A total of 216 patients were included in the present study. These patients (176 male and 40 females; age range, 23-77 years) were recruited from Shouyi Campus and East Campus of the Renmin Hospital of Wuhan University (Wuhan, China). To account for potential demographic variations between the two campuses, they were divided into two independent cohorts: Cohort 1 comprised 131 patients (110 males and 21 female; age range, 23-77 years), and cohort 2 comprised 85 patients (66 males and 19 females; age range, 28-75 years). Paired tumorous and matched adjacent non-tumorous tissues (located at least 3 cm away from the tumor margin) were collected from these patients who underwent surgical resection at the Renmin Hospital of Wuhan University (Wuhan, China) from January 2022 to December 2023. The inclusion criteria were as follows: i) Pathologically confirmed primary HCC; and ii) availability of complete clinicopathological and follow-up data. The exclusion criteria were: i) Patients who had received preoperative anti-cancer treatments (such as chemotherapy, radiotherapy, targeted therapy, or local embolization); and ii) presence of other primary malignancies. The present study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Renmin Hospital of Wuhan University (Wuhan, China; approval no. WDRY2022-K013). Written informed consent was obtained from all patients prior to surgery and sample collection.

Huh7 (cat. no. CL-0120) and Hep-3B (cat. no. CL-0102) cell lines were purchased from Wuhan Pricella Biotechnology Co. MHCC 97h (cat. no. CBP60227) and HLF (cat. no. CBP60589) cell lines were purchased from Nanjing Kebai Biotechnology Co., Ltd. Snu449 (cat. no. CC0105) cells were purchased from Saiku Biotechnology Co. 97h is a highly metastatic cell line derived from a patient with HCC lung metastasis. Huh7 is a well-differentiated, non-metastatic cell line with mutant p53. Hep-3B is a poorly differentiated cell line deficient in p53 expression and harboring an integrated hepatitis B virus genome. HLF is a highly metastatic cell line with mutant p53, while Snu449 is derived from a primary tumor and also carries a mutant p53. 97h, Huh7, Hep-3B and HLF cells were cultured in DMEM supplemented with 10% FBS, while Snu449 cells were maintained in RPMI-1640 medium (all Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS. All cell lines were incubated under standard conditions (37°C, 5% CO₂).

For lentiviral production, 293T cells (Wuhan Pricella Biotechnology; cat. no. CL-0001) were co-transfected with 10 μg 2nd-generation RRP9, CCNA2 or their respective negative control lentiviral plasmids and helper plasmids (psPAX2 and pMD2.G; ratio 4:3:1; MiaoLing Plasmid) using Lipo8000™ (Beyotime Biotechnology; cat. no. C0533) at 37°C for 10 h. Viral particles collected at 48 h were used to infect HCC cells at an MOI of 10 for 24 h at 37°C with 5% CO₂. Stable transductants were selected with 10 μg/ml puromycin, maintained at 2 μg/ml and used for subsequent experiments 7 days post-transduction.

740 Y-P is a cell-permeable peptide that mimics a tyrosine-phosphorylated segment of the platelet-derived growth factor receptor, thereby directly activating PI3K by binding its SH2 domains (34,35). Cells were treated with 20 μM 740 Y-P or 10 μM PI3K/AKT/mTOR-IN-2 for 24 h at 37°C (25,26).

Tissue specimens, fixed in 4% paraformaldehyde at room temperature for 24 h, were embedded in paraffin and cut into 1.5 μm thick sections. The sections were deparaffinized in xylene and rehydrated through a descending alcohol series. Antigen retrieval was performed by heating in Tris-EDTA antigen repair solution (pH 9.0) at 95°C for 15 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 15 min, followed by membrane permeabilization using 0.2% Triton X-100 for 45 min. Non-specific binding was blocked with 10% donkey serum (Beijing Solarbio Biotechnology; cat. no. SL050) at room temperature for 1 h. Subsequently, sections were incubated at 4°C for 12-16 h with primary antibodies, including anti-RRP9 (Santa Cruz Biotechnology; 1:50; cat. no. sc-100592) or Ki-67 (Wuhan Pricella Biotechnology; 1:50; cat No. 27309-1-AP). Sections were incubated with an HRP-conjugated secondary antibody (Thermo Fisher Scientific Inc.; 1:500; cat. no. 31460) using the MaxVision detection reagent at room temperature for 30 min. Immunoreactivity was visualized using a DAB Chromogen detection kit (Abcam). Nuclei were counterstained with hematoxylin at room temperature for 5 min. Slides were examined and imaged using a light microscope (Olympus Corporation), with quantitative analysis performed using ImageJ software (version 1.8.0; National Institutes of Health).

To evaluate RRP9 expression in the clinical cohorts, a tissue microarray was constructed using the collected formalin-fixed, paraffin-embedded (FFPE) HCC and matched adjacent non-tumor tissues. Due to the large sample size exceeding the maximum capacity of a single recipient block, the cohort samples were distributed across three independent TMA blocks to preserve tissue integrity and ensure high-quality sectioning. To minimize technical variations and ensure direct comparability of the data, all three TMA blocks were prepared, sectioned, and immunohistochemically stained simultaneously using an identical protocol.

To evaluate apoptosis in the xenograft tumor tissues, TUNEL) assay was performed using a TUNEL Apoptosis Assay Kit (Beyotime Biotechnology; cat. no. C1086) according to the manufacturer's instructions.

Total protein was extracted from cultured cells and HCC tissues using RIPA lysis buffer (Beyotime Biotechnology) supplemented with phosphatase and protease inhibitors (including PMSF). Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein (20 μg per lane) were separated via 10% SDS-PAGE. Proteins were then electrotransferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and incubated overnight at 4°C with primary antibodies against the target proteins and the internal reference, RRP9 (Santa Cruz Biotechnology; 1:50; cat. no. sc-100592); E-cadherin (Proteintech Group, Inc.; 1:5,000, cat. no. 22018-1-AP), N-cadherin (Proteintech Group, Inc.; 1:5,000, cat. no. 20874-1-AP), Vimentin (Proteintech Group, Inc.; 1:5,000, cat. no. 10366-1-AP), Snail-1 (Proteintech Group, Inc.; 1:5,000, cat. no. 13099-1-AP), Bax (Proteintech Group, Inc.; 1:5,000, cat. no. 50599-2-Ig), Bcl-2 (Proteintech Group, Inc.; 1:5,000, cat. no. 12789-1-AP), PI3K (Proteintech Group, Inc.; 1:5,000, cat. no. 20584-1-AP), AKT (Proteintech Group, Inc.; 1:5,000, cat. no. 10176-2-AP), mTOR (1:5,000, cat. no. 20657-1-AP) and GAPDH (1:5,000; cat. no. 60004-1-Ig; all Proteintech Group, Inc). Following three washes with TBST containing 0.1% Tween-20, the membranes were incubated with HRP-conjugated secondary antibody (1:5,000; Proteintech Group, Inc.), HRP-Goat Anti-Mouse Secondary Antibody (Proteintech Group, Inc.; 1:5,000, cat. no. RGAR001) and HRP-Goat Anti-Rabbit Secondary Antibody (Proteintech Group, Inc.; 1:5,000, cat. no. RGAM001) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (Proteintech Group, Inc.). Densitometric analysis was performed using ImageJ software (version 1.8.0).

Cells were lysed in cold IP buffer (Beyotime Biotechnology; cat. no. P0013) containing protease inhibitors. Total cell lysates were incubated with primary antibodies against RRP9, CCNA2, or normal IgG (as a negative control) at 4°C overnight. Protein A/G magnetic (Beyotime Biotechnology; cat. no. P2108; 25 μl) beads were added to capture the immune complexes. After washing three times with lysis buffer, the precipitated proteins were eluted by boiling in SDS loading buffer and analyzed by western blotting.

After establishing stable RRP9-overexpression and RRP9-knockdown models, the cells were seeded into 96-well culture plates at an initial density of 8×10³ cells/well in quadruplicate. The cells were cultured in DMEM supplemented with 10% FBS and incubated under standard conditions (37°C, 5% CO₂). CCK-8 solution (Biosharp Life Sciences) was added at 0, 24, 48 and 72 h according to the manufacturer's protocol. Following 120 min incubation, optical density was measured at 450 nm using a multi-mode microplate reader.

Stable RRP9-overexpressing and -knockdown and control HCC cells were plated in 6-well culture dishes at a density of 1,000 cells/well and maintained for 14 days under standard conditions. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The colonies were stained with 2% crystal violet for 20 min at room temperature, followed by rinsing with distilled water. Images were captured using a digital imaging system. The number of colonies (clusters of >50 cells) was quantified using ImageJ software (version 1.8.0).

To evaluate cell migration and invasion, Transwell chambers with 8.0 μm porous membranes (Corning, Inc.) were used. For the migration assay, 4×10⁵ cells in serum-free medium [DMEM (Thermo Fisher Scientific Inc.; cat. no. 11965092) for HLF, 97h, and Huh7 cells; RPMI-1640 (Thermo Fisher Scientific Inc.; cat. no. 11875119) for Snu449 cells] was added to the upper chamber, while the lower chamber was filled with culture medium supplemented with 10% FBS. In the invasion assay, the upper chamber was pre-coated with Matrigel at 37°C for 2 h prior to seeding 5×10⁵ cells in the aforementioned serum-free media; the lower chamber contained medium with 10% FBS as a chemoattractant. After 24-48 h incubation at 37°C, cells that had migrated through the membrane were fixed with 4% paraformaldehyde at room temperature for 20 min. The cells were stained with 0.1% crystal violet solution at room temperature for 20 min and quantified using a light microscope.

Migration of Snu449, HLF, 97h and Huh7 cells was evaluated using a wound healing assay. A uniform scratch was made in a cell monolayer at >90% confluence using a sterile pipette tip. Following three washes with PBS, the cells were maintained in serum-free medium [HLF, 97h and Huh7 cell cultivated in DMEM, Snu449 cultivated in RPMI-1640 (Thermo Fisher Scientific Inc.; cat. no. 11875119)] for 48 h at 37°C. Images were captured at 0 and 48 h using a light microscope (Olympus). The relative wound closure rate was determined by measuring the wound width at both time points and calculating the percentage reduction using Adobe Photoshop software (version 26.8.0; Adobe Systems, Inc.).

Total RNA was extracted from HLF cells of the wild-type (WT) and RRP9-overexpressing (RRP9-OE) groups using TRIzol. Three independent biological replicates were performed for each group to ensure statistical reliability. RNA quality and quantity were assessed using a Fragment Analyzer, Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.), or Qseq-400 (BiOptic, Inc.). The DESeq2 software package (v1.34.0) was employed to identify differentially expressed genes (DEGs) for subsequent investigations. Detailed RNA sequencing procedures, including extraction, library preparation, quality control, and data analysis, are provided in Supplementary Material 1. Venn diagrams were generated using the VennDiagram R package (version_1.8; github.com/) and Gene Ontology (GO; geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG; kegg.jp/) enrichment analyses were performed using the cluster-Profiler R package (version 4.18.4; https://github.com/).

Differentially expressed genes (DEGs) [adjusted P-value <0.05 and |log2(fold-change)|>1] identified by RNA sequencing were imported into the STRING database (cn.string-db.org/) with a minimum interaction score of 0.9. The data were processed in Cytoscape 3.10.3 (cytoscape.org/), and core targets of the PPI network were identified by calculating the degree values.

Female immunocompromised BALB/c nude mice (age, 4 weeks; weight, 16-18 g) were obtained from Bainter Biotechnology (Wuhan, China). The mice were housed under specific pathogen-free conditions at 22±2°C and 50±10% humidity, with a 12-h light/dark cycle and ad libitum access to food and water. The animals (n=18) were randomly divided into three experimental groups (n=6): WT, OE and OE + 740 Y-P. Tumor xenografts were generated by subcutaneously implanting 5×10⁶ HLF cells suspended in 100 μl PBS into the right flank region. Tumor growth was monitored weekly, with specimens collected after 4 weeks. Tumor dimensions were recorded and volumes were calculated using the standard ellipsoid formula: V=(L × W²)/2, where L is the longest diameter and W is the perpendicular width. The humane endpoints included a maximum tumor volume of 1,500 mm³, severe tumor ulceration or >20% loss of initial body weight. During the 4-week experimental period, none of the mice reached these humane endpoints, and no animals were prematurely euthanized. All procedures involving laboratory animals were conducted in accordance with protocols approved by the Animal Research Ethics Committee of Wuhan University People's Hospital (Wuhan, China; approval no. WDRM20250303).

The experimental data were processed and visualized using SPSS software (version 22.0; IBM Corp.) and GraphPad Prism (version 9.0; Dotmatics). Comparisons between two groups were performed using paired or unpaired t-test. For comparisons involving >2 groups, a one-way ANOVA followed by Tukey's honestly significant difference post hoc test was used. χ² test was used to analyze the association between target gene expression and clinicopathological characteristics. Overall survival curves were plotted using the Kaplan-Meier method, and the differences between the survival curves were evaluated using the log-rank test. All data are presented as the mean ± standard deviation of ≥3 independent experiments. P<0.05 was considered to indicate a statistically significant difference.

The expression of RRP9 in the samples was assessed using western blotting, revealing lower RRP9 expression in cancer compared with adjacent non-tumor tissues (Fig. 1A). Tissue microarray analysis of both cohorts showed reduced RRP9 expression in HCC tissues compared with adjacent non-tumor tissue (Figs. 1B and S1), consistent with western blot results. Based on RRP9 expression in HCC tissue, the patients were categorized into high- and low-expression groups using the median score of 133 as the cut-off value. The demographic and clinicopathological characteristics of these groups are summarized in Table I. Lower RRP9 expression in HCC was associated with more tumor nodules, Barcelona Clinic Liver Cancer stage, tumor size and Tumor-Node-Metastasis stage (27,28) (Table I). Kaplan-Meier survival analysis revealed that patients with higher RRP9 expression had significantly longer overall survival (Fig. 1C).

Western blot analysis was performed to assess RRP9 expression in HCC cell lines. RRP9 levels were lower in HLF and Snu449 cells, while higher levels were observed in Huh7 and 97h cells (Fig. 2E). RRP9 was overexpressed in cell lines with low basal expression (HLF and Snu449) to evaluate its tumor-suppressive potential, and knocked down in high-expressing lines (Huh7 and 97H) to assess its tumor-promoting effects. Western blotting confirmed the successful construction of these cell lines (Figs. 2F and G, S3A-D). The CCK-8 assay demonstrated RRP9-OE reduced short-term proliferative activity in Snu449 and HLF cells (Fig. 2A and B), while RRP9 knockdown increased short-term proliferative activity in Huh7 and 97h cells (Fig. 2C and D). RRP9-OE inhibited colony formation (Fig. 2H and I), while RRP9 knockdown promoted colony formation (Fig. 2J and K). These results suggested that RRP9 can inhibit short-term and long-term proliferation of HCC cells.

The effect of RRP9 on the migration and invasion of HCC cells was evaluated using wound healing and Transwell assay. Wound healing assays showed that RRP9-OE inhibited migration in Snu449 and HLF cells, while RRP9 knockdown enhanced migration in Huh7 and 97h cells (Fig. 3A-D). Transwell assay confirmed RRP9-OE inhibited migration and invasion in Snu449 and HLF cells, whereas RRP9 knockdown decreased migration and invasion in Huh7 and 97h cells (Fig. 3E-H).

EMT marker expression was analyzed by western blotting, revealing that RRP9-OE decreased the levels of Snail-1, N-cadherin and vimentin in Snu449 and HLF cell lines, while increasing E-cadherin expression (Fig. 4A). By contrast, RRP9 knockdown in Huh7 and 97H cells significantly increased the expression of Snail-1, N-cadherin, and vimentin, while decreasing E-cadherin levels (Fig. 4B). To investigate the impact of RRP9 on apoptosis, the expression of Bax and Bcl-2 was measured. The results demonstrated a decrease in the expression of anti-apoptotic factor Bcl-2 and an increase in the pro-apoptotic factor Bax in Snu449 and HLF cells following RRP9 OE (Fig. 4C). Conversely, RRP9 knockdown in Huh7 and 97H cells significantly increased the expression of the anti-apoptotic factor Bcl-2 and decreased the expression of the pro-apoptotic factor Bax (Fig. 4D). These results suggested that RRP9 suppressed HCC cell invasion and migration, promoted apoptosis and inhibited EMT.

To elucidate the mechanism underlying the action of RRP9 action in HCC, transcriptome sequencing was performed on WT and RRP9-OE HLF cells. The sequencing results revealed a number of DEGs following RRP9 OE (Fig. 5A). The Venn diagram indicated that 15,926 genes were shared between the WT and OE groups (Fig. 5B). Gene Ontology enrichment analysis highlighted significant enrichment of DEGs in processes associated with the cell cycle, 'sites of DNA damage' and 'extracellular exosome' (Fig. 5C). Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that RRP9-OE influenced pathways such as 'PI3K-Akt signaling', 'pathways in cancer', 'AMPK signaling pathway', 'mTOR signaling pathway', 'MAPK signaling pathway' and 'Ras signaling pathway' (Fig. 5D). To identify core regulatory nodes within the RRP9-mediated transcriptomic changes, 465 DEGs were selected for PPI network analysis. These DEGs were imported into the STRING database and a core target network containing 25 targets and 96 edges was constructed using Cytoscape 3.10.3. Cyclin A2 (CCNA2; degree=10) was associated with RRP9 (Fig. 5E). Given that CCNA2 is a well-established key regulator of the cell cycle and its dysregulation is frequently implicated in cancer progression (29-31), it was selected for experimental validation to explore its functional connection to RRP9 in HCC.

Given the key role of the PI3K/AKT/mTOR signaling pathway in cancer, the expression of PI3K, AKT, mTOR, phosphorylated (p-)PI3K, p-AKT and p-mTOR was assessed by western blotting. The ratios of p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR were decreased in RRP9-OE Snu449 and HLF cell lines, suggesting inhibition of the PI3K/AKT/mTOR pathway (Fig. 6A). Conversely, RRP9 knockdown in Huh7 and 97H cells significantly increased the ratios of p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR (Fig. 6B). To investigate the association between RRP9 and CCNA2, proteins were extracted from RRP9-OE, RRP9-knockdown, and wild-type cells. Western blot analysis revealed that RRP9-OE decreased CCNA2 protein levels (Fig. 6C), whereas RRP9 knockdown led to increased CCNA2 expression (Fig. 6D). Co-immunoprecipitation (Co-IP) experiments confirmed that RRP9 interacted with CCNA2 in both 293T and 97h cell lines (Fig. S2). Following the validation of CCNA2 knockdown and overexpression via western blotting (Fig. S3E and F), we observed that CCNA2 overexpression increased the ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR, whereas CCNA2 knockdown exerted the opposite inhibitory effect, demonstrating that CCNA2 positively regulates the PI3K/AKT/mTOR signaling pathway (Fig. S2D and E).

It was hypothesized that RRP9 regulates EMT in HCC cells via the PI3K/AKT/mTOR pathway. Therefore, Snu449 and HLF cells were treated with the PI3K activator 740 Y-P and Huh7 and 97h cells with the PI3K/AKT/mTOR pathway inhibitor PI3K/AKT/mTOR-IN-2. Western blotting showed that 740 Y-P activated the PI3K/AKT/mTOR pathway and the inhibitory effect of RRP9-OE on EMT was reversed following treatment with 740 Y-P (Fig. 7A). To confirm that RRP9 knockdown regulated EMT via the PI3K/AKT/mTOR pathway, Huh7 and 97h cells were treated with PI3K/AKT/mTOR-IN-2. Western blotting demonstrated that PI3K/AKT/mTOR-IN-2 inhibited the promotion of EMT induced by RRP9 KD (Fig. 7B). These results suggested that RRP9 regulates EMT in HCC cells through the PI3K-AKT/mTOR signaling pathway.

It was hypothesized that RRP9 affected the migration and invasion of HCC cells via the PI3K/AKT/mTOR signaling pathway. Snu449 and HLF cells overexpressing RRP9 were treated with 740 Y-P. The wound healing and Transwell assays revealed that 740 Y-P reversed the inhibitory effect of RRP9-OE on the migration and invasion of HCC cells (Fig. 8A-D).

Huh7 and 97h cells with RRP9 knockdown were treated with PI3K/AKT/mTOR-IN-2; wound healing and Transwell assays indicated that PI3K/AKT/mTOR-IN-2 inhibited the enhanced migratory and invasive capacity induced by RRP9 knockdown (Fig. 9A-D). In summary, RRP9 regulated HCC cell migration, invasion and EMT by regulating the PI3K/AKT/mTOR signaling pathway.

To investigate the role of RRP9 in tumor progression, an in vivo model was constructed. Nude mice were divided into three groups: WT, OE and OE + 740 Y-P. HLF cells, which exhibited one of the lowest basal levels of RRP9 protein, were selected for xenograft experiments to maximize the phenotypic difference between the OE and WT groups. RRP9-OE significantly inhibited tumor growth in a subcutaneous xenograft model, with marked decreases in both tumor weight and volume. However, 740 Y-P reversed the inhibitory effect of RRP9-OE on tumor growth (Fig. 10A-C, E), suggesting that RRP9 suppressed tumor growth in vivo via the PI3K/AKT/mTOR signaling pathway. TUNEL staining showed that RRP9 promoted apoptosis in subcutaneous tumors (Fig. 10D). Western blot analysis confirmed effective OE of RRP9 in xenograft tumors, with increased E-cadherin and decreased N-cadherin expression in the OE group compared with the WT group (Fig. 10F). Immunohistochemical analysis revealed that the OE group exhibited decreased expression of the proliferation marker Ki-67 and the mesenchymal marker N-cadherin, alongside increased expression of the epithelial marker E-cadherin, compared with the WT group (Fig. 10G). These results indicated that RRP9 effectively suppressed tumor growth in vivo.