Dataset source and preprocessing

The expression levels of RAC1 in 424 LIHC samples with 50 adjacent normal tissue samples were analyzed based on the dataset of The Cancer Genome Atlas (TCGA-LIHC) mRNA sequencing (RNA-seq) database. Clinical characteristics such as tumor statue, histologic grades, pathologic stages and vascular invasion were assessed with respect to their respective clinical patterns. Differential transcriptional expression was compared to Student`s t-test.

Immune infiltration algorithm

Based on the ssGSEA algorithm provided in R-packet-GSVA [1.46.0], the provided markers of 24 immune cells were used to calculate the immune infiltration of the corresponding data.

Diagnostic analysis

The Receiver Operating Characteristic (ROC) curve was applied to assess the specificity and sensitivity of gene prediction accuracy, using the area under the ROC curve (AUC) as a diagnostic value based on the “pROC” package in the statistical software (version 1.18.0) was used.

Survival analysis

The Kaplan-Meier curve was performed to compare overall survival (OS) between the differential expression groups of RAC1 including 424 LIHC samples in the TCGA database and KM Plotter, respectively. The correlation between RAC1 expression and survival was analyzed to discover the significant prognostic factors. The hazard ratio (HR) with 95% confidence interval (CI) and log-rank P value were also calculated.

Correlation analysis

Gene expression correlation analysis was performed for given sets of mRNA expression data in TCGA-LIHC were used for analysis. The correlation coefficient was determined by the Spearman method.

Immunohistochemical staining (IHC)

We have obtained the IHC result of hepatocellular carcinoma sample (ID: 2766) from The Human Protein Atlas (https://www.proteinatlas.org/). Meanwhile, we also explored the RAC1 protein level in LIHC by IHC using a commercial tissue microarray (Bioaitech Co., Xi’an, China, cat #D202Lv01) with 10 LIHC and 10 normal liver tissues. IHC procedure was conducted as follows: tissues were first dewaxed and hydrated, followed by antigen retrieval and blocking of endogenous peroxidase activity. The slides were then incubated with the primary antibody overnight at 4 °C. After incubation with the secondary antibody, the slides were stained with diaminobenzidine (DAB). For the evaluation and scoring, two pathologists independently assessed the staining results. RAC1 protein expression was scored based on both the percentage of positively stained tumor cells and the staining intensity. Specifically, the percentage of immunoreactive tumor cells was scored as 1 (< 10%), 2 (10–25%), 3 (26–49%), and 4 (≥ 50%). Staining intensity was visually assessed and scored as 1 (negative), 2 (light yellow), 3 (light brown), and 4 (dark brown). The final immunoreactivity score for each case was obtained by multiplying the percentage score by the intensity score.

Cell culture

The LIHC cell lines utilized in this study, HepG2 and Huh7, were procured from the China Center for Type Culture Collection. The complete culture medium formulation used for cell culture consisted of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were cultured in an incubator maintained at 37 °C with 5% CO2. Cells in the logarithmic growth phase were used for subsequent experiments.

Transfection of small interfering RNA

The siRNAs used in this study were sourced from Genscript Co., Ltd., located in Nanjing, China. For the transfection of siRNAs into liver hepatocellular carcinoma (LIHC) cell lines, we utilized Lipofectamine 2000 (Invitrogen, Carlsbad, USA), following the manufacturer’s instructions.

The transfection procedure began with seeding LIHC cells onto cell culture plates. The cells were cultured in appropriate growth medium until they reached approximately 50% confluence. At this stage, the siRNAs were prepared for transfection. Each siRNA was diluted in an opti-MEM medium and mixed with Lipofectamine 2000 reagent in a separate tube. The siRNA-Lipofectamine 2000 mixture was then incubated at room temperature for 20 min to allow for the formation of siRNA-lipid complexes.

Following incubation, the siRNA-lipid complex was added to the cells in their culture plates. The final concentration of siRNA in the transfection mixture was set to 100 nM. The cells were then incubated with this transfection mixture for 48 h to ensure sufficient uptake and effective gene silencing.

The siRNAs used in this study had the following sequences:

siRAC1-1: 5’-AAGACAAGCCGATTGCCGACG-3’.

siRAC1-2: 5’-AAGCCGATTGCCGACGTGTTC-3’.

Quantitative real-time polymerase chain reaction

The cDNA was amplified using SYBR-Green PCR Master Mix (TAKARA, Tokyo, Japan) on a QuantStudio 5 system (ABI, Carlsbad, USA) under the following conditions: an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. The level of RAC1 mRNA was calculated by 2−ΔΔCt method using GAPDH as internal control.

The sequences of the specific primers used for amplification were as follows:

GAPDH forward, 5’-AGATCCCTCCAAAATCAAGTGG-3’;

GAPDH reverse, 5’- GGCAGAGATGATGACCCTTTT-3’;

RAC1 forward, 5’-ATGTCCGTGCAAAGTGGTATC − 3’;

RAC1 reverse, 5’-CTCGGATCGCTTCGTCAAACA-3’.

Western blotting

After transfecting cells with siRNA for 72 h, cells were collected and lysed using RIPA buffer. Equal amounts of protein from each group were separated by 12% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA at room temperature for 1 h, followed by incubation with anti-RAC1 antibody (Abcam, ab155938) and then with HRP-conjugated secondary antibody (Abcam, ab288151). Signal detection was performed using ECL chemiluminescence reagents, and results were recorded with a chemiluminescent imaging system. The membranes were then stripped of antibodies and re-blocked, followed by sequential incubation with anti-GAPDH antibody (Abcam, ab8245) and HRP-conjugated secondary antibody (Abcam, ab205719). ECL detection and imaging were subsequently performed.

Wound healing assay

The migratory ability of different groups of LIHC cells was evaluated using a wound-healing assay. When the cells covered 95% of the bottom of a six-well plate, a 10 µL pipette tip was used to create a straight scratch in the monolayer. The cells were then washed twice with PBS and replenished with complete medium. After imaging, the cells were cultured for an additional 24 h before being imaged again. The migration index was calculated using Image J software with the following formula:

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Proliferation assay

The proliferation capability of different LIHC cell groups was assessed using the CCK-8 assay. When the cells reached 50% confluence, culture medium containing 10% CCK-8 solution was added to each well and incubated at 37 °C for 30 min. Absorbance at 450 nm was then measured for each well. After the measurement, the medium was replaced with fresh culture medium, and cells were cultured further. Proliferation was assessed at various time points, including 1, 2, 4, 8, 12, 24, 36, 48, and 96 h. The proliferation rate of each cell line was calculated relative to the absorbance value at the 0-hour time point.

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Subcutaneous tumor xenograft model of LIHC cells

After 72 h of transfection, the cells were collected for tumor xenograft implantation. The LIHC xenograft model was established by subcutaneously injecting different groups of LIHC cells into the right armpits of six-week-old BALB/c nude mice (1 × 10^7 cells per mouse; three animals per group). Tumor size was recorded every other day, and tumor volume was calculated according to the formula: width^2 × length/2. Twelve days after tumor cell implantation, all mice were euthanized, and tumor tissues were collected.

Statistical analysis

The Wilcoxon rank sum test was used to assess the significant differential expression levels of the RAC1 in LIHC with the threshold of gene expression being selected as the median method. Correlation of gene expression was evaluated by Spearman’s correlation coefficient. Univariate Cox analysis was used to screen for potential risk factors, and multivariate Cox analysis was used to verify the independent variate of RAC1 expression on overall survival. All statistical analyzes were performed with the R statistical software (version 4.2.1). A P-value of less than 0.05 is considered statistically significant.