2.1 TCGA data collection and analysis
The TCGA database was utilized to gather information on HTR1D gene expression in hepatocellular carcinoma tissues. The data was then analyzed to determine the correlation between HTR1D gene expression and various clinicopathological factors of HCC patients, including age, gender, TNM stage classification, TP53 mutation status, etc. Additionally, the correlation between HTR1D gene expression and patients' survival time was also examined.
2.2 Clinical sample collection
Four liver cancer samples and four paraneoplastic control samples were collected from fresh tissues during surgeries at the Department of Hepatobiliary Surgery, Haikou People's Hospital, between June 2020 and February 2023. The samples were promptly stored in liquid nitrogen after resection. Pathologists evaluated each sample independently to determine whether it was HCC or paraneoplastic control tissue. Clinicopathological information on the patients, such as age, sex, TNM stage, and survival time, was retrieved from hospital records. In addition, 88 paraffin-embessed tissue sections were obtained for further analysis.
2.3 Immunohistochemical analysis
Human liver microarrays (HLivH180Su30) were purchased from Shanghai Xinchao Biotechnology Co. Immunohistochemical (IHC) staining with a 5HT1D antibody was then performed on the microarrays using a staining kit from Bios Biological Technology Co., Ltd.
2.4 Quantitative real-time fluorescent PCR (qRT-PCR)
Total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific), and RNA concentration and quality were assessed using NanoDrop 2000 (Thermo Fisher Scientific). RNA was reverse transcribed using the PrimeScript RT Kit (Takara, Japan), and the cDNA region of interest was amplified using TB Green™ Fast qPCR Mix (Takara). After normalization to GAPDH protein levels, relative gene expression levels were determined using the 2-ΔCt method.
2.5 Cell culture
HuH-7 cells and HEP3B cells were purchased from Guangzhou Gineo Biotechnology Co. Cells were cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 1% penicillin-streptomycin (Thermo Fisher Scientific, USA), and 5 µg/mL plasmocin (InvivoGen, France). Cells were maintained at 37°C in a 5% CO2 atmosphere.
2.6 Cell transfection
Cells were collected and transferred to cell culture vessels and added to 6-well plates at a rate of 2 mL per well. The inoculated 6-well plate was then placed in a cell culture environment. When the cell count of approximately 60%-70% is observed, the transfection process can begin. The first step is to mix the transfection reagent, small RNA preservation solution, and transfection medium according to the product guide to form the transfection compound solution, then gently drop the compound solution into the wells to be transfected one by one, and then place the cells back into the cell culture incubator to continue incubation, and then wait for 48–72 h, and then decide on the next step according to the needs of the experiment.
2.7 CCK8 assay for cell viability
Cells were continuously monitored for 0, 24, 48, 72 and 96 h after treatment. At the end of the assay, all the culture medium was removed, the cells were washed once with PBS, 10 µL of CCK8 solution and 90 µL of complete culture medium were added and incubated for 30 minutes at 37°C. At the end of the incubation, the OD value of the cells was detected by enzyme labeling at 450 nm. The proliferation rate of the cells or the multiplicity of the NC group was calculated, and then the proliferation curve was plotted
2.8 Flow cytometry to detect apoptosis
Flow cytometry was used to detect apoptosis. The cells were treated according to the experimental protocol and then removed from the incubator. The supernatant was discarded and the cells were rinsed with PBS three times to remove surface material. The cells were digested (following the steps outlined in section 3.2.3, using 0.25% trypsin without EDTA), and then collected by centrifugation at 4°C for 10 minutes at 1000 r/min. After discarding the supernatant, the cells were washed with PBS twice. The cells were then centrifuged again at 1000 r/min for 10 minutes at 4°C, the supernatant was removed, and the cells were resuspended in flow buffer. The cell density was adjusted to approximately 5×104 /mL. A mixture of 5 µL Annexin V-FITC staining solution and 5 µL PI staining solution was added to the cell suspension, which was then incubated in a dark, room temperature environment on a centrifuge tube rack for 15 minutes to complete the staining. A total of 50,000 cells were analyzed using a flow cytometer, following the specific excitation channel recommended in the kit, to determine the apoptotic percentage of each group.
2.9 Transwell
Transwell assays were performed to assess cell migration capacity. Specifically, cells were transfected with different plasmids and then inoculated into the top compartment with serum-free medium, while medium containing 10% FBS was added to the lower compartment. After 48 h of incubation, the remaining cells in the top compartment were removed with a cotton swab and the cells in the lower compartment were stained with 0.1% crystal violet for 10 minutes. Finally, the migrated cells were measured and photographed under a microscope.
2.10 Assessment of Clone Formation Ability
The gelatin-coated 6-well plate was seeded with 10,000 cells per well, followed by transfection once cells had adhered to the plate. Cells were cultured for 7 days with a second transfection performed on day 4 to inhibit the HTR1D gene. Afterward, cells were washed with PBS, fixed with 4% paraformaldehyde, stained with crystal violet, and photographed. Cell counting was done using image J software.
2.11 Western Blot Analysis
HCC cells were lysed in RIPA buffer containing protease inhibitors on ice for 30 minutes. The lysates were centrifuged, and the supernatant was collected for protein concentration measurement using the Micro BCA Protein Assay Kit. The supernatant was then boiled with 5x Sample Buffer, separated by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with skimmed milk, incubated with primary antibodies overnight, followed by secondary antibodies the next day. Protein signals were visualized using ECL reagents and Bio-Rad Image Lab. Antibodies used include Anti-RhoA, Anti-Cyclin D1, GSK3β, GAPDH, Phospho-Akt(Ser473), and Phospho-PI3K P85 α/β/P55γ.
2.12 Animal Models
The HEP3B cell line was revived, expanded, and cultured. When the cell count reached the required tumorigenic level, the cells were adjusted to logarithmic growth phase and then harvested, resuspended in saline, and adjusted to a concentration of 1 × 107 cells/mL. Injections were administered in the axillary subcutaneous region of the upper limb of nude mice in a 200 µL volume, with 5 mice in each group.
(1) NC group: Intratumoral injection of sh-NC every 2 days at 10 nmol/each.
(2) sh-HTR1D group: Intratumoral injection of sh-HTR1D every 2 days at 10 nmol/each.
(3) Sorafenib group: Intratumoral injection of Sorafenib (2 mg/kg) every 2 days.
(4) sh-HTR1D/Sorafenib group: Intratumoral injection of si-HTR1D (2 mg/kg) and sh-HTR1D (10 nmol/each) every 2 days.
2.13 Statistical Analysis
All experimental data were presented as mean ± standard deviation and statistical analysis was conducted using SPSS 19.0 software. T-test was used for comparison between two groups, while one-way analysis of variance was used for comparison among multiple groups. A P-value of less than 0.05 was considered statistically significant and indicative of a significant difference.