Mitochondria-associated gene SLC25A32 as a novel prognostic and immunotherapy biomarker: From pan-cancer multi-omics analysis to breast cancer validation

doi:10.21203/rs.3.rs-3322997/v1

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Mitochondria-associated gene SLC25A32 as a novel prognostic and immunotherapy biomarker: From pan-cancer multi-omics analysis to breast cancer validation

https://doi.org/10.21203/rs.3.rs-3322997/v1

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Background:

Mutations in SLC25A32 in humans cause riboflavin-responsive exercise intolerance, which is associated with various neurological and metabolic diseases. However, its specific mechanism of action in tumour development is poorly understood owing to the lack of multi-omics integrated analysis of SLC25A32 in pan-cancer.

Methods:

We used various analytical tools to comprehensively investigate the transcription, protein level, and promoter methylation of SLC25A32. Furthermore, the GSCA and cBioPortal databases were used to evaluate the inheritance impact and epigenetic alterations of SLC25A32 in pan-cancer. SLC25A32 expression and the prognostic significance of copy number alterations in multiple cancers were compared using the UCSCXenaShiny and GEPIA2.0 platforms, and its specific function in breast cancer was experimentally verified.

Results:

SLC25A32 is abnormally expressed at the transcriptional and protein levels in most cancer types, with aberrant DNA promoter methylation and significant gene amplification in most tumours. SLC25A32 is significantly associated with the survival prognosis of some cancers, immune infiltrating cells, tumour stemness, and immune-related markers. SLC25A32 knockdown decreased breast tumour cell proliferation, invasion, and metastasis.

Conclusions:

This study aimed to reveal SLC25A32 as a novel prognostic biomarker for pan-cancer prediction and immunotherapy efficacy and specifically describes its underlying mechanism of action in breast cancer. SLC25A32 is widely differentially expressed in pan-cancer with prognostic significance and is correlated with immune infiltration. Additionally, it can affect breast cancer occurrence and development.

SLC25A32

multi-omics bioinformatics

immune infiltration

triple-negative breast cancer

epithelial-mesenchymal transition

Cancer prevalence and death are steadily rising, which is detrimental to societal advancement and human health [1]. Breast cancer is the most commonly diagnosed cancer. Therefore, improving timely diagnosis and comprehensive treatment should be prioritised [2]. Targeted therapy and immunotherapy have also gained popularity in tumour treatment recently. However, more appropriate prognostic biomarkers and therapeutic targets are needed to support their combination therapy [3].

The solute carrier transporter (SLC) family comprises over 300 membrane-bound proteins essential in regulating substrate transport exchange, drug absorption, and efflux [4]. SLC25A32 is located on chromosome 8q22.3 and contains seven exons [5]. It encodes the mitochondrial flavin adenine dinucleotide (FAD) transporter, an inner membrane carrier that imports FAD from the cytoplasm into the mitochondria. SLC25A32 dysfunction leads to mitochondrial FAD deficiency and impaired mitochondrial acyl-CoA dehydrogenase and electron transfer chain, further disrupting muscle and brain functions [6]. Studies on SLC25A32 have focused on neurological and metabolic diseases, where its mutations in humans lead to riboflavin-responsive motor intolerance [7]. However, earlier studies have suggested that SLC25A32 is a mitochondrial folate transporter protein [8]. SLC25A32 dysfunction disrupts folate-mediated one-carbon metabolism by reducing mitochondrial 1C unit production. The biallelic loss-of-function variant of SLC25A32 exists in human foetuses with neural tube defects (NTDs). SLC25A32-trapped knockout mice also had brain NTD at the embryonic stage [9]. In addition, metabolic analysis suggests that disrupted SLC25A32 dysfunction is associated with multiple biochemical processes of ATP generation, including fatty acid β oxidation, catabolism of essential amino acids (leucine, isoleucine, valine, tryptophan, and lysine), and choline degradation [10]. SLC25A32 inhibition leads to respiratory chain dysfunction of FAD-dependent complex II enzymes, reactive oxygen species (ROS) induction, and reduced glutathione (GSH) depletion, which impairs cancer cell proliferation. Beyond the controversial substrate specificity of SLC25A32, its role in tumour progression is rarely discussed. Additionally, the pan-cancer systematic analysis and specific mechanism of action in the immune microenvironment need further characterisation and exploration.

Data collection, mining, and processing

We obtained the mRNA expression profile of SLC25A32 and data related to prognosis, stage, DNA promoter methylation, copy number fragments, and clinical features of 33 cancers from The Cancer Genome Atlas (TCGA) database [11]. Ethics committee or institutional review board approval was not required because this was a bioinformatics study. In addition, special sources of data were annotated.

Transcriptional and protein-level differential expression analysis of SLC25A32 in pan-carcinoma

First, at the transcriptional level, SLC25A32 expression in healthy and corresponding tumour tissues was collected using Tumour Immune Estimation Resource 2.0 (TIMER2.0) [12], GEPIA2.0, and UALCAN databases. The GEPIA2.0 database simultaneously revealed the association between SLC25A32 and the pathological stages of different cancers [13]. Furthermore, the UALCAN database compared DNA promoter methylation levels of SLC25A32 in various cancer types [14], and RNA-seq data from TCGA were associated with methylation levels in UCSCXenaShiny [15]. In addition, the difference in SLC25A32 protein expression levels was compared between the healthy and corresponding tumour tissues using the Clinical Proteomics Cancer Analysis Consortium (CPTAC) data in the UALCAN database, and the post-translational modification of SLC25A32 was studied using the PhosphoSitePlus database [16].

Genetic and mutational situation analysis of SLC25A32 in pan-carcinoma

Gene Set Cancer Analysis (GSCA) is a database that aggregates information on tumour genomic gene sets [17]. We initially studied the copy number variation (CNV) of SLC25A32 in different cancers, cross-verified the correlation between SLC25A32 DNA methylation and transcriptional expression levels in different cancers via the GSCA database, and summarised gene expression and drug sensitivity using the Genomics of Drug Sensitivity in Cancer (GDSC) database.

The correlation between SLC25A32 mutations, the site, copy number alteration (CNA), and the mRNA levels in SLC25A32 and CNV were analysed using the cBioPortal platform [18]. We further analysed the relationship between SLC2532, different CNA type groups, and pan-cancer tumour prognosis using the UCSCXenaShiny.

SLC25A32 prognostic analysis of patients in pan-cancer

In computing the survival heat map for overall and disease-free survival (OS and DFS, respectively) in pan-cancer using the GEPIA2.0 database, P < 0.05 was considered significant, and approaching red indicates the highly-expressed SLC25A32 group in the corresponding cancer type with a worse prognosis, revealing multiple specific tumours. Furthermore, SLC25A32 OS, progression-free survival (PFS), and disease-specific survival (DSS) data in pan-cancer were visualised via a forest map using the UCSCXenaShiny platform.

Therapeutic analysis of SLC25A32 immune infiltration in pan-carcinoma

Using the TIMER 2.0 database [12], multiple algorithms analysed the effect of SLC25A32 on the immune cell infiltration in 33 cancers [19]. Furthermore, we summarised the correlation between SLC25A32 expression, methylation, CNA conditions, and immunotherapy-related markers using the TISIDB database [20].

Using pan-cancer samples, stemness scores, tumour mutation burden (TMB), and microsatellite instability (MSI) scores from the TCGA database, the immunedeconv software package (version 2.0.3) revealed the relationship between tumour-infiltrating immune cells (TIC) and immunotherapy-related indicators and SLC25A32 gene expression data in pan-cancer [21].

SLC25A32 function and analysis of biological tumour function at the pan-cancer single-cell level

The CancerSEA platform, including various cancer single-cell sequencing data, can more accurately reveal the various biological cancer functions [22]. We analysed the relationship between SLC25A32 expression and various cancers’ biological tumour behaviours through a t-SNE diagram that displays the SLC25A32 expression profile in several TCGA cancer single cells.

SLC25A32 functional enrichment analysis and co-expressed genes

The molecules that interact with SLC25A32 in pan-cancer were selected using BioGRID [23]. In addition, the LinkedOmics database was also used to analyse the heat map of positive and negative gene co-expression with SLC25A32 in RNA-seq data of patients with breast cancer in TCGA [24]. Furthermore, Pearson's correlation coefficient was used to plot the volcano plot of co-expressed genes. Using the Metascape database for functional enrichment analysis of co-expressed genes in breast cancer [25], including gene ontology (GO) and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway.

The gene set enrichment analysis (GSEA) was performed using the clusterProfiler package for R (version 3.14.3) [26]. Enriched pathways were classified based on normalised enrichment scores and corrected p-values. The clusterProfiler package was similarly used for GO/KEGG analysis of the DEGs between breast cancer and healthy tissue samples [27–28]. The results revealed that these genes exhibit multiple biological processes, including signalling pathways, molecular functions, and cellular components, which the ggplot2 package for R (version 3.3.3) annotated and visualised [29].

Protein-protein interaction network analysis

Protein interaction network analysis was simultaneously performed using Metascape Online [25]. The results revealed a protein-protein interaction network comprising genes co-expressed with SLC25A32 in breast cancer, with different colour blocks demonstrating possible mechanisms of action affecting the tumour.

Cell culture

Healthy mammary epithelial cell lines were cultured in an MCF-10A-specialised medium. Breast cancer cell lines, including MCF-7, MDA-MB-231, BT-549, and HCC1937, were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and the Cancer Institute of Southern Medical University (Guangzhou, China) and grown in DMEM supplemented with 10% fetal bovine serum (Gibco, USA). All cells were grown at a constant temperature of 37°C in a 5% CO₂ culture in the incubator.

Quantitative real-time polymerase chain reaction (PCR)

The premix was tested on a CFX-96 real-time PCR instrument (Bio-Rad, USA). GAPDH was used as a normalisation control. The primers used are as follows: GAPDH-F: GGAGCGAGATCCCTCCAAAAT, GAPDH-R:GGCTGTTGTCATACTTCTCATGG, SLC25A32-F:TACGGGGACTTTATCAAGGAGT, and SLC25A32-R:AAGGCGAGTTTTTGTTACCCATA. We used the 2^−ΔΔCt method to quantitatively compare the cyclic threshold (Ct) value results.

Cell transfection

Transfection was performed using the siRNA kit designed and synthesised by Guangzhou Ruibo Bio (Guangzhou, China). With transfection in 6-well plates (NEST Biotechnology, China), cells in the exponential growth phase rose to 50–60% using lipo3000 (Invitrogen Biotechnology, Shanghai, China). Cells were harvested for RNA extraction 24–48 h post-transfection or functional experiments and at 72 h for western blot analysis of extracted proteins.

Protein-based western blot analysis

The cell lysate was prepared using a 100:1:1 ratio of phosphatase inhibitor, protease inhibitor, and total protein extraction. The lysate was subjected to electrophoresis with 10% SDS-PAGE gel, using PVDF at 350 mA, and blocked with 5% skimmed milk. The primary antibodies included: SLC25A32, E-ca, N-ca, vimentin, MMP 9, β -catenin, β -tubulin, and GAPDH.

Cell proliferation assay

Cells were harvested at 24–48 h post-transfection, and digested cells were seeded into 96-well plates at a density of 2,000 cells per well. After 6 h of apposition, 10 µL of CCK8 reagent was added per well, and absorbance was measured at 450 nm, with measuring time points at 0, 1, 24, 48, 72, and 96 h. Three duplicate wells were set for each group. The experiments were repeated more than thrice.

Migration and invasion assays

Cells in 6-well plates attained 100% confluence at 48 h post-transfection. They were scratched using the pipette gun head, placed in PBS, and photographed at 0, 24, and 48 h. The wound healing area was then determined. All assays were performed in triplicates, and representative images were selected.

Transwell (BD Biosciences, NJ, USA) assays were used to detect cell invasion abilities.

Statistical analyses

Some statistical analyses and visualisation were performed using R (version 4.2.1), and public databases were analysed by default. Regarding breast cancer, the Wilcoxon rank sum and signed rank tests were used to detect the significance of SLC25A32 expression in unpaired and paired tissues, respectively. All tests in this study were two-sided, and a P-value of < 0.05 was considered significant.

The transcriptome expression level of SLC25A32 varied in most cancers compared with healthy samples

The differential expression of SLC25A32 in multiple tumours and corresponding healthy tissues at transcript levels was investigated using the TIMER2.0, GEPIA, and UALCAN databases. The GEPIA2.0 database presents the differential expression of SLC25A32 in paired tumours and healthy tissues, validating the evidence that SLC25A32 was significantly up-regulated in most tumours, including lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), oesophageal carcinoma (ESCA), pancreatic adenocarcinoma (PAAD), stomach adenocarcinoma (STAD), and thymoma (THTM) (Fig. 1A). The unpaired expression analysis provided by TIMER2.0 revealed that SLC25A32 was significantly up-regulated in 10 cancer types: breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), ESCA, head and neck squamous cell carcinoma (HNSC), liver hepatocellular carcinoma (LIHC), lung squamous cell carcinoma (LUSC), pheochromocytoma and paraganglioma (PCPG), rectum adenocarcinoma (READ), and STAD; down-regulated in the five cancer types: glioblastoma multiforme (GEM), kidney chromophobe (KICH), kidney renal papillary cell carcinoma (KIRP), thyroid carcinoma (THCA), and uterine corpus endometrial carcinoma (UCEC) (Fig. 1B). SLC25A32 expression analysis in the TCGA database revealed similar differential results (Fig. 1C).

The GEPIA2.0 database revealed the differential expression of SLC25A32 in different pathological stages of various cancer types. SLC25A32 was significantly associated with the pathological stage of adrenocortical carcinoma (ACC), KICH, KIRP, lung adenocarcinoma (LUAD), and uterine carcinosarcoma (USC) (Fig. 1D). The cancer types without a significant effect of SLC25A32 expression on the disease pathology stage are presented in Fig. S1.

SLC25A32 Promoter methylation differed across multiple cancer types

Aberrant expression of oncogenes is usually correlated with DNA methylation levels [30]. Therefore, we investigated the promoter methylation level of SLC25A32 in pan-cancer. In BRCA, bladder urothelial Carcinoma (BLCA), HNSC, LUAD, Sarcoma (SARC), THCA, and UCEC SLC25A32 promoter methylation differed between normal tissues (Fig. 2A). However, according to the TCGA database, a correlation exists between promoter methylation and transcriptome levels of SLC25A32 in UCS, SKCM, CESC, LIHC and BLCA (Fig. 2B). Thus, DNA promoter methylation might be a reason for the aberrant transcriptome expression of SLC25A32. The cancer types with no significant differences in SLC25A32 promoter methylation are presented in Fig. S2, indicating that other factors cause transcriptomic level differences besides DNA promoter methylation abnormalities. We then analysed the genetic and epigenetic mutations of SLC25A32 in pan-cancer.

Genetic alterations of SLC25A32 affected their expression at the transcriptomic level and correlated with tumour prognosis

Notably, we summarised the association between SLC25A32 expression and related drug sensitivity in various cancer types based on the anticancer drug susceptibility database Genomics of Drug Sensitivity in Cancer (GDSC). Cetuximab and Afatinib treatment sensitivity was positively correlated with SLC25A32. In contrast, the treatment sensitivity of Dabrafenib and I-BET-762 demonstrated a negative correlation (Fig. S2).

Using UCSCXenaShiny, we further explored the prognostic relationship of several CNA species in SLC25A32 and different cancer types, and in the total pan-cancer data, OS and PFS were lower in groups with deleted and duplicated SLC25A32 than in the normal group, and most significantly different tumour types. Moreover, the SLC25A32 duplicated group had more adverse OS and PFS prognosis data. In addition, the KIRC OS and KIRP PFS in the SLC25A32 normal group were between the SLC25A32 deletion and duplication groups (Fig. 3A–B). TCGA pan-cancer data from the cBioPortal platform was used to study the genetic alterations of SLC25A32, and the gene copy number of SLC25A32 was analysed in pan-cancer. SLC25A32 was significantly amplified in most cancer types (Fig. S3A–B). Additionally, its gene amplification was significantly associated with the upregulation of mRNA expression (Fig. S3C–F). In addition to altered DNA promoter methylation in pan-cancer, genetic and epigenetic alterations are also involved in the upstream mechanism.

The protein expression level of SLC25A32 similarly varied in the pan-cancer and normal samples

Analysing the SLC25A32 protein expression level data from the CPTAC database in the UALCAN platform revealed that SLC25A32 protein expression increased over SLC25A32 in OV, UCEC, lung cancer, HNSC, and liver cancer and decreased over SLC25A32 in PAAD and Glioblastoma (Fig. 4A, D). To further analyse the SLC25A32 proteomics data, we investigated the possible post-translational modification sites of SLC25A32 and the mutation frequency in various tumour types using the PhosphpSitePlus database. According to previous data, SLC25A32 mainly has two modification species: phosphorylation and ubiquitination (Fig. 4B). Notably, we obtained mutation frequency data that are almost consistent with the UALCAN database because both have high SLC25A32 mutation frequency (Fig. 4C) in lung adenocarcinoma, endometrial, HNSC, stomach, breast, colorectal, Glioblastoma, and Ovarian serous cystadenocarcinoma (OV). The proteomic data complement the transcriptome view that SLC25A32 has differential mRNA and protein expression in pan-cancer and healthy tissues and may be involved in tumour development. However, the regulation and specific mechanisms of SLC25A32 in tumour progression require further investigation.

SLC25A32 has a prognostic value as a novel biomarker in pan-cancer

Using the GEPIA database to investigate the prognostic significance of SLC25A32 in multiple cancer types, OS and DFS map of SLC25A32 revealed that patients with lower class tumours that highly express SLC25A32 have more adverse OS, including BLCA, BRCA, HNSC, KICH, KIRP, LAML, LUAD, MESO, SARC, and UVM (Fig. 5A). In addition, high SLC25A32 expression was associated with poor DFS data in patients with BLCA, KICH, KIRP, and SARC (Fig. 5B). Furthermore, the OS, progressive-free interval (PFI), and disease-specific survival (DSS) data of SLC25A32 in different cancer types were analysed using the UCSCXenaShiny database (Fig. S4). Both results indicate that SLC25A32 is significantly associated with prognosis in most cancer types and is a risk factor for cancer prognosis.

SLC25A32 expression is closely associated with the tumour immune microenvironment and immunotherapy

In the immune microenvironment, immune infiltration is closely related to tumour progression [31]. Thus, we calculated the correlation between SLC25A32 expression and different immune cell infiltration in multiple cancer types. Tumour-associated fibroblast infiltration was positively correlated with SLC25A32 expression in CHOL, HNSC, KIRP, MESO, Thymoma (THYM), UCS, and PAAD. Contrastingly, a negative correlation was observed with SLC25A32 expression in DLBC and Brain Lower Grade Gliom (LGG) (Fig. 6A). A heatmap of B-cell infiltration revealed that B-cell infiltration was positively correlated with SLC25A32 expression in CHOL, LGG, ACC, DLBC, KICH, PAAD, and PCPG and negatively correlated in CESC, HNSC, LUSC, and STAD (Fig. 6B). Similarly, CD8 T cell infiltration was positively correlated with SLC25A32 expression in DLBC, PAAD, and UVM; however, it was negatively correlated with SLC25A32 expression in CESC, HNSC, and UCEC (Fig. 6C). Notably, CD4 T cell infiltration determined using the XECLL algorithm revealed that Th1 cell infiltration was negatively correlated with SLC25A32 expression in almost all cancer types. In contrast, Th2 cell infiltration was positively correlated with SLC25A32 expression. This indicated that the Th1/Th2 ratio was significantly negatively correlated with SLC25A32 expression in almost all cancer types (Fig. 6D). In addition, the correlation analysis between SLC25A32 expression and 20 kinds of immune infiltrating cells in pan-cancer revealed that SLC25A32 expression was negatively correlated with Treg cells, CD8 T cells, plasma cells, natural killer (NK) activated cells, mast resting cells, and B cell memory in pan-cancer. A significant positive correlation was observed with CD4 T cell memory, mast activated, M1 and M0 macrophages, and B cell naïve (Fig. 6E).

Therefore, SLC25A32 expression is closely associated with immune cell infiltration in various cancer types. Additionally, it may serve as a novel immune-related biomarker in tumorigenesis and progression. Subsequently, we investigated the relationship between SLC25A32 expression, methylation, and CNA levels and three immunomodulators (Fig. S5).

We then utilised the relationship between SLC25A32 expression and TMB and MSI in most cancer types to investigate the relationship between SLC25A32 expression and immunotherapy. TMB and MSI are critical in deciding whether to proceed with immune checkpoint therapy [32]. Our findings revealed that SLC25A32 expression was positively correlated with TMB in some cancers, including DLBC, KICH, LUAD, and STAD. However, a significant negative correlation was lacking between SLC25A32 expression and TMB in UVM, KIRP, and READ (Fig. 6F). SLC25A32 expression level was positively correlated with MSI in most cancers, such as UCEC, SKCM, LUAD, and Glioblastoma multiforme (GBM) (Fig. 6G). Tumour stemness is closely associated with the development of drug resistance and tumour cell proliferation during treatment [33]. Therefore, we extracted the correlation between SLC25A32 expression and stemness scores of different tumour types. Figure 6 reveals a significant positive correlation between stemness scores and SLC25A32 expression in most tumours, including STAD, LAML, COAD, ESCA, and STAD. LUSC, LUAD, BLCA, and LGG; Stemness scores were negatively correlated with SLC25A32 expression in some tumours, such as THYM, CHOL, and KICH (Fig. 6H).

SLC25A32 expression at the single-cell level is closely associated with biological tumour behaviour

We analysed the correlation data using the CancerSEA database to explore the relationship between SLC25A32 expression at the single-cell level and the biological behaviour and function of most tumours. SLC25A32 expression is positively correlated with the malignant biological functions of most tumours, including metastasis, differentiation, inflammation, angiogenesis, apoptosis, cell proliferation, stemness, and epithelial-mesenchymal transition (EMT). However, a significant negative correlation was observed with the biological functions of cellular DNA damage repair and cell cycle (Fig. 7G). In addition, we explored the correlation of SLC25A32 with biological behaviours and functions in single-cell datasets of different cancers. SLC25A32 significantly promoted angiogenesis and differentiation but inhibited DNA repair in the RB-EXP0073 dataset (Fig. 7A). In the ALL-EXP0046 dataset, it was positively correlated with apoptosis (Fig. 7B). A negative correlation was observed with DNA damage and repair in UM-EXP0074 single-cell data (Fig. 7C). Moreover, SLC25A32 expression at the single-cell level was visualised using t-SNE plots (Fig. 7D–F). In conclusion, SLC25A32 expression may be closely associated with the malignant biological functions of most cancer types, promoting tumour cell metastasis and proliferation and inhibiting DNA damage repair.

Co-expression genes, functional enrichment, and protein-protein interaction network analysis of SLC25A32 in breast cancer

Evaluation of the genes co-expressed with SLC25A32 revealed the possible mechanism of SLC25A32 in tumour progression (Fig. S6). Thereafter, we investigated the specific regulatory mechanism of SLC25A32 in breast cancer progression. Enrichment analysis was used to evaluate the molecular mechanism of SLC25A32 action. GSEA revealed that SLC25A32 was enriched in PI3KAKTMTORSIGNALING _PATHWAY and EXTRACELcLULAR_ MATRIX_ ORGANIZATION pathways in breast cancer (Fig. 8A–B). Previous studies revealed that in addition to the structural role, the extracellular matrix also affects cell behaviour, such as proliferation, adhesion, and migration [34]. Furthermore, SLC25A32 was enriched in the β-catenin repressor gene marker (BCAT_BILD_ET_AL_DN), suggesting that SLC25A32 might regulate β-catenin activity (Fig. 8C). In addition, string and bubble plots visualised the GO/KEGG analysis and revealed other functional roles of SLC25A32 in breast cancer (Fig. 8D–E). The LinkedOmics platform and Metascape were used to further analyse the gene and functional enrichment of SLC25A32 co-expressed in breast cancer (Fig. S7).

SLC25A32 promoted the proliferation, invasion, and migration of breast cancer cells

Bioinformatics analysis revealed the role of SLC25A32 in breast cancer, and we further explored its biological function. PCR confirmed that SLC25A32 expression was relatively highest in MDA-MB-231 and BT-549 cells; therefore, we selected both cell lines for subsequent experiments (Fig. 9A). The two siRNA knockdown vectors were transfected into 231 and 549 cells, and the knockdown efficiency was verified using RT-PCR and western blotting. The two fragments could be successfully transfected in 231 and 549 cells (Fig. 9B–E). The effect of SLC25A32 on cell proliferation was determined using the CCK8 assay. SLC25A32 knockdown by both fragments significantly reduced the proliferation ability of 231 and 549 cells (Fig. 9F-G). Plate cloning experiments demonstrated similar results (Fig. 9H–I). Subsequently, the migration and invasion abilities of the 231 and 549 cells was reduced when SLC25A32 was down-regulated in triple-negative breast cancer (TNBC) cells (Fig. 10A-C), indicating that SLC25A32 can affect the biological functions of TNBC cells proliferation and migration.

SLC25A32 promoted EMT of breast cancer cells

Based on biological behaviours and functions at the single-cell level, SLC25A32 may positively correlate with malignant biological behaviours such as metastasis, tumour differentiation, angiogenesis, and EMT. Moreover, enrichment analysis revealed that SLC25A32 was enriched in PI3K/AKT/mTOR and β-catenin signalling-associated pathways and extracellular matrix. We hypothesised that SLC25A32 might similarly affect tumour cell metastasis and EMT in TNBC cells. EMT is involved in the malignant progression of tumours; therefore, we examined the changes in EMT-associated indicators in transfected TNBC cells. Western blotting revealed that EMT marker protein expression was significantly changed after the SLC25A32 knockdown. N-cadherin, β-catenin, vimentin, and MMP9 expressions were significantly down-regulated in both TNBC cells, while E-cadherin expression was increased (Fig. 10D–E). Therefore, SLC25A32 promoted the invasion, metastasis, and EMT of TNBC cells.

The mitochondrial carrier family SLC25, one of the solute carrier transporters embedded in its inner membrane, is ubiquitous in eukaryotes. It transports multiple compounds in the mitochondrial inner membrane, participates in multiple metabolic pathways, and regulates cellular functions as a pathway connecting the mitochondrial matrix and cytoplasm [35–36]. However, studies on the role of its family member SLC25A32 in malignancy are insufficient. Here, a systematic bioinformatics analysis combined with experiments can validate SLC25A32 as an innovative pan-cancer prognostic and immune-related biomarker and reveal its targetable mechanism of action in breast cancer. Folate metabolism is regulated by SLC25A32 in Chinese hamster ovary cells [37], and monocarbon and folate metabolism have been previously linked to tumour growth [38]. Thus, SLC25A23 might participate in carcinogenesis and the growth of most malignancies. However, further investigation is required to understand the precise consequences and regulatory mechanisms.

Epigenetic abnormalities may alter gene expression levels. Simultaneously, targeting epigenetic regulation to reprogram cancer to a healthy state has gained increasing attention [39]. The correlation suggests that promoter methylation may cause the altered SLC25A32 expression in some cancers. Genetic alteration affects gene expression [40]. Therefore, we analysed the gene mutations of SLC25A32 and CNV in pan-cancer. In most cancers, SLC25A32 CNV types are mainly gene amplifications, and CNV alterations exhibit some correlation with their expression level. These results indicate that SLC25A32 alteration at the genetic level may also affect tumour growth and prognosis. Thus, the specific mechanism of CNV action on pan-cancer prognosis requires further studies.

Evidence exists that genes influence tumour growth and prognosis by changing the immune microenvironment and cell infiltration [41]. Tumour-infiltrating CD8 + T cells demonstrated antitumor activity in previous studies and had favourable effects on the survival of patients with breast cancer [42]. CD4 + T cells have two subsets—Th 1 and Th 2 cells—and some clinical data observed unbalanced Th 1/Th 2 levels in patients with breast cancer, where Th 2 cells released the cytokines IL-4 and IL-10 and suppressed the host immune system, exhibiting a tumour-promoting effect [43]. In contrast, Th 1 cell infiltration resulted in a better prognosis and lower recurrence rate [44]. Thus, SLC25A32 might influence tumour progression by regulating immune cell infiltration and function. Immune-related markers are crucial in tumour immunity [45]. Hence, we investigated the relationship of mRNA expression, methylation, and CNA levels of SLC25A32 with the three TISIDB immune markers. Our findings suggest that the correlation between SLC25A32 methylation, CNV levels, and immune-related markers in some cancers provides new ideas for tumour immunotherapy response regulation.

This study has limitations. First, the part of bioinformatics analysis that only uses cross-analysis of multiple public databases, despite multiple crossovers and repeated alignments, and did not use clinical samples specifically collected for sequencing analysis, making our data more targeted. Second, despite conducting multiple functional experiments to verify the possible role and mechanism of SLC25A32 in breast cancer, in vitro experiments and immune infiltration of SLC25A32 and immunotherapy are lacking. Moreover, the regulatory mechanism of SLC25A32 in specific signalling pathways remains unclear. Our integrative analysis and experimental validation provide insight for future studies.

Overall, this study integrated expression, genetics, prognosis, immunity, and functional perspectives of SLC25A32, a novel pan-cancer prognosis gene, and immunotherapy-related biomarkers. We preliminarily demonstrated the influence and underlying mechanism of SLC25A32 on biological tumour behaviour in breast cancer via functional and pathway experiments on proliferation, invasion, and migration.

Author information

Shiqi Zuo, Siyuan He and Yingying Zhang contributed equally to this work.

Authors and Affiliations:

Cancer Center, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong 510310, China

Shiqi Zuo, Siyuan He, Yingying Zhang, Yao Tang and Ziqing Wu

Guangdong Provincial Key Laboratory of Molecular Tumor Pathology, Guangzhou 510515, China

Shiqi Zuo and Ziqing Wu

Department of Pathology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515, China

Shiqi Zuo and Ziqing Wu

Corresponding authors

Correspondence to Ziqing Wu and Yao Tang.

Ethics approval and consent to participate

Ethics committee or institutional review board approval was not required because this was a bioinformatics study.

Consent for publication

Not applicable.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

This study was funded by the National Natural Science Foundation of China (NSFC) (no.81372769).

Author Contributions

All authors contributed to the study conception and design. Yao Tang and Ziqing Wu designed and supervised the project. Material preparation, data collection and analysis were performed by Shiqi Zuo, Siyuan He and Yingying Zhang. The first draft of the manuscript was written by Shiqi Zuo and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgment

Not applicable.

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No competing interests reported.

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Mitochondria-associated gene SLC25A32 as a novel prognostic and immunotherapy biomarker: From pan-cancer multi-omics analysis to breast cancer validation

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Abstract

Background:

Methods:

Results:

Conclusions:

Figures

Introduction

Materials and Methods

Data collection, mining, and processing

Transcriptional and protein-level differential expression analysis of SLC25A32 in pan-carcinoma

Genetic and mutational situation analysis of SLC25A32 in pan-carcinoma

SLC25A32 prognostic analysis of patients in pan-cancer

Therapeutic analysis of SLC25A32 immune infiltration in pan-carcinoma

SLC25A32 function and analysis of biological tumour function at the pan-cancer single-cell level

SLC25A32 functional enrichment analysis and co-expressed genes

Protein-protein interaction network analysis

Cell culture

Quantitative real-time polymerase chain reaction (PCR)

Cell transfection

Protein-based western blot analysis

Cell proliferation assay

Migration and invasion assays

Statistical analyses

Results

The transcriptome expression level of SLC25A32 varied in most cancers compared with healthy samples

SLC25A32 Promoter methylation differed across multiple cancer types

The protein expression level of SLC25A32 similarly varied in the pan-cancer and normal samples

SLC25A32 has a prognostic value as a novel biomarker in pan-cancer

SLC25A32 expression is closely associated with the tumour immune microenvironment and immunotherapy

SLC25A32 expression at the single-cell level is closely associated with biological tumour behaviour

Co-expression genes, functional enrichment, and protein-protein interaction network analysis of SLC25A32 in breast cancer

SLC25A32 promoted the proliferation, invasion, and migration of breast cancer cells

SLC25A32 promoted EMT of breast cancer cells

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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