1. Human samples
The research included ten patients with HCC who received surgical intervention at the First Affiliated Hospital of Sun Yat-sen University. The diagnosis of HCC was confirmed through postoperative pathological examination. Samples of healthy tissue were collected from a site that was 2cm next to the cancerous tissue. First Affiliated Hospital of Sun Yat-sen University's Ethics Committee approved the collection of patient clinical samples with informed consent.
2. Cell culture
The human liver cell lines Lo2, human HCC cell lines (Hep1, Huh7, LM3) and Hep1-6 were acquired from the General Surgery Laboratory at the First Affiliated Hospital of Sun Yat-sen University. The cell lines were cultured in a humidified atmosphere of 5% CO2 using DMEM medium containing 10% fetal bovine serum (FBS) and 1% antibiotics.
3. Reverse transcription and quantitative real-time PCR (qRT-PCR)
Trizol reagent was utilized to extract the total RNA, followed by DNase I treatment to eliminate any remaining genomic DNA. Subsequently, the Roche RT-PCR kit (Transcriptor cDNA Synth) was utilized for reverse transcription. Follow the manufacturer's instructions for Kit, Roche. The analysis of gene expression was conducted on a Roche LightCycler 480 PCR instrument using the SYBR Green real-time PCR mixture, with GAPDH serving as the internal control.
4. Vector construction, lentivirus production, and transfections
GeneCopoeia constructed the shRNA complementary oligonucleotides that target circHIPK3 and the overexpression vector for miR-381-3p. A lentiviral vector with no content was employed as a negative control. 293T cells were transfected to produce lentivirus. Hep1 and Huh7 cells were placed in a 6-well dish and exposed to the relevant lentivirus in the presence of 10ug/ml polybrene. Following a 48-hour period, Hep1 and Huh7 cells with downregulated circHIPK3 or overexpressed miR-381-3p were subjected to stable selection by adding puromycin at a concentration of 2ug/ml. Tsingke synthesized the inhibitor of miR-381-3p. All transfections were performed using the lipo3000 reagent.
5. CCK-8 assay
The cells were placed in DMEM supplemented with 10% FBS and distributed at a concentration of 1000 cells per well in a 96-well plate. Following a 24-hour period, 10 microliters of CCK-8 solution were introduced into every well and subsequently incubated in darkness for an extra 2 hours. The measurement of absorbance was conducted at a wavelength of 450 nm, with the initial day's absorbance value serving as a reference. Measurements were taken continuously from the second to the fourth day. Every measurement was conducted three times, and the experiment was repeated no less than three occasions.
6. Colony formation assay
The cells were placed in a 6-well plate with a density of 1000 cells per well and left to incubate for a duration of two weeks, with a change of media every three days. Following that, the cells were rinsed using PBS, treated with methanol for fixation, and then subjected to staining with crystal violet. Colony counting was performed using ImageJ software.
7. Cell scratch assay
The cells were grown in a 6-well dish and given time to reach full coverage. After the cells achieved confluence, a 200μl pipette tip was used as a guide to draw a straight line in each well. After removing the culture medium, the wells were rinsed three times with sterile PBS. The addition of medium without serum was followed by placing the plate under a microscope for imaging, which was regarded as the starting point (0-hour) for scratch formation. Afterwards, the dish was placed in an environment with a temperature of 37°C and a CO2 concentration of 5%. The cells were observed once more under the microscope after a period of 24 hours.
8. Transwell migration assay
A total of 75,000 cells were suspended in 200 microliters of DMEM culture medium without serum and then placed in the upper chamber of a Transwell insert. DMEM culture medium containing 10% FBS filled the lower chamber. The cells were placed in an incubator set at a temperature of 37°C with a 5% concentration of CO2 for a duration of 24 hours. Following incubation, the cells that did not migrate in the upper chamber were eliminated. Poly-methanol was used to fix the cells located beneath the membrane, which were then stained with 0.5% crystal violet and captured using a 100x microscope. Cell counting was performed using ImageJ software.
9. Luciferase reporter assay
Hep1 and Huh7 cells were seeded in a 24-well plate and left overnight to perform the luciferase reporter gene assay. Next, the cells were transfected with Lipofectamine 3000 transfection reagent, along with 50 nM miR-381-3p mimic, and co-transfected with pmirGLO-circHIPK3-WT reporter plasmid and its corresponding mutant vectors. Geneyuan provided both the plasmids and the mimics. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System following the manufacturer's guidelines after a 36-hour period of cell culture. Firefly/Renillafluorescence ratio was used to calculate relative luciferase activity.
10. Western blotting
Proteins were extracted from cells or tissues using RIPA lysis buffer, which included inhibitors for proteinase and phosphatase. Following the addition of 5 times the amount of SDS, the samples underwent denaturation at a temperature of 95 degrees Celsius for a duration of 10 minutes. Afterwards, the proteins were isolated using a 10% SDS-polyacrylamide gel and subsequently transferred onto a PVDF membrane. Following the blocking process using non-fat milk at room temperature for 1 hour, the membrane was then subjected to separate overnight incubation at 4°C with rabbit anti-YAP antibody and mouse anti-GAPDH antibody. Afterwards, the membrane was exposed to secondary antibodies at ambient temperature for a duration of 1 hour. Use ECL chemiluminescent substrate for detection and image the samples using Tanon’s gel chemiluminescence imaging system.
11. Statistical analysis
GraphPad Prism9 software was utilized for conducting all statistical data analysis. The results are presented as mean ± standard deviation. The significance of variations was assessed using t-test or one-way analysis of variance (ANOVA) followed by the relevant Tukey's post hoc examination. Statistical significance was determined when the value of P was less than 0.05.