SLC25A45 as a prognostic biomarker promotes malignant progression via mutant p53 in hepatocellular carcinoma

Introduction

Hepatocellular carcinoma (HCC) accounts for approximately 80% of all primary liver malignancies (1). It remains one of the most intractable malignancies worldwide, with a dismal 5-year overall survival (OS) rate as low as 18% (2). The etiology of HCC is complex; it is primarily driven by risk factors such as chronic hepatitis B virus/hepatitis C virus infections, alcohol-associated liver disease, and metabolic dysfunction-associated steatotic liver disease (3-5). Of all major cancers, HCC is the one in which mortality rates have not declined over the past decade (6). Consequently, the molecular mechanisms of pathogenic HCC advancement need to be elucidated to identify emerging targets for intervention and prevention.

In patients with early-stage localized HCC, especially those presenting with a single tumor nodule, radical surgical resection remains the optimal curative treatment option (7). Unfortunately, a large proportion of HCC cases are detected at advanced clinical stages, rendering them ineligible for curative therapies (8-10). This highlights the critical need to optimize early detection strategies to alleviate the economic burden and improve clinical outcomes. Currently, abdominal ultrasound is the standard screening modality for HCC; however, its sensitivity remains suboptimal (approximately 50%) (11), a limitation further exacerbated in obese individuals. Additionally, traditional serum biomarkers such as alpha-fetoprotein (AFP) lack the requisite sensitivity for effective surveillance (12). Elevated serum des-gamma-carboxy prothrombin has shown diagnostic value, particularly in AFP-negative patients with chronic hepatitis B; however, the overall diagnostic landscape remains inadequate (13). Accordingly, the identification of novel, highly sensitive molecular markers has important clinical value for the early detection, recurrence surveillance, and prognostic assessment of HCC.

Mitochondria function as essential metabolic platforms that orchestrate diverse biosynthetic and energy metabolism processes (14), with their metabolic plasticity heavily dependent on specialized transporters (15). The solute carrier 25 (SLC25) family, which comprises the bulk of mitochondrial metabolite transporters (14,16), is responsible for transporting diverse molecules, including nucleotides, amino acids, and fatty acids (17), across the mitochondrial membrane. Recently, SLC25A45, an orphan member of the SLC25 family, was identified as a mitochondrial trimethyllysine carrier (18) involved in the homeostasis of methylated amino acids and carnitine (19,20). Notably, the liver is the primary organ responsible for systemic metabolic homeostasis. Metabolic reprogramming, particularly the dysregulation of carnitine and amino acid metabolism, is a hallmark of HCC. To meet the high energy and biosynthetic demands of rapid proliferation, HCC cells frequently exploit mitochondrial transporters to remodel their metabolic networks.

Despite the established metabolic functions of SLC25A45, its specific biological role, underlying mechanisms, and clinical significance in the pathogenesis of human cancers, particularly in metabolically active HCC, remain poorly understood. To address this gap in the literature, this study sought to investigate the oncogenic role of SLC25A45 in HCC. We first evaluated the expression and clinical significance of SLC25A45 in HCC using large-scale bioinformatics databases, and then validated these findings in HCC tumor tissues. Next, we performed in vitro assays with HCC cell lines to characterize the effects of SLC25A45 on tumor progression and elucidate the underlying molecular mechanisms. Collectively, the study findings establish SLC25A45 as a potential biomarker and therapeutic target in HCC, providing novel insights into its role in HCC and supporting its potential for clinical translation. We present this article in accordance with the MDAR and REMARK reporting checklists (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0725/rc).

Methods

Reagents and antibodies

Anti-SLC25A45 antibody (14160-1-AP, rabbit polyclonal antibody) was obtained from the Proteintech Group (). Anti-p53 antibody (sc-126, mouse monoclonal antibody) was supplied by Santa Cruz Biotechnology (). Anti-Bcl-2 antibody (R381702, rabbit monoclonal antibody) and anti-caspase-3 antibody (R23315, rabbit monoclonal antibody) were acquired from Chengdu ZEN Bioscience (Chengdu, China). Anti-β-Tubulin antibody (GB15140-100, mouse monoclonal antibody), anti-glyceraldehyde-3-phosphate dehydrogenase antibody (GB15004-100, rabbit monoclonal antibody), as well as horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (GB23303) and HRP-conjugated goat anti-mouse secondary antibody (GB23301) were provided by Wuhan Servicebio Technology (Wuhan, China).

Acquisition of clinical specimens

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The Affiliated Hospital of Guizhou Medical University (No. 2025-548). Written consent was obtained from all patients before enrollment.

The inclusion criteria were as follows: (I) age ≥18 years; (II) hepatectomy and postoperative pathological diagnosis of HCC; and (III) written informed consent from all patients or their legal guardians before tissue collection. The exclusion criteria were as follows: (I) metastatic liver cancer; (II) any preoperative anti-tumor treatment before hepatectomy, including transarterial chemoembolization, radiotherapy, chemotherapy, targeted therapy, or immunotherapy; and/or (III) concomitant systemic malignancies other than HCC.

A total of 40 patients with HCC who underwent surgical resection at the Department of Hepatobiliary Surgery of the same hospital provided paired tumor and adjacent normal tissue samples. Tumor and paired adjacent normal tissues were collected during surgical resection. For immunohistochemistry (IHC), tissue samples were fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin blocks, and stored at 4 ℃. For Western blot analysis, fresh tissues were immediately frozen in liquid nitrogen and stored at −80 ℃. All 40 paired specimens were used for IHC staining to evaluate SLC25A45 expression, while six pairs of specimens were reserved for Western blotting to confirm the protein levels. Histopathological examination confirmed the diagnosis of HCC for each specimen, and key clinical variables, including age, gender, tumor size, and tumor-node-metastasis (TNM) stage, were recorded.

Acquisition of research findings

RNA-sequencing (RNA-seq) data and clinicopathological information from The Cancer Genome Atlas (TCGA) database were obtained using the University of California, Santa Cruz (UCSC) Xena browser (developed at the UCSC). RNA-seq data normalized to transcripts per million (TPM) were obtained and subjected to a log₂(TPM+1) transformation. A pan-cancer analysis was performed across 26 cancer types, including HCC. To externally validate SLC25A45 expression in liver cancer, the GSE62232 and GSE36346 datasets were downloaded from the Gene Expression Omnibus (GEO). Subsequently, SLC25A45 expression analysis, survival analysis, clinicopathological correlation assessment, and drug susceptibility assessment were performed to preliminarily investigate the functional role and prognostic assessment efficacy of SLC25A45 in HCC.

Pan-cancer profiling of SLC25A45

A uniformly standardized comprehensive tumor dataset was obtained from the UCSC database. SLC25A45 transcriptional data (Ensembl ID: ENSG00000162241) were collected across samples, yielding expression profiles for 26 cancer types. The Wilcoxon rank-sum test was used to compare standardized expression levels between tumor and normal tissues for each cancer type. For cancer types showing significant differential expression, Kaplan-Meier survival analysis was performed to investigate the association between SLC25A45 expression levels and OS.

Biological and clinical significance of SLC25A45 in HCC

The Wilcoxon rank-sum test was used to compare SLC25A45 expression levels between tumor and adjacent normal tissues from patients with HCC. For further validation, paired t-tests were performed using 50 pairs of tumor and adjacent normal tissues from TCGA database. Before the analysis, the Shapiro-Wilk test was performed to assess normality, and outliers were identified using the 1.5× interquartile range criterion based on the paired differences. Receiver operating characteristic (ROC) curves were generated using the “pROC” package in R, and the area under the curve (AUC) was calculated to evaluate the diagnostic performance of SLC25A45 expression for identifying HCC in TCGA database.

To further validate the elevated expression of SLC25A45 in HCC, RNA-seq data from the GSE62232 and GSE36376 datasets were retrieved from the GEO database and subjected to ROC curve analysis. The “maxstat” R package was then used to determine the optimal cut-off value. Kaplan-Meier survival analysis was then performed to evaluate the associations between SLC25A45 expression levels and OS, disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI) in patients with HCC, with P<0.05 considered statistically significant. Finally, the associations between SLC25A45 expression levels and clinicopathological characteristics, including AFP level, vascular invasion, tumor stage, histological grade, and N stage, were evaluated.

Pathway enrichment analyses

To preliminarily investigate the potential functions of SLC25A45, the Gene Expression Profiling Interactive Analysis (GEPIA) website was used to identify the top 100 genes co-expressed with SLC25A45 in HCC. Functional enrichment profiling of the co-expressed genes was performed using Metascape. Next, HCC cases in TCGA cohort were divided into high and low SLC25A45 expression groups based on the median transcriptional cut-off value. Differentially expressed genes (DEGs) between the two groups were identified using the “limma” R package, with thresholds of |log₂ fold change| ≥2.0 and false discovery rate (FDR) <0.01. Heatmaps and volcano maps were generated using the “ggplot2” package in R. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the identified DEGs were performed using the “clusterProfiler” package in R. Additionally, gene set enrichment analysis (GSEA) was performed using the software available from the Broad Institute (software.broadinstitute.org), with statistical significance defined as an uncorrected P<0.05 and FDR <0.25.

Cell culture and transfection

The normal human liver epithelial cell line THLE-2 and the HCC cell lines, including Huh7, Huh1, PLC/PRF/5, and Hep3B, were obtained from the American Type Culture Collection (ATCC). The cell lines MHCC-97H (Cat. No. STCC10113G), HCC-LM3 (Cat. No. STCC10111G), and Li-7 (Cat. No. STCC10107G) were purchased from Wuhan Servicebio Technology. All cell lines were cultured and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and penicillin-streptomycin. Cells were incubated at 37 ℃ in a humidified environment containing 5% CO₂. The PLC/PRF/5, Huh7, and Huh1 cell lines were transfected with SLC25A45 small interfering RNAs (siRNAs), which were purchased from TranSheepBio (Shanghai, China). The siRNA sequences were as follows: siSLC25A45-1: CUCAUCAAAGUCCGGCUACAAdTdT; siSLC25A45-2: CCUCAGCUACGAAUAUCUCCUdTdT; and siNC: CCCGUUUGACACUGUAAAGCUdTdT. The transfection procedure was performed in accordance with the manufacturer’s instructions for Lipo8000TM Transfection Reagent (C0533, 1.5 mL; Beyotime, Shanghai, China).

Cell proliferation

Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8). Cells were seeded at a density of 3,000–5000 per well in 96-well plates and transfected with siSLC25A45-1, siSLC25A45-2, and siNC, respectively. Following 24–72 h of culture, the cells were washed three times with phosphate-buffered saline (PBS), and subsequently incubated with CCK-8 reagent for 1 h. Colorimetric detection was performed by measuring absorbance at 450 nm using a microplate reader (Cytation5, BioTek, USA).

Colony forming assay

Cells were transfected with siSLC25A45-1, siSLC25A45-2, or siNC and seeded in six-well plates at an inoculation density of 500–1,000 cells per well. After incubation for 14–21 days, the cells were fixed with 4% paraformaldehyde for 15 min, followed by staining with 0.1% crystal violet. The experimental results were observed and photographed using a camera.

Western blotting

After the indicated treatments described in the figure legends, cells were collected by trypsinization and centrifugation. Total protein was extracted using Radioimmunoprecipitation assay buffer (Sangon Biotech, Shanghai, China). Cellular total protein content was assessed using a bicinchoninic acid protein quantification kit (EK-5001-500T, ECOTOP Scientific). A total of 20 µg of protein from the supernatant was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes. The membranes were then blocked with 5% skim milk, formulated with 1× Tris-buffered saline with 0.1% Tween-20 (TBST), for 1 h, and then cultured with primary antibodies at 4 ℃ overnight. Next, following thorough rinsing in TBST buffer, the membranes were cultured with diluted secondary antibodies at room temperature. Finally, protein signals were detected using enhanced chemiluminescence reagents and a chemiluminescence detection instrument.

IHC

A total of 40 pairs of HCC tissues and matched adjacent non-tumorous tissues were collected from the Department of Hepatobiliary Surgery at The Affiliated Hospital of Guizhou Medical University. All specimens were paraffin-embedded and subjected to IHC staining to evaluate SLC25A45 expression. The tissue sections were sequentially heated, deparaffinized, and gradually rehydrated, and then subjected to antigen retrieval in citrate buffer. The tissue sections were immersed in 3% H₂O₂ solution to block intrinsic peroxidase enzymatic activity and then blocked with 10% sheep serum in PBS. After three washes, the sections were exposed to primary antibody targeting SLC25A45 overnight at 4 ℃. Following three washes with PBS, the sections were processed with the secondary antibodies for 40 min at 37 ℃. The sections were then incubated with 3,3’-diaminobenzidine for color development, counterstained with hematoxylin, dehydrated, mounted, and observed under a light microscope (DM2500, Leica, Germany).

The IHC staining was scored based on staining intensity and the proportion of positive cells. Staining intensity was scored as follows: 0 = negative, 1 = weak, 2 = moderate, and 3 = strong. The proportion of positive cells was scored as follows: 0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, and 4 = >75%. The final IHC score was calculated as the product of the staining intensity and positive cell proportion scores. Scores ≤4 indicated low expression, and scores >4 indicated high expression.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated using the NcmSpin Rapid RNA Extraction Kit (M5106, Suzhou NcmBio, Suzhou, China) in accordance with the manufacturer’s instructions. Reverse transcription for complementary DNA synthesis was performed using random primers and a reverse transcription kit (M7100, Suzhou NcmBio). Quantitative polymerase chain reaction (qPCR) amplification was performed using SYBR^® Premix Ex Taq™ (B690016, Sangon Biotech), and the amplification process was monitored using a DNA Engine Peltier thermal cycler. The qPCR amplification conditions were as follows: initial denaturation at 95 ℃ for 2 min, followed by 40 cycles of denaturation at 95 ℃ for 10 s and annealing/extension at 60 ℃ for 30 s. The primer sequences used for qPCR are detailed in Table S1.

In silico statistical analysis

For unpaired samples, differences in gene expression levels between groups were assessed using the Wilcoxon rank-sum test. For paired samples, the Shapiro-Wilk test was first used to assess data normality, followed by the paired t-test as appropriate. All bioinformatics analyses and data visualization were performed using R Studio (version 4.5.1) and Sangerbox 3.0 (http://www.sangerbox.com/tool).

Statistical analysis

All quantitative data were analyzed using GraphPad Prism to detect differences between experimental groups. Differences between two groups were assessed using the two-tailed Student’s t-test, while differences among multiple groups were assessed using one-way analysis of variance. A P value <0.05 was considered statistically significant.

Results

Pan-cancer profiling of SLC25A45 expression and its elevation in HCC

The expression profile and prognostic value of SLC25A45 across various cancer types were analyzed. As shown in Figure 1A, differential SLC25A45 expression was observed in 12 types of cancer in TCGA database. Notably, aberrantly high SLC25A45 expression was observed in five tumor types, including kidney renal papillary cell carcinoma (KIRP), prostate adenocarcinoma (PRAD), uterine corpus endometrial carcinoma (UCEC), liver hepatocellular carcinoma (LIHC), and pheochromocytoma and paraganglioma (PCPG). We further assessed the association between aberrantly high SLC25A45 expression and patient survival in these five tumor types using Kaplan-Meier analysis. The findings revealed that high SLC25A45 expression was not significantly associated with OS in KIRP, PRAD, UCEC, or PCPG (Figure 1B-1E). Conversely, high SLC25A45 expression was significantly associated with poor OS in patients with HCC (Figure 1F). In addition to OS, the survival analysis revealed that HCC patients with high SLC25A45 expression also presented with significantly shorter DSS and PFI (Figure 1G,1H). The DFI analysis in LIHC showed a borderline P value of 0.05 and did not reach statistical significance; however, a potential trend toward an association between high SLC25A45 expression and shorter DFI was observed, potentially reflecting the considerable effect size and large sample size of the cohort (Figure 1I).

Figure 1 SLC25A45 expression in HCC. (A) Aberrant SLC25A45 expression was detected across 26 cancer types. (B-F) Elevated SLC25A45 expression was not significantly associated with prognosis in KIRP, PRAD, UCEC, or PCPG, while high SLC25A45 expression was associated with poor prognosis in LIHC. (G-I) Kaplan-Meier curves comparing DSS, PFI, and DFI between the high- and low-SLC25A45 expression groups. (J) SLC25A45 expression in 50 paired HCC and adjacent non-tumorous tissues from TCGA database. (K,L) SLC25A45 expression in HCC validated using two independent GEO cohorts. (M-O) ROC curves evaluating the diagnostic performance of SLC25A45 for distinguishing between HCC and non-tumorous tissues in TCGA, GSE36376, and GSE62232 cohorts. *, P<0.05; **, P<0.01; ****, P<0.0001. AUC, area under the curve; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma; CHOL, cholangiocarcinoma; CI, confidence interval; COAD, colon adenocarcinoma; DFI, disease-free interval; DSS, disease-specific survival; ESCA, esophageal carcinoma; FPR, false positive rate; GBM, glioblastoma multiforme; GEO, Gene Expression Omnibus; HCC, hepatocellular carcinoma; HNSC, head and neck squamous cell carcinoma; HR, hazard ratio; KICH, kidney chromophobe; KIPAN, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LGG, low grade glioma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PFI, progression-free interval; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; ROC, receiver operating characteristic; STAD, stomach adenocarcinoma; STES, stomach and esophageal carcinoma; TCGA, The Cancer Genome Atlas; THCA, thyroid carcinoma; TPR, true positive rate; UCEC, uterine corpus endometrial carcinoma.

To investigate the expression profile and clinical relevance of SLC25A45 in HCC, we performed expression analyses using TCGA and GEO datasets. Analysis of 50 paired HCC and adjacent non-tumorous tissue samples from TCGA database confirmed significantly elevated SLC25A45 expression in tumor tissues (Figure 1J). Moreover, analyses of two independent GEO cohorts (GSE36376 and GSE62232) further validated the aberrant overexpression of SLC25A45 in HCC, consistent with TCGA results (Figure 1K,1L). Meanwhile, ROC curve analysis showed that SLC25A45 expression exhibited an AUC of 0.748 in TCGA database and AUC values of 0.64 and 0.73 in the two independent GEO datasets, respectively, supporting the potential diagnostic value of SLC25A45 as an indicator for HCC (Figure 1M-1O). Collectively, these findings support the clinical value and potential biological significance of aberrant SLC25A45 expression in HCC.

High SLC25A45 expression contributes to the malignant progression of HCC

SLC25A45 expression was stratified based on different clinicopathological classifications in HCC. Based on clinical stage, histological grade, and T stage, three subgroups were defined: normal tissues, stage I tumors, and stage II–IV tumors. SLC25A45 expression progressively increased with tumor progression (Figure 2A-2C). Further, SLC25A45 expression was significantly higher in HCC tissues with vascular invasion than in those without vascular invasion (Figure 2D). Patients were further stratified based on AFP indicators into three groups: normal, ≤400 µg/L, and >400 µg/L. SLC25A45 expression increased significantly as AFP levels increased (Figure 2E).

Figure 2 SLC25A45 upregulation was significantly associated with the initiation and malignant progression of HCC. (A-C) SLC25A45 expression in HCC patients stratified by tumor stage, histological grade, and T stage. (D,E) Box plots illustrating the association between SLC25A45 expression and vascular invasion status and AFP levels. (F) Forest plots illustrating the results of univariate and multivariable Cox regression analyses based on SLC25A45 expression. (G) Western blot analysis of SLC25A45 protein expression in matched tumor and non-tumorous tissues, with the corresponding statistical analysis shown below. Three independent experiments were performed. (H) Western blot analysis of SLC25A45 protein expression in THLE-2 and human HCC cell lines, with the corresponding statistical analysis shown below. Three independent experiments were performed. (I) Representative IHC staining and IHC scores assessing SLC25A45 expression in HCC and adjacent non-tumorous liver tissues. Scale bar, 50 µm. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; NS, not significant. AFP, alpha-fetoprotein; BMI, body mass index; CI, confidence interval; HCC, hepatocellular carcinoma; HR, hazard ratio; IHC, immunohistochemistry; N, normal; T, tumor.

Univariable and multivariable Cox regression analyses revealed that high SLC25A45 expression was an independent adverse prognostic indicator in HCC (P=0.049 and P=0.03, respectively) (Figure 2F). Western blot analysis was then performed to evaluate SLC25A45 expression in six of the 40 paired HCC and adjacent non-tumorous tissue samples collected from the Department of Hepatobiliary Surgery at The Affiliated Hospital of Guizhou Medical University, as well as in the normal human liver epithelial cell line THLE-2 and six HCC cell lines. SLC25A45 expression was significantly higher in both the HCC tissues and HCC cell lines than in the corresponding normal controls (Figure 2G,2H), with the highest expression detected in Huh7 and PLC/PRF/5 cells. Consequently, these two cell lines were selected for subsequent SLC25A45 silencing and functional verification. Staining of 40 paired clinical specimens from HCC patients admitted to our hospital further confirmed the upregulation of SLC25A45 in HCC tissues (Figure 2I).

Collectively, these findings demonstrate that SLC25A45 is abnormally upregulated in HCC and is associated with HCC occurrence, supporting its potential as a candidate molecular signature for HCC.

Depletion of SLC25A45 suppresses the malignant growth and colony formation abilities of HCC cells

To examine the biological role of SLC25A45 in mediating the malignant phenotypes of HCC cells, SLC25A45 expression was silenced in Huh7 and PLC/PRF/5 cells using two specific siRNAs targeting SLC25A45. Western blotting and RT-qPCR (Figure 3A,3B) confirmed that both siRNA sequences (siSLC25A45-1 and siSLC25A45-2) exerted optimal knockdown efficacy and were therefore selected for subsequent functional assays. CCK-8 assays demonstrated that SLC25A45 silencing significantly inhibited the proliferation of both PLC/PRF/5 and Huh7 cells (Figure 3C). Further, colony formation assays revealed that cells transfected with SLC25A45 siRNAs formed significantly fewer colonies than cells in the negative control group (Figure 3D). Wound-scratch assays demonstrated that SLC25A45 knockdown significantly reduced the migratory ability of HCC cells (Figure 3E). Transwell invasion assays further demonstrated that SLC25A45 knockdown significantly suppressed the invasive capacities of PLC/PRF/5 and Huh7 cells (Figure 3F). In short, these findings identified SLC25A45 as a critical regulator that promotes the malignant progression of HCC cells.

Figure 3 SLC25A45 downregulation suppresses the growth, colony formation, and malignant progression of HCC cells. (A,B) Western blot and RT-qPCR assays were performed to validate the knockdown efficiency of SLC25A45 in HCC cells. (C) Cell proliferation of HCC cells transfected with siNC or SLC25A45 siRNA was assessed using the CCK-8 assay. (D) SLC25A45 silencing significantly suppressed the cell colony formation ability of HCC cells. (E) Effects of SLC25A45 depletion on cell migration were examined using scratch assay in HCC cells. Scale bar, 100 µm. (F) Transwell assays were performed to assess the effects of SLC25A45 knockdown on the migratory and invasive abilities of HCC cells. Scale bar, 100 µm. **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; HCC, hepatocellular carcinoma; OD, optical density; RT-qPCR, reverse transcription quantitative polymerase chain reaction; siNC, small interfering RNA negative control; siRNA, small interfering RNA.

SLC25A45 promotes malignant progression of HCC potentially by modulating p53

To examine the potential mechanisms underlying the role of SLC25A45 in HCC malignant progression, GSEA was performed using hallmark gene signatures and TCGA-LIHC samples were stratified into SLC25A45 high- and low-expression subgroups. The GSEA identified three major downstream regulatory pathways: suppression of the p53 pathway; activation of the G1/S phase transition pathway; and activation of the DNA repair pathway (Figure 4A-4G).

Figure 4 SLC25A45 promotes HCC tumorigenesis and progression, which is closely associated with the p53 signaling pathway. (A) Heatmap analysis of SLC25A45, with samples divided into high- and low-expression groups. (B) Volcano plot of DEGs for SLC25A45. (C,D) GO functional and KEGG pathway enrichment analyses. (E-G) GSEA of SLC25A45. (H) Western blot analysis of SLC25A45, p53, caspase-3, and Bcl-2 in the Huh1 cell line following transfection with siNC or SLC25A45 siRNA. (I) Western blot analysis of SLC25A45, p53, caspase-3, and Bcl-2 expression in the Huh7 and PLC/PRF/5 cell lines following transfection with siNC or SLC25A45 siRNA. (J) Cell proliferation of Huh1 cells transfected with siNC or SLC25A45 siRNA was assessed using the CCK-8 assay. (K) SLC25A45 silencing promoted the cell colony formation ability of Huh1 cells. *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; DEG, differentially expressed gene; FDR, false discovery rate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GO, Gene Ontology; GSEA, gene set enrichment analysis; HCC, hepatocellular carcinoma; KEGG, Kyoto Encyclopedia of Genes and Genomes; OD, optical density; siNC, small interfering RNA negative control; siRNA, small interfering RNA; WT, wild type.

As a central tumor suppressor, the “guardian of the genome”, p53 maintains genomic integrity by regulating diverse DNA-damage-response cascades (21). However, p53 mutations occur in more than half of human cancers and may confer tumor-promoting gain-of-function operational characteristics, contributing to poor clinical outcomes and chemoresistance (22). Our research group previously investigated the regulatory mechanisms governing mutant p53 stability as a potential strategy for anti-tumor therapy (23-25). Accordingly, we sought to characterize the relationship between SLC25A45 expression and p53 stability in HCC.

To investigate the effect of SLC25A45 on p53 expression, we used the wild-type p53-expressing Huh1 cell line. Western blot analysis showed that SLC25A45 knockdown significantly increased p53 levels in Huh1 (p53WT) cells (Figure 4H). Conversely, SLC25A45 knockdown significantly decreased mutant p53 expression in Huh7 (p53Y220C) and PLC/PRF/5 (p53R249S) cells. Additionally, Bcl-2 protein expression was markedly downregulated, while Caspase-3 was notably upregulated (Figure 4I). The same phenotypic trend was further supported by both CCK-8 and colony formation assays in Huh1 (p53WT) cells (Figure 4J,4K), consistent with the observations in the Huh7 (p53Y220C) and PLC/PRF/5 (p53R249S) cells (Figure 3C,3D). Collectively, these findings suggest that SLC25A45 may promote tumorigenesis and progression of HCC by modulating the p53 signaling pathways, specifically by suppressing wild-type p53 expression while facilitating the accumulation of mutant p53 protein.

Discussion

This study showed that SLC25A45 is elevated in HCC tissues and may serve as an indicator of poor prognosis. Further, SLC25A45 knockdown may suppress the malignant biological traits of HCC cells via the p53 biological signaling pathway. These findings highlight the potential contribution of SLC25A45-mediated signaling to HCC progression, suggesting that SLC25A45 may serve as a potential prognostic indicator and a functional therapeutic target in HCC.

SLC family members play critical roles in the development of diverse human disorders, including chronic diseases, mental disorders, cardiovascular diseases, and malignant tumors. During HCC progression, these transporters contribute to metabolic disturbance in tumor cells, mainly affecting carbohydrate, lipid, and amino acid metabolism, as well as the production of nucleic acid precursors (26). For example, SLC2A3 and SLC16A1 promote glycolysis, while SLC27A5 plays a pivotal role in glutamine metabolism (26,27). As a crucial mitochondrial solute carrier, SLC25A45 mediates the transport of intracellular metabolites. Aberrant expression of this gene may perturb core metabolic routes, with prominent effects on lipid and amino acid metabolism. Such metabolic reprogramming further modulates multiple downstream biological processes during tumor development, including steady-state messenger RNA maintenance, protein biosynthesis, and pre-RNA maturation (28), although the precise molecular mechanisms warrant further investigation.

Overall, despite extensive research on SLC family members across malignancies, the contribution of SLC25 members to HCC pathogenesis remains largely unexplored. A few studies have revealed that SLC25A39 and SLC25A19 are upregulated in HCC and associated with unfavorable prognosis (29,30), while SLC25A35 has been characterized as a key oncogenic factor through mitochondrial metabolic reprogramming (31), suggesting its potential as a druggable target. In colon cancer, SLC5A7 has been demonstrated to directly interact with p53, thereby weakening the p53-MDM2 interaction and enhancing p53 expression in wild-type p53 cells (32). Concurrently, synthetic material-based precision delivery systems designed to simultaneously target p53 aberrations and SLC transporters have shown potential for restoring chemosensitivity and suppressing tumor progression (33). Notably, with p53 mutations present in more than half of HCC patients (34), exploring the regulatory axis between SLC25 family members and p53 in HCC could fill a critical knowledge gap and pave the way for more effective personalized therapeutic regimens.

The current study had several limitations. All functional experiments were conducted in vitro, and the in vivo role of SLC25A45 in HCC progression needs to be verified using nude mouse xenograft models. Mechanistically, our findings were primarily based on bioinformatic enrichment and protein expression analyses; future studies employing dual-luciferase reporter and co-immunoprecipitation assays are necessary to clarify the direct regulatory interplay between SLC25A45 and p53. Additionally, the relationship between SLC25A45 (as a mitochondrial protein) and the nuclear p53 pathway, including their respective subcellular localization, remains to be fully elucidated. Moreover, the dependence of SLC25A45 function on p53 mutation status should be further investigated by incorporating additional HCC cell lines with distinct p53 genotypes, such as Hep3B (p53null) and Hep3B (p53R175H).

Consistent with public database analyses, our clinical samples and in vitro experiments confirmed the aberrant upregulation of SLC25A45 in HCC cells. Elevated SLC25A45 expression was significantly associated with advanced tumor grade, microvascular invasion, lymph node metastasis, and poor OS. The initiation and malignant progression of HCC are a multi-stage pathological process (35), in which microvascular invasion (36) and lymph node metastasis (37) often occur as critical events facilitating intrahepatic metastasis during HCC progression (38). Concordantly, our in vitro assays demonstrated that SLC25A45 silencing significantly inhibited the proliferation, migration, and invasion of HCC cells. Further studies indicated that SLC25A45 may modulate HCC progression through the p53 signaling pathway.

Several therapeutic strategies have been proposed for the treatment of tumors harboring mutant p53, ranging from reconstitution of wild-type p53 protein conformation and expression to targeted depletion of mutant p53 (39,40), with the latter representing a more direct therapeutic approach. Building on our group’s previous research (23), the present study revealed a novel, context-dependent regulatory mechanism for the first time, whereby SLC25A45 knockdown simultaneously promotes the accumulation of wild-type p53 while attenuating mutant p53 expression. Collectively, these observations indicate that SLC25A45 may primarily exert oncogenic effects in HCC by modulating the p53 pathway.

Conclusions

SLC25A45 may serve as a prognostic biomarker and therapeutic target in HCC. Our findings demonstrate that SLC25A45 promotes the tumorigenesis and progression of HCC, and this regulatory effect may be dependent on the p53 signaling pathway.

Acknowledgments

The authors would like to acknowledge all personnel contributing to the establishment of the Xena, TCGA and GEO databases.

Footnote

Reporting Checklist: The authors have completed the MDAR and REMARK reporting checklists. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0725/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0725/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0725/prf

Funding: This work was supported by the Nature Science Foundation of Nation (No. 82560359), Guizhou Provincial Science and Technology Projects (QKHJC-2024-Youth 268), Guizhou Provincial Basic Research Program (Natural Science) (QKHJC-zk-2025, No. 462), Doctor Start-up Fund of The Affiliated Hospital of Guizhou Medical University (gyfybsky-2022-23 and gyfybsky-2024-39), Science and Technology Foundation Project of the Guizhou Provincial Health Commission (gzwkj2025-301), and Key Laboratory for Cancer Prevention and treatment of Guizhou Province (QKHPT[2025]031).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2026-0725/coif). All authors report that this work was supported by the Nature Science Foundation of Nation (No. 82560359), Guizhou Provincial Science and Technology Projects (QKHJC-2024-Youth 268), Guizhou Provincial Basic Research Program (Natural Science) (QKHJC-zk-2025, No. 462), Doctor Start-up Fund of The Affiliated Hospital of Guizhou Medical University (gyfybsky-2022-23 and gyfybsky-2024-39), Science and Technology Foundation Project of the Guizhou Provincial Health Commission (gzwkj2025-301), and Key Laboratory for Cancer Prevention and treatment of Guizhou Province (QKHPT[2025]031). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The Affiliated Hospital of Guizhou Medical University (No. 2025-548). Written consent was obtained from all patients before enrollment.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: L. Huleatt)