SLC25A3 promotes hepatocellular carcinoma progression by inducing mitochondrial dysfunction and chromatin remodeling.

Introduction
Liver cancer is one of the most prevalent malignancies of the digestive system worldwide, with more than 900,000 new cases annually, accounting for 4.7% of all newly diagnosed cancers globally, ranking sixth. Despite these numbers, liver cancer causes over 800,000 deaths each year, representing 8.3% of cancer-related fatalities, second only to lung cancer (1). Hepatocellular carcinoma (HCC), the most common subtype of liver cancer, accounts for over 90% of cases. In recent years, advancements in surgical techniques, along with improvements in diagnostic and therapeutic approaches, have significantly improved the overall prognosis of HCC patients (2,3). However, for those with advanced, inoperable HCC, current treatment options remain limited, and prognosis remains poor. Therefore, there is an urgent need to identify novel therapeutic targets and develop synergistic treatment strategies to enhance therapeutic efficacy, improve patients’ quality of life, and extend survival.
Mitochondria, the energy powerhouses of cells, generate large amounts of energy through oxidative phosphorylation. They also regulate cellular physiological functions and apoptosis by maintaining cellular homeostasis (4,5). However, the classic metabolic pathway in tumors, known as the Warburg effect, leads tumor cells to primarily rely on anaerobic glycolysis for energy production, even in the presence of oxygen, thereby inhibiting oxidative phosphorylation in the mitochondria. This adaptation arises because cancer cells often face hypoxic conditions and nutrient deficiencies, compelling them to undergo metabolic reprogramming to meet the high demands for energy and biosynthetic materials (6,7). Recent studies suggest that mitochondria play an active role in the cancerization process through various mechanisms, primarily by influencing oxidative phosphorylation and mitochondria-associated apoptosis processes (8-10). The transition mechanisms between anaerobic glycolysis and mitochondrial oxidative phosphorylation in HCC remain poorly understood, highlighting the need for systematic investigation to identify new therapeutic targets.
Numerous studies have shown that alterations in the activity and expression levels of ion channel proteins located on the cell membrane and organelle surfaces can modulate mitochondrial oxidative phosphorylation and apoptosis, subsequently influencing hepatic fibrosis and the progression of HCC (11,12). Based on this, mitochondrial-related gene sets were obtained from the Molecular Signatures Database (MSigDB) and subsequently analyzed using data from The Cancer Genome Atlas (TCGA) database, along with experimental validation. In this study, we identified solute carrier family 25 member A3 (SLC25A3), a mitochondrial membrane protein, as being significantly overexpressed in HCC and associated with unfavorable prognosis.
SLC25A3 is a phosphate transporter located on the inner mitochondrial membrane, playing a vital role in oxidative phosphorylation and influencing mitochondrial metabolism and adenosine triphosphate (ATP) production (13). Previous studies have reported that mitochondrial metabolites can be transferred to the cytoplasm and nucleus, where they act as second messengers and induce significant (epi)genetic alterations (14-16). However, it remains unclear whether SLC25A3 can impact mitochondrial metabolism, epigenetic modifications, and the specific mechanisms involved in HCC.
In this study, we performed a series of bioinformatics analyses, experimental validations, and multi-omics approaches to investigate the potential carcinogenic role of SLC25A3 in HCC, providing new insights into potential therapeutic targets. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1946/rc).

Methods

Bioinformatics analysis
Mitochondrial-related gene sets were downloaded from the MSigDB database (17). The expression levels, DNA methylation status, and prognostic significance of these genes in HCC were analyzed using the TCGA database.

Clinical samples
Paired tumor and peri-tumor tissues were collected from fifty HCC patients who underwent hepatectomy at The Second Xiangya Hospital of Central South University. 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 Second Xiangya Hospital of Central South University (No. Z0741-02) and informed consent was taken from all the patients.

Immunohistochemistry (IHC) staining
Liver tissues from human specimens were fixed in 10% formalin, embedded in paraffin, and subsequently sectioned. IHC was conducted according to previously established protocols (18). Briefly, the tissue sections were incubated overnight at 4 ℃ with anti-SLC25A3 antibody (1:100, #10420-1-AP, Proteintech, Chicago, USA) on the first day. Both staining intensity and the proportion of tumor cells were independently scored by two pathologists. Staining intensity was rated on a four-point scale (strongly stained, moderately stained, weakly stained, no staining, corresponding to 3, 2, 1, 0 points, respectively). The proportion of tumor cells was evaluated on a five-point scale (>70%, 35–70%, 10–35%, <10%, 0%, corresponding to 4, 3, 2, 1, 0 points). The final score was determined by multiplying the scores for staining intensity and tumor cell proportion.

Western blotting
Total protein was extracted from tissues or cells using radioimmunoprecipitation assay (RIPA) buffer containing 1% phenylmethanesulfonyl fluoride (PMSF), following the manufacturer’s instructions. Western blotting was performed as described previously, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control (19). The following antibodies were used: SLC25A3 (1:1,000, #10420-1-AP, Proteintech) and GAPDH (1:5,000, #10494-1-AP, Proteintech).

Cell culture and transfection
HCC cell lines, including Huh7, HCC-LM3, MHCC-97H, PLC/PRF/5, and HepG2, along with a normal hepatic cell line, L02, were sourced from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) at 37 ℃ in a 5% CO2 incubator. Plasmids overexpressing SLC25A3 (SLC25A3-OE), shRNA targeting SLC25A3 (SLC25A3-KD), and their respective negative controls (OE-NC and KD-NC) were purchased from Genechem (Shanghai, China). Transient transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. Transfection efficiency was assessed via western blot analysis. The sequences of the shRNA targeting SLC25A3 were as follows: shRNA-1 (SLC25A3-KD-1): 5'-GAAGAUUAGGGAAGUAGAA-3', shRNA-2 (SLC25A3-KD-2): 5'-GGAAGCAGGAACAGCAAA-3'.

Cell Counting Kit-8 (CCK-8) assay
HCC-LM3 and Huh7 cells were seeded in 96-well plates at a density of 1,500 cells per well and cultured for 1, 2, and 3 days. At each time point, 10 µL of CCK-8 reagent (Dojindo, Japan) was added to each well. The plates were incubated in the dark at 37 ℃ for 2 hours, and the optical density was measured at 450 nm. All experiments were performed in triplicate.

Colony formation assay
HCC-LM3 and Huh7 cells were seeded in six-well plates at a density of 1,000 cells per well for HCC-LM3 and 500 cells per well for Huh7 cells, and cultured for 10 days. Colonies were fixed with 4% paraformaldehyde and stained with crystal violet. The number of colonies was subsequently counted. All experiments were performed in triplicate.

Oxygen consumption rate (OCR) and ATP content measurements
Mitochondrial OCR was measured using a Seahorse Bioscience XF-24 analyzer (Agilent, SCC, USA). Briefly, HCC-LM3 and Huh7 cells were seeded into XF-24 microplates and calibrated overnight. The following day, cells were maintained at 37 ℃ in a non-CO2 incubator for 1 hour. Basal OCR was measured, and OCR values were normalized to protein content. Total ATP levels were measured using an ATP Assay Kit (Beyotime, Beijing, China), following the manufacturer's instructions.

Mitochondrial membrane potential
Mitochondrial membrane potential was assessed using a JC-1 staining kit (Beyotime). Cells were washed with phosphate-buffered saline (PBS) and then incubated with JC-1 staining buffer for 20 minutes at 37 ℃. After two washes with binding buffer, cells were analyzed using fluorescence microscopy.

Mice
SLC25A3 knockout (SLC25A3-KO) mice and wild-type (WT) littermates, free of pathogens, were purchased from Jackson Laboratory (Maine, USA). All mice were maintained in a pathogen-free environment and provided with a standard diet at the Animal Experimental Center of the Second Xiangya Hospital, Central South University. All animal experiments were performed under a project license (No. 20240856) granted by the Animal Care and Use Committee of the Second Xiangya Hospital of Central South University, in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration. Genomic DNA from tail samples of the two groups of mice was extracted, and genotyping was performed via electrophoresis.

Diethylnitrosamine (DEN)-induced mice HCC models
Two-week-old male SLC25A3-KO mice and WT littermates (range from 4–8 g, 12 per group) were administered a single intraperitoneal injection of DEN (Sigma-Aldrich, Salt Lake City, USA) at 10 mg/kg body weight, dissolved in 0.9% saline. Mice were sacrificed 35 weeks post-injection. Macroscopic lesions and tumor counts in liver tissues were recorded. Serum markers including aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), and direct bilirubin (DBIL) were measured using an automatic biochemical analyzer (Abbott, Chicago, USA).

Transmission electron microscopy (TEM)
Liver tissues from SLC25A3-KO and WT mice were sectioned into small chips and examined by TEM to analyze the ultrastructure of liver cells.

RNA sequencing (RNA-seq)
Liver tissues from SLC25A3-KO and WT mice were subjected to bulk RNA-seq (Rainbow-Genome, Shanghai, China). Three independent biological replicates were performed for each group. Differentially expressed genes (DEGs) between the SLC25A3-KO (KO group) and WT (normal control, NC group) mice were identified using a |log2 fold change (FC)| >1 and a P value <0.05. Functional analysis and gene set enrichment analysis (GSEA) were subsequently conducted based on the RNA-seq data.

Assay for transposase-accessible chromatin using sequencing (ATAC-seq)
Nuclei from SLC25A3-KO and WT mouse liver cells were extracted according to the manufacturer’s protocol (Rainbow-Genome). The transposition reaction was carried out using the Tn5 enzyme. Transposed DNA fragments were then recovered using the MinElute PCR Purification Kit. Library construction was performed using 1X NEBNext High Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA) for PCR amplification, and the amplified products were purified with the MinElute PCR Purification Kit. Finally, sequencing was carried out using the Illumina NovaSeq 6000 platform with a PE150 sequencing strategy.

Statistical analyses
Statistical evaluations were conducted using SPSS version 19.0. To compare two groups, Student’s t-test was utilized. For comparisons involving three or more groups within the same experiment, one-way analysis of variance (ANOVA) was applied. Data are presented as the mean ± standard error of the mean (SEM). A P value of less than 0.05 was considered statistically significant.

Results

SLC25A3 is significantly upregulated in HCC and is associated with poor prognosis
Mitochondrial-related gene sets were retrieved from the MSigDB database and analyzed using the TCGA database. Among these gene sets, SLC25A3 was found to be highly expressed in the liver, and it was selected for further investigation (Figure 1A). Compared to normal liver tissue, SLC25A3 was significantly overexpressed in HCC tissues (Figure 1B), with higher expression levels observed at various stages of HCC (Figure 1C). DNA methylation analysis of the SLC25A3 gene region revealed a significant reduction in DNA methylation levels in HCC tissues compared to normal liver tissue (Figure 1D,1E). This suggests that the upregulation of SLC25A3 expression in HCC may be due to reduced DNA methylation. Furthermore, high expression of SLC25A3 in HCC patients was associated with a poor prognosis (Figure 1F). These findings provide preliminary evidence for the potential oncogenic role of SLC25A3 in HCC.

Investigation of SLC25A3 expression in HCC based on clinical samples and cell lines
To investigate the potential oncogenic role of SLC25A3 in HCC, we conducted western blot analysis. The results revealed a significant upregulation of SLC25A3 protein expression in HCC tissues compared to adjacent peri-tumor tissues (Figure 2A), a finding that was further corroborated by IHC staining (Figure 2B,2C). Additionally, we examined the expression of SLC25A3 in five human HCC cell lines (Huh7, HCC-LM3, MHCC-97H, PLC/PRF/5, and HepG2) alongside the normal human liver cell line L02. In line with the tissue data, SLC25A3 expression was significantly higher in all five HCC cell lines compared to the L02 cell line (Figure 2D). These findings provide further clinical evidence supporting the upregulation of SLC25A3 in HCC.

SLC25A3 knockout impairs hepatocarcinogenesis by inducing morphological changes in mitochondria
To investigate the role of SLC25A3 in HCC onset and progression, in vivo animal experiments were conducted. Genotyping confirmed that both SLC25A3-KO and WT mice were homozygous, and successful knockout of SLC25A3 was achieved (Figure 3A,3B). A DEN-induced HCC model was established in both SLC25A3-KO and WT mice. After successful modeling, liver tissues were collected for analysis. Compared to the WT group, SLC25A3-KO mice exhibited smaller liver-to-body weight ratios, fewer tumors, smaller tumor diameters, and less liver damage (Figure 3C,3D). Electron microscopy analysis revealed that hepatocytes in SLC25A3-KO mice had smaller mitochondria (Figure 3E). These results suggest that SLC25A3 deficiency alleviates DEN-induced HCC, likely by regulating mitochondrial function.

SLC25A3 promotes hepatocarcinogenesis by inducing mitochondrial dysfunction
To explore the effects of SLC25A3 on the biological behavior and mitochondrial function of HCC cells, a series of in vitro experiments were conducted. HCC-LM3 cells, with relatively low expression of SLC25A3, and Huh7 cells, with relatively high expression of SLC25A3, were selected for subsequent experiments. Plasmids overexpressing SLC25A3 (SLC25A3-OE) and its negative control (OE-NC), along with shRNA targeting SLC25A3 (SLC25A3-KD-1, SLC25A3-KD-2) and its negative control (KD-NC), were transfected into these two cell lines. The efficiency of overexpression and knockdown was confirmed by western blot analysis, with SLC25A3-KD-2 demonstrating superior knockdown efficiency, which was used for further analysis and referred to as SLC25A3-KD (Figure 4A). The results of cell viability assays indicated that overexpression of SLC25A3 significantly enhanced cell proliferation and colony formation, while knockdown of SLC25A3 produced the opposite effect (Figure 4B-4D). To further investigate mitochondrial dysfunction, we performed experiments to assess mitochondrial metabolism, including measurements of ATP content, OCR, and mitochondrial membrane potential. Overexpression of SLC25A3 resulted in a significant increase in ATP levels and basal OCR in both HCC-LM3 and Huh7 cells, while SLC25A3 knockdown produced a decrease in these parameters (Figure 4E,4F). Moreover, overexpression of SLC25A3 significantly elevated mitochondrial membrane potential, suggesting enhanced oxidative phosphorylation and reduced apoptosis, whereas knockdown of SLC25A3 had the opposite effects (Figure 4G). These findings suggest that SLC25A3 promotes hepatocarcinogenesis, at least in part, by inducing mitochondrial dysfunction.

Multi-omics sequencing analysis to investigate the impact of SLC25A3 on epigenetic modifications and gene expression
To further elucidate the oncogenic role of SLC25A3 in HCC, bulk RNA-seq was performed using liver tissues from SLC25A3-KO and WT mice. GSEA revealed that functional differences between SLC25A3-KO and WT livers were associated with mitochondrial metabolic processes, such as oxidative phosphorylation and fatty acid metabolism (Figure 5A). DEGs were identified between the two groups. Functional analysis of the DEGs showed that those significantly downregulated in the SLC25A3-KO group were involved in oxidative phosphorylation and energy metabolism (Figure 5B,5C). Epigenetic modifications, such as DNA methylation, histone modifications, and chromatin conformational changes, can affect gene expression without altering the DNA sequence and contribute to cancer progression (20,21). Our previous study showed that non-coding RNA SNORA55, derived from the nucleus, can influence mitochondrial function (12). However, it remains unclear how SLC25A3 regulates epigenetic modifications through mitochondrial energy metabolism. To address this preliminarily, ATAC-seq was performed, revealing that SLC25A3-KO induces chromatin conformational changes in various chromatin regions (Figure 5D). Further analysis showed that the genes corresponding to these regions were closely linked to metabolic processes (Figure 5E). Joint analysis of ATAC-seq and RNA-seq data identified genes with expression changes due to chromatin conformational alterations, which were also associated with mitochondrial metabolic processes (Figure 5F,5G). Collectively, these results suggest that SLC25A3 promotes HCC progression by modulating mitochondrial metabolism and epigenetic modifications.

Discussion
The development of HCC is a complex biological process driven by multiple factors, including gene regulation, transcription, translation, protein modification and degradation, and the accumulation of metabolic products, all of which encompass the entire central dogma of molecular biology (22). Traditionally, the hallmark metabolic pathway in tumors has been anaerobic glycolysis, commonly known as the Warburg effect. However, in recent years, the pivotal role of metabolic reprogramming in the progression of various diseases, including cancer, has become increasingly recognized (4,23,24). In the context of HCC, we analyzed the expression profiles of mitochondrial-related genes and identified SLC25A3, which was significantly overexpressed in HCC tissues and correlated with poor prognosis.
SLC25A3 is a mitochondrial membrane transporter protein responsible for the transport of phosphate into the mitochondrial matrix, either via proton cotransport or exchange with hydroxyl ions (13). Recent studies have implicated SLC25A3 in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), osteonecrosis, and hypertrophic cardiomyopathy, primarily through its influence on cuproptosis or mitochondrial ion homeostasis (25-27). However, the potential oncogenic role of SLC25A3 in HCC remains largely unexplored. In this study, we demonstrate that SLC25A3 is highly expressed in HCC and that its knockout in mice significantly inhibits the development of DEN-induced HCC. To investigate the impact of SLC25A3 on mitochondrial function, we performed electron microscopy on liver tissues from SLC25A3-KO and WT mice. We observed smaller mitochondria in the liver cells of SLC25A3-KO mice, suggesting altered mitochondrial dynamics.
Nuclear-mitochondrial communication, encompassing both anterograde (nucleus-to-mitochondria) and retrograde (mitochondria-to-nucleus) signaling, has been extensively and studied in various physiological pathological processes (5,28). In our previous work, we demonstrated that the non-coding RNA SNORA55, derived from the nucleus, can regulate mitochondrial function and promote HCC progression (12). However, it remains unclear whether SLC25A3 modulates mitochondrial-nuclear communication. With recent technological advancements, the role of chromatin conformation, DNA methylation, histone modifications, and m6A modifications in disease progression has been increasingly elucidated, particularly their involvement in various cancers (20,21,29). ATAC-seq technology enables the detection of chromatin open sites, providing insight into the epigenetic landscape of samples. By linking chromatin openness to gene expression profiles, ATAC-seq offers powerful data mining capabilities. To further investigate the mechanisms underlying SLC25A3’s role in HCC, we conducted bulk RNA-seq and ATAC-seq on liver tissues from SLC25A3 knockout and WT mice. Differential expression and functional enrichment analyses of the RNA-seq data revealed that DEGs were closely associated with energy metabolism and oxidative phosphorylation processes. Integrating the RNA-seq and ATAC-seq results, we observed that SLC25A3 knockout induced changes in chromatin conformation, which, in turn, altered the expression of genes related to mitochondrial metabolism. This suggests that SLC25A3 may regulate chromatin remodeling through its effects on mitochondrial dysfunction.
There are several limitations in this study. First, the specific mechanism by which SLC25A3 promotes hepatocarcinogenesis requires further investigation through in-depth experimental research. Additionally, the potential of SLC25A3 as a therapeutic target for HCC necessitates further drug screening and clinical studies.

Conclusions
In this study, we identified SLC25A3, a mitochondrial membrane protein, as being highly expressed in HCC and associated with poor prognosis. Our experiment results demonstrated that SLC25A3 knockout significantly inhibits the occurrence and progression of HCC, potentially through modulation of mitochondrial function and epigenetic modifications. To the best of our knowledge, this is the first report of SLC25A3’s oncogenic role in HCC, providing new insights into potential therapeutic approaches for HCC.

Supplementary
The article’s supplementary files as