGF-β 3’UTR gene sequence and pMIR-REPORT plasmid were digested with SacI/HindIII restriction enzyme. DNA fragments were recovered by DNA purification kit (Guangzhou Megi, China) according to the manufacturer’s instructions, and an ultramicro ultraviolet spectrophotometer detected the concentration. The 3’UTR fragment recovered by restriction enzyme digestion was ligated with pMIR-REPORT plasmid fragment at a volume of 3:1. Plasmids were extracted with a plasmid extraction kit (Axygen, USA) according to the manufacturer’s instructions, and plasmid concentrations were measured with an ultramicro ultraviolet spectrophotometer. The extracted plasmids were sequenced to verify the successful construction of the TGF-β 3’UTR reporter vector.
After 293T cells were grown to the logarithmic phase, the cell suspension was prepared, seeded in 12-well plates, and cultured in an incubator at 37℃ with 5% CO2. When the cell density reached more than 60% confluence, PMIR-TGF-β reporter Gene and CV045 plasmid (Renilla Luciferase) (Jikai, Shanghai) were transfected according to the instructions of X-treme GENE 9 DNA transfection reagent. Dual luciferase assays were performed 24 h to 48 h after transfection. According to the instructions of the dual luciferase reporter gene detection system kit (Promega, USA), the fluorescence values of firefly luciferase and renilla luciferase were detected by GloMax Navigator microplate luminescence detector, respectively, and the data were analyzed.
Statistical analysis
SPSS 26.0 and GraphPad Prism 8.0 software were used for data analysis and drawing. Quantitative data were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used to compare the data between multiple groups of samples, and a two-tailed unpaired t-test was used to compare the data between two groups of samples.
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