Application of SLC25A20 as a diagnostic, prognostic evaluation and treatment stratification marker for lung adenocarcinoma

Abstract

The application belongs to the field of biotechnology and tumor molecular diagnosis, and discloses application of a reagent for detecting SLC25A20 expression level in preparation of a product for lung adenocarcinoma diagnosis, prognosis evaluation, malignant progression risk evaluation and treatment stratification. The application finds that SLC25A20 is significantly highly expressed in lung adenocarcinoma tissues and lung adenocarcinoma cells, and the increase of the expression level is related to poor clinical prognosis of lung adenocarcinoma patients; the increase of SLC25A20 is related to lung adenocarcinoma cell proliferation, migration, invasion, enhanced tumorigenic ability in vivo and active energy metabolism of fatty acid oxidation related mitochondria. By detecting the mRNA or protein expression level of SLC25A20 in a sample of a subject, whether the subject has lung adenocarcinoma can be judged, the prognosis risk of the patient can be evaluated, the patient with high malignant progression risk can be identified, and the lung adenocarcinoma patient suitable for receiving fatty acid oxidation inhibition, mitochondrial metabolism inhibition or SLC25A20 related metabolic targeted intervention can be screened. The application further provides a kit comprising a reagent for detecting SLC25A20 expression level and application thereof.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and tumor molecular diagnostics, and specifically discloses the application of reagents for detecting SLC25A20 expression levels in the preparation of products for the diagnosis, prognostic assessment, malignant progression risk assessment and treatment stratification of lung adenocarcinoma. Background Technology

[0002] Lung adenocarcinoma (LUAD) is one of the most common histological subtypes of non-small cell lung cancer. Despite advancements in targeted therapy, immunotherapy, and comprehensive treatment strategies, patients with advanced LUAD still face challenges such as recurrence, metastasis, treatment tolerance, and tumor heterogeneity. Therefore, identifying biomarkers that can be used for early auxiliary diagnosis, prognostic assessment, risk assessment of malignant progression, and treatment stratification in LUAD is of great significance for improving the level of precision medicine for patients.

[0003] Tumor metabolic reprogramming is a key characteristic of cancer. Lung adenocarcinoma cells can meet their energy demands for rapid proliferation and metastasis by altering lipid metabolism, fatty acid oxidation, mitochondrial oxidative phosphorylation, and redox homeostasis. Compared to traditional tumor gene mutation biomarkers, biomarkers reflecting metabolic dependence can provide new dimensions for patient subtyping and treatment selection.

[0004] SLC25A20 (NCBI Gene ID: 788, Ensembl: ENSG00000178537, UniProt Accession Number: 043772), also known as carnitine-acylcarnitine translocase (CACT), is located on the short arm of chromosome 3 in humans, region 2, band 1, subband 3, and subsubband 1 (3p21.31). Within the cellular apparatus, it is located on the inner mitochondrial membrane and participates in acylcarnitine / carnitine exchange, a crucial step in the entry of long-chain fatty acids into the mitochondria and their β-oxidation. Previous research has primarily focused on the role of SLC25A20 in congenital metabolic disorders; its application in the development, prognosis, and treatment stratification of lung adenocarcinoma has not been fully explored.

[0005] Therefore, developing a lung adenocarcinoma detection, prognostic assessment, and treatment stratification scheme based on SLC25A20 expression level would help identify lung adenocarcinoma patients with high SLC25A20 expression, active fatty acid oxidation, and active mitochondrial energy metabolism, providing new technical means for precision diagnosis and treatment of lung adenocarcinoma. Summary of the Invention

[0006] To address the technical problem of the lack of effective metabolic-related diagnostic, prognostic, and treatment stratification biomarkers for lung adenocarcinoma in the prior art, this invention provides the application of SLC25A20 as a biomarker for the diagnosis, prognostic assessment, malignant progression risk assessment, and treatment stratification of lung adenocarcinoma.

[0007] This invention is the first to discover and verify that SLC25A20 is significantly overexpressed in lung adenocarcinoma tissues and cells, and its high expression is closely related to poor patient prognosis and malignant progression (proliferation, migration, invasion, and enhanced tumorigenic capacity in vivo). Simultaneously, high expression of SLC25A20 can promote active mitochondrial energy metabolism related to fatty acid oxidation, lipid metabolism reprogramming, and alterations in redox homeostasis. Based on these findings, detecting the expression level of SLC25A20 can be used for the diagnosis, prognostic assessment, malignant progression risk assessment, and treatment stratification of lung adenocarcinoma.

[0008] Specifically, the present invention includes the following technical solutions: In a first aspect, the present invention provides the use of a reagent for detecting SLC25A20 expression levels in the preparation of products for the diagnosis, prognostic assessment, risk assessment of malignant progression, and / or treatment stratification of lung adenocarcinoma.

[0009] Furthermore, the SLC25A20 expression level includes the SLC25A20 mRNA expression level and / or the SLC25A20 protein expression level.

[0010] Furthermore, the product is used to compare the SLC25A20 expression level in a subject's sample with a normal control, adjacent normal tissue control, healthy population control, or a preset threshold; when the SLC25A20 expression level is elevated, it indicates to the subject that: (i) the risk of developing lung adenocarcinoma is increased, the prognosis of lung adenocarcinoma is poor, and / or the risk of malignant progression is high; and / or (ii) the subject is suitable for treatment stratification assessment.

[0011] Furthermore, the samples are selected from lung adenocarcinoma tumor tissue, adjacent normal tissue, lung tissue biopsy samples, puncture samples, surgically resected samples, cytological samples, blood, serum, plasma, exosomes, circulating tumor cells, pleural effusion, sputum, or bronchoalveolar lavage fluid.

[0012] Further, the reagents are selected from: (a) primers, probes, chip probes, sequencing adapters, digital PCR reagents, reverse transcription reagents, or amplification reagents for detecting SLC25A20 mRNA; and / or (b) antibodies, antibody fragments, immunohistochemical reagents, immunofluorescence reagents, enzyme-linked immunosorbent assay (ELISA) reagents, chemiluminescence assay reagents, or mass spectrometry assay reagents for detecting SLC25A20 protein. The products can be kits, chips, detection systems, diagnostic models, prognostic assessment models, or treatment stratification models.

[0013] Furthermore, the prognostic assessment includes assessing overall survival, progression-free survival, risk of recurrence, risk of metastasis, risk of tumor proliferation, risk of invasion, and / or risk of tumorigenic capacity.

[0014] Furthermore, the treatment stratification includes screening lung adenocarcinoma patients suitable for metabolic intervention therapy; the metabolic intervention therapy is selected from fatty acid oxidation inhibition therapy, mitochondrial oxidative phosphorylation inhibition therapy, lipid metabolism intervention, SLC25A20-related metabolic pathway intervention, chemotherapy combined with metabolic intervention, targeted therapy combined with metabolic intervention, or immunotherapy combined with metabolic intervention.

[0015] Furthermore, the fatty acid oxidation inhibition therapy includes treatment that inhibits CPT1-dependent fatty acid entry into mitochondria, and / or treatment that inhibits SLC25A20-mediated acylcarnitine / carnitine exchange-related metabolic processes.

[0016] In a second aspect, the present invention provides a kit for the diagnosis, prognostic assessment, risk assessment of malignant progression, and / or treatment stratification of lung adenocarcinoma, the kit comprising a reagent for detecting the expression level of SLC25A20, and at least one complementary component selected from internal control reagents, sample processing reagents, control standards, and instructions for use.

[0017] Thirdly, the present invention provides a method for screening candidate therapeutic drugs or treatment regimens for lung adenocarcinoma, comprising the following steps: applying the drug or treatment regimen to be tested to lung adenocarcinoma cells or samples with high SLC25A20 expression; detecting one or more indicators selected from SLC25A20 expression level, cell proliferation, colony formation, migration, invasion, apoptosis, ATP level, oxygen consumption rate, lipid droplet accumulation, ROS level, and fatty acid oxidation-related indicators; and selecting drugs or treatment regimens that can reduce SLC25A20-related malignant phenotypes or metabolic phenotypes as candidate therapeutic drugs or treatment regimens compared with untreated controls.

[0018] Compared with the prior art, the present invention has the following outstanding advantages: (1) This invention provides the application of SLC25A20 as a biomarker for the diagnosis and risk assessment of lung adenocarcinoma. By detecting the expression level of SLC25A20 mRNA or protein in patient samples, it can help identify patients with lung adenocarcinoma or high-risk individuals.

[0019] (2) This invention provides the application of SLC25A20 as a prognostic biomarker for lung adenocarcinoma. High expression of SLC25A20 indicates a poor prognosis for patients with lung adenocarcinoma and can be used to assess clinical outcomes such as overall survival, progression-free survival, recurrence risk, and metastasis risk.

[0020] (3) This invention provides the application of SLC25A20 as a risk marker for malignant progression of lung adenocarcinoma. Increased expression of SLC25A20 is associated with enhanced proliferation, migration, invasion, and tumorigenicity of lung adenocarcinoma cells in vivo, and can be used to determine the degree of tumor malignancy.

[0021] (4) This invention provides the application of SLC25A20 as a biomarker for treatment stratification in lung adenocarcinoma. High expression of SLC25A20 is associated with enhanced mitochondrial energy metabolism related to fatty acid oxidation, and can be used to screen patients who may benefit from fatty acid oxidation inhibition, mitochondrial metabolism inhibition, or related metabolic targeted therapies, thereby achieving precise treatment stratification.

[0022] (5) The detection method of the present invention is compatible with multiple technology platforms such as qRT-PCR, Western blot, immunohistochemistry, immunofluorescence, ELISA, digital PCR, sequencing, microarray and mass spectrometry, which facilitates clinical translation and reagent kit development. Attached Figure Description

[0023] Figure 1 The following are the results of SLC25A20 expression detection in lung adenocarcinoma tissues, adjacent normal tissues, lung adenocarcinoma cells, and normal bronchial epithelial cells, as well as the survival analysis results of lung adenocarcinoma patients stratified by SLC25A20 expression levels: A. Representative protein immunoblotting analysis and quantitative detection: expression levels of SLC25A20 protein in paired LUAD tumor tissues and adjacent normal tissues, with GAPDH as a loading control; B. qRT-PCR analysis: expression levels of SLC25A20 mRNA in paired LUAD tumor tissues and adjacent normal tissues; C. Representative immunohistochemical staining: showing the expression of SLC25A20 in paired LUAD tumor tissues and adjacent normal tissues, displaying representative magnified images and quantitative results of IHC scores, scale bar: 50 μm; D. qRT-PCR analysis: detection of SLC25A20 mRNA expression levels in BEAS-2B cells and LUAD cell lines; E. Western blot analysis and quantification of proteins: The expression level of SLC25A20 protein in BEAS-2B cells and LUAD cell lines was detected, with GAPDH as a loading control; F. Kaplan–Meier survival analysis: Overall survival and progression-free survival of LUAD patients were stratified according to SLC25A20 expression level, and the association between SLC25A20 expression and clinical outcomes (including overall survival (OS, left figure) and progression-free survival (FFP, right figure)) was analyzed using the KM-Plotter database; Figure 2To detect the proliferation, colony formation, migration, invasion, and apoptosis of lung adenocarcinoma cells after SLC25A20 knockout: A, B. Verification of SLC25A20 knockout in H1975 and H1650 cells by Western blotting and qRT-PCR analysis, with β-actin as a control in Western blotting; C. Growth curves of EV and SLC25A20-KO H1975 and H1650 cells, determined by direct live cell counting at specified time points; D. Colony formation assay of EV and SLC25A20-KO H1975 and H1650 cells, showing representative colony images and quantitative results of colony number; E. Scratch healing assay showing the migration ability of EV and SLC25A20-KO H1975 and H1650 cells, showing representative images at 0 hours and 24 hours and quantitative results of scratch closure area, scale bar: 100 μm; F. Transwell invasion assays showed the invasive ability of EV and SLC25A20-KO H1975 and H1650 cells, presenting representative images and quantitative results of invasive cells in each field of view. Scale bar: 500 μm. G. Representative flow cytometry images and quantification of apoptotic cells: apoptotic cells in EV and SLC25A20-KO H1975 and H1650 cells were detected by Annexin V / PI staining. H. Representative immunofluorescence staining and Western blotting analysis of cleaved caspase-3 in EV and SLC25A20-KO H1975 and H1650 cells. Cell nuclei were counterstained with DAPI. GAPDH was used as a loading control for Western blotting. Scale bar: 20 μm. Figure 3 To assess the changes in lung adenocarcinoma cell proliferation, colony formation, migration, invasion, and EMT-related markers after SLC25A20 overexpression, A and B were performed using Western blotting and qRT-PCR to verify SLC25A20 overexpression in A549 and H1299 cells, with β-actin as a control. C. Growth curves of EV cells and A549 and H1299 cells overexpressing SLC25A20 were evaluated using direct viable cell counting. D. Representative colony formation images and quantification of colony numbers for EV cells and A549 and H1299 cells overexpressing SLC25A20. E. Representative scratch healing images and quantification of scratch closure area for EV cells and A549 and H1299 cells overexpressing SLC25A20 (scale bar: 100 μm). F. EV cells and A549 and H1299 cells overexpressing SLC25A20. Representative Transwell invasion images of cells and quantitative data on the number of invading cells, scale bar: 500 μm; Figure 4 Results of the effects of SLC25A20 knockout or overexpression on the in vivo growth of lung adenocarcinoma xenografts and Ki67 expression: A. Tumor growth curves of subcutaneous xenografts derived from control cells and SLC25A20-KO cells, and control cells and SLC25A20-OE cells. Tumor volume was measured weekly after cell injection, n = 6; B. Representative images of ex vivo subcutaneous xenografts in each group, scale bar: 1 cm, n = 6; C. Endpoint tumor weight of xenografts in each group, n = 6; D. Representative immunohistochemical staining and quantification of SLC25A20 and Ki67 in xenografts derived from control cells and SLC25A20-KO cells, scale bar: 50 μm; E. Representative immunohistochemical staining and quantification of SLC25A20 and Ki67 in xenografts derived from control cells and SLC25A20-OE cells, scale bar: 50 μm; Figure 5 RNA sequencing analysis results of changes in the transcriptional program related to lipid metabolism, mitochondrial respiratory chain, oxidative phosphorylation, and ATP synthesis induced by SLC25A20 overexpression: A. Volcano plot of differentially expressed genes between SLC25A20 overexpression group and control A549 cells based on RNA-seq analysis, with red and blue dots representing significantly upregulated and downregulated genes, respectively; B. Number of significantly upregulated and downregulated genes in SLC25A20 overexpression A549 cells compared to control cells; C. Heatmap of representative differentially expressed genes in control and SLC25A20 overexpression A549 cells; D. Enrichment analysis of the gene ontology biological processes of differentially expressed genes in SLC25A20 overexpression A549 cells, showing the most significantly enriched biological processes; E, F. Gene set enrichment analysis showing the enrichment of gene sets related to ATP synthase synthesis and oxidative phosphorylation in SLC25A20 overexpression A549 cells; G. Differentially expressed genes were selected in the control group and SLC25A20-overexpressing A549 cells and verified by qRT-PCR; H. Western blot analysis of FASN and HMOX1 protein expression in the control group and SLC25A20-overexpressing A549 cells, with GAPDH or β-actin as loading controls; Figure 6Results of changes in metabolome profiles, lipid droplet accumulation, ATP levels, oxygen consumption, and ROS levels caused by SLC25A20 overexpression or knockout: A. PCA score plots of metabolomics profiles of control and SLC25A20-overexpressing A549 cells, with each point representing an independent sample; B. Volcano plot showing differentially expressed metabolites between control and SLC25A20-overexpressing A549 cells, with red and blue dots representing significantly increased and decreased metabolites, respectively, and representative differentially expressed metabolites marked; C. Number of significantly increased and decreased metabolites in SLC25A20-overexpressing A549 cells compared to control cells; D. Heatmap showing representative differentially expressed metabolites between control and SLC25A20-overexpressing A549 cells, with metabolite categories and statistical parameters labeled on the right; E. Relative abundance of NADPH and Dephospho-CoA in control and SLC25A20-overexpressing A549 cells; F. G. Representative BODIPY 493 / 503 staining images and quantification of lipid accumulation in SLC25A20 overexpressing cells and SLC25A20 knockout cells relative to their respective control cells; cell nuclei counterstained with DAPI. H. Intracellular ATP levels in control and SLC25A20 overexpressing A549 cells. I. Oxygen consumption curves of control and SLC25A20 overexpressing A549 cells measured by Seahorse extracellular flux analysis. J. Quantification of basal respiration, ATP-related respiration, and maximal respiration calculated from the oxygen consumption curves in I. K. Intracellular ATP levels in control and SLC25A20 knockout cells. L. Oxygen consumption curves of control and SLC25A20 cells measured by Seahorse extracellular flux analysis. Oxygen consumption curves of knockout cells; M. Quantification of basal respiration, ATP production, and maximal respiration calculated from the oxygen consumption curves in L; Figure 7Effects of fatty acid oxidation inhibitor treatment on SLC25A20-related mitochondrial respiration, ATP levels, cell proliferation, colony formation, migration, invasion, and lipid droplet accumulation: A. Intracellular ATP levels in SLC25A20-overexpressing A549 cells treated with solvent or etomoxib; B. Oxygen consumption curves of SLC25A20-overexpressing A549 cells treated with solvent or etomoxib, measured by Seahorse extracellular flux analysis; C–E. Quantification of basal respiration, ATP-related respiration, and maximal respiration calculated from the oxygen consumption curves in B; F. Cell viability of SLC25A20-overexpressing A549 cells at different time points after treatment with solvent or etomoxib; G. Colony formation assay of SLC25A20-overexpressing A549 cells treated with solvent or etomoxib, showing representative images and quantitative colony numbers; H. Scratch healing assay of SLC25A20-overexpressing A549 cells treated with solvent or etomoxib, showing 0 I. Representative images of SLC25A20 cells overexpressing A549 treated with solvent or etomoxicillin and transwell invasion assay, showing representative images and quantification of the number of invading cells per field of view, scale bar: 100 μm; J. Representative BODIPY 493 / 503 stained images of SLC25A20 cells overexpressing A549 treated with solvent or etomoxicillin and quantification of neutral lipid accumulation, with cell nuclei counterstained with DAPI, scale bar: 10 μm; Figure 8 A schematic diagram illustrating how SLC25A20 promotes lung adenocarcinoma progression by coordinating fatty acid oxidation-related mitochondrial energy metabolism, lipid metabolism reprogramming, and redox homeostasis. Figure 9 This is a ROC curve. Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments. These embodiments are only for illustrating the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Equivalent substitutions or conventional improvements made by those skilled in the art based on the disclosure of the present invention regarding the detection platform, sample type, threshold setting method, statistical model, or reagent composition should all fall within the scope of protection of the present invention.

[0025] Example 1 SLC25A20 is highly expressed in lung adenocarcinoma tissues and cells, and its diagnostic / prognostic significance.

[0026] Tumor tissues and matched adjacent normal tissues were collected from patients with pathologically confirmed lung adenocarcinoma. The expression levels of SLC25A20 mRNA (NM_000387.6) and protein (NP_000378.1) were detected by qRT-PCR and Western blot, and the expression of SLC25A20 protein in the tissues was detected by immunohistochemistry. The specific methods are as follows: 1) Sample Collection: A 28:28 matched sample ratio was used for lung adenocarcinoma patients and adjacent non-cancerous tissues, covering pathological stages including IA, IB, IIA, IIB, IIIA, and IIIB. Twenty-eight patients who underwent surgical resection of lung adenocarcinoma (LUAD) at Jiangsu Cancer Hospital between January 2025 and March 2026 were included. Strict inclusion criteria were used: (i) histological confirmation of LUAD by two independent senior pathologists; and (ii) no history of neoadjuvant radiotherapy or chemotherapy preoperatively. For each patient, matched tissue pairs were collected, including primary tumor tissue and adjacent non-cancerous tissue (located at least 2 cm from the tumor margin to ensure clear boundaries). Twenty-eight matched samples were successfully obtained. After resection, tissue specimens were rinsed with sterile saline, immediately rapidly frozen in liquid nitrogen, and stored at -80°C. 2) Western blot assay for proteins Total protein was extracted from adjacent non-tumor tissues and LUAD tissues, and Western blot analysis was performed using SLC25A20 antibody (1:1000, 19363-1-AP, Proteintech) and GAPDH antibody (1:5000, 10494-1-AP, Proteintech) or β-Actin antibody (1:5000, A5441, Sigma). The specific procedure was as follows: primary antibody: incubated overnight at 4°C, secondary antibody: incubated at room temperature for 1 hour, expected 33 kDa.

[0027] 3) qRT-PCR detection Genomic DNA was extracted from adjacent non-tumor tissues and LUAD tissues and qRT-PCR was performed using the primers shown in Table 1. The specific procedure was as follows: 95°C for 3 min; 8 cycles: 98°C for 15 s, 60°C for 15 s, 72°C for 30 s; and finally extended at 72°C for 5 min.

[0028] Table 1: Primer sequences (SEQ ID NO.1-6) 4) Immunohistochemistry For TMA H-score, the expression is scored by pathologists in a blinded manner, which is the optical density level (0 for no brown, 1 for light brown and fine brown, 2 for moderate chromogen deposition, 3 for obvious chromogen deposition) multiplied by the percentage of cells at each staining level, resulting in a total h-score ranging from 0 to 300.

[0029] The results showed that, compared with matched adjacent normal tissues, the expression level of SLC25A20 in lung adenocarcinoma tissues was significantly increased ( Figure 1 Consistent with this, immunohistochemical (IHC) staining of the independent LUAD cohort also showed that SLC25A20 protein levels were significantly elevated in tumor tissues compared to matched non-tumor tissues. Figure 1 C). Although SLC25A20 is documented as a mitochondrial localization protein in the MitoCarta 3.0 database, its specific distribution in LUAD cells has not been characterized. By co-staining SLC25A20 and mitochondria in A549 cells and U2OS cells (a commonly used human osteosarcoma cell line for subcellular localization imaging) using immunofluorescence (IF), we confirmed that SLC25A20 is localized in mitochondria.

[0030] Furthermore, SLC25A20 expression was higher in a series of human LUAD cell lines (H1299, A549, H1975, and H1650) than in the normal human bronchial epithelial cell line (BEAS-2B). Figure 1 D, E).

[0031] Furthermore, survival analysis of public gene chip expression datasets using the online Kaplan-Meier Plotter tool showed that SLC25A20 upregulation was associated with shortened overall survival and progression-free survival in LUAD patients. Figure 1 F).

[0032] Survival analysis using publicly available data on SLC25A20 expression in lung adenocarcinoma patients revealed poorer overall survival and progression-free survival in patients with high SLC25A20 expression. These results suggest that SLC25A20 can serve as a biomarker for the diagnosis, auxiliary diagnosis, and prognostic assessment of lung adenocarcinoma.

[0033] Example 2 The animal experiments were approved by the ethics committee of this institute, approval number IACUC-2604116.

[0034] SLC25A20 expression levels are associated with the risk of malignant progression of lung adenocarcinoma.

[0035] A lung adenocarcinoma model with SLC25A20 knockout was constructed using the CRISPR / Cas9 system to eliminate SLC25A20 expression in H1975 and H1650 cells. Figure 2 A, B), and the levels of SLC25A20 protein and mRNA were effectively eliminated by qRT-PCR and Western blot, respectively. Figure 2 A, B). The specific method is as follows: 1) Cell proliferation experiment To perform a CCK8 cell proliferation assay, lung adenocarcinoma cells (1000 cells / well) were seeded into 96-well plates. After 24 h of growth, 10 μL of CCK8 solution was added to each well, and the plates were incubated at 37°C for 2 h. Cell viability was measured at 490 nm using a microplate reader.

[0036] 2) Transwell migration and invasion experiments Migration and invasion experiments were performed using an 8 μm pore size Transwell laboratory. For invasion assays, the upper cavity was pre-coated with matrix gel. Cells were seeded in serum-free medium in the upper cavity, and medium containing 10% FBS was added to the lower cavity as a chemical inducer. After incubation for 18–30 h, cells that had migrated or invaded to the lower membrane surface were fixed with 4% PFA, stained with 0.5% crystal violet, and photographed under an inverted microscope.

[0037] Functional analysis showed that SLC25A20 knockout significantly inhibited the proliferation and colony formation of H1975 and H1650 cells. Figure 2 C, D). Furthermore, the loss of SLC25A20 significantly impaired the cell migration and invasion abilities of these cells. Figure 2 E, F).

[0038] Considering that inhibition of LUAD growth may stem from cell cycle arrest and / or increased apoptosis, we subsequently used flow cytometry to assess the effects of SLC25A20 knockout on cell cycle distribution and apoptosis. The specific methods are as follows: 3) Cell cycle analysis Cell cycle distribution was analyzed using a cell cycle analysis kit (C-6031, Everbright Pharmaceuticals, USA). In short, LUAD cells with different SLC25A20 expression levels were collected and fixed in 80% cold ethanol at -20°C for at least 2 hours. After washing, the cells were incubated with 0.5 mL of propidium iodide (PI) staining solution in the dark at 4°C for 20 minutes. Cell cycle distribution was then assessed using flow cytometry (Beckman Coulter, Fullerton, CA).

[0039] 4) Apoptosis detection Apoptosis was detected using the Annexin V-FITC / PI apoptosis assay kit (F-6012, Everbright Pharmaceuticals, USA). In short, cells were collected, resuspended in binding buffer, and stained with 5 μL of FITC-conjugated Annexin V and 5 μL of PI in the dark at room temperature for 15 minutes. The apoptosis rate was then analyzed by flow cytometry (Beckman Coulter, Fullerton, CA).

[0040] Notably, SLC25A20 knockout significantly induced G2 / M phase arrest and triggered apoptosis in H1975 and H1650 cells. Furthermore, the detection of activated cleaved-caspase-3 supported the conclusion that SLC25A20 deficiency leads to programmed cell death. Figure 2 (G, H). In summary, these data indicate that SLC25A20 promotes LUAD cell proliferation and inhibits apoptosis in vitro.

[0041] Next, we transfected A549 and H1299 cells with the constructed SLC25A20 overexpression plasmid to overexpress SLC25A20 ( Figure 3 A, B). As shown in the figure, SLC25A20 was overexpressed in A549 and H1299 cells, and compared with the control group, cell proliferation and colony formation were significantly increased. Figure 3 C, D), cell migration and invasion abilities are also enhanced ( Figure 3 (E, F). Given the close association between EMT-related phenotypic changes and cancer cell migration and invasion, we evaluated the effect of SLC25A20 overexpression on the expression of selected EMT-related biomarkers using immunofluorescence staining and Western blot analysis. SLC25A20 overexpression was associated with increased Vimentin expression and decreased E-cadherin expression. These results indicate that SLC25A20 overexpression promotes LUAD cell migration and invasion, accompanied by changes in EMT-like biomarkers, including increased Vimentin expression and decreased E-cadherin expression.

[0042] In a subcutaneous xenograft tumor model in nude mice, 8-week-old male BALB / c nude mice were randomly assigned to experimental groups. A single-cell suspension containing 2 × 10^6 cells (SLC25A20-OE / NC or SLC25A20-KO / NC) (containing 100 μL sterile PBS) was subcutaneously injected into the flank of each mouse. Tumor growth kinetics were monitored every 5 days using digital calipers. At the endpoint, the subcutaneous tumor was excised, photographed, weighed, and fixed in 4% paraformaldehyde for subsequent immunohistochemical analysis. Tumor growth kinetics and endpoint weight showed that knockout of SLC25A20 significantly inhibited the growth of LUAD in H1975 cells. Figure 4Conversely, in A549 cells, SLC25A20 overexpression promoted tumor progression. Immunohistochemical analysis of SLC25A20 and the proliferation marker Ki67 showed that SLC25A20 knockout significantly reduced the percentage of Ki67-positive cells (AC). Figure 4 D). Conversely, SLC25A20 overexpression led to a significant increase in proliferation activity (D). Figure 4 (E). In summary, these data indicate that SLC25A20 lacks the ability to suppress lung tumor growth in vivo, further supporting its oncogene role in LUAD progression. These results suggest that SLC25A20 expression levels can be used to assess the risk of malignant progression in lung adenocarcinoma.

[0043] Example 3 SLC25A20 serves as a stratification marker for lung adenocarcinoma treatment.

[0044] RNA sequencing analysis was performed on SLC25A20-overexpressing lung adenocarcinoma cells. The specific methods are as follows: 1) Reads containing adapter contamination, low-quality bases, and unidentified bases were removed using the fastp software (https: / / github.com / OpenGene / fastp) with default parameters. Sequence quality was validated using fastp. Reads were mapped to the human reference genome (GRCh38) using HISAT2 (https: / / ccb.jhu.edu / software / hisat2). Mapped reads from each sample were assembled using StringTie (https: / / ccb.jhu.edu / software / stringtie) with default parameters. Subsequently, the transcriptomes of all samples were merged using gffcompare (https: / / github.com / gpertea / gffcompare / ) to reconstruct a comprehensive transcriptome.

[0045] After generating the final transcriptome, StringTie was used to estimate mRNA expression levels by calculating Fragments Per Kilobase of Transcript Per Million Mapped Reads (FPKM). The calculation formula is as follows: FPKM = [Total Exon Fragments / Mapped Reads (millions) × Exon Length (kB)] . Differentially expressed mRNAs with fold changes >2 or fold changes <0.5 were selected, and the parameter f-test was performed using R-bounding() to compare nested linear models (p-value <0.05).

[0046] The metabolomics analysis method is as follows: 2) Metabolites were extracted from cell debris by sonication in an ice bath for 1 h with 1 mL of pre-cooled methanol / acetonitrile / water (v / v, 2:2:1). The mixture was incubated at -20℃ for 1 h, centrifuged at 16000 g for 20 min at 4℃, transferred to sample vials, and analyzed by LC-MS by Shanghai Bioprofile Technology Co., Ltd.

[0047] In addition, to ensure the data quality of the metabolic analysis, quality control (QC) samples were prepared by pooling all representative samples from the entire dataset into equal portions, which were then used for data normalization. The preparation and analysis of QC samples used the same procedures as for each batch of experimental samples. The dried extracts were then dissolved in 50% acetonitrile. Each sample was filtered through a disposable 0.22 µm cellulose acetate filter, transferred to a 2 mL HPLC vial, and then stored at -80°C for analysis.

[0048] Principal component analysis (PCA) revealed distinct metabolomics characteristics between the control group and slc25a20 overexpressing cells. Figure 6 A), volcano plot analysis identified differential metabolites between the two groups ( Figure 6 B). Compared with the control group, the slc25a20 overexpression group showed upregulation of 28 metabolites and downregulation of 75 metabolites (B). Figure 6 C). Metabolic pathway enrichment analysis showed that SLC25A20 overexpression was associated with alterations in steroid / cholesterol biosynthesis, lipid synthesis, and FAO-related metabolic pathways. Figure 6 (D, E). To elucidate how enriched pathways can be inferred from metabolomics datasets, we mapped representative differentially expressed metabolites to their corresponding metabolic categories. Cholesterol-related intermediates were localized to steroid / cholesterol biosynthesis, lipid-related metabolites to lipid synthesis or lipid remodeling, and altered acylcarnitine species were interpreted as metabolites associated with z-fatty acid oxidation (Supplementary Table 2). This annotation provides a clearer link between differentially expressed metabolite profiles and pathway-level interpretation of SLC25A20-related metabolic remodeling. Results showed that SLC25A20 overexpression altered transcriptional programs related to lipid metabolism, cholesterol / sterol biosynthesis, mitochondrial respiratory chain, oxidative phosphorylation, and ATP synthesis, and upregulated lipid metabolism-related genes such as FASN, FADS2, SCD, and DHCR7.

[0049] Given the crucial role of SLC25A20 in mitochondrial metabolite transport, we further analyzed functional readouts related to lipid homeostasis and cellular energy status. Notably, dephosphorylated coenzyme A, a key intermediate in coenzyme A biosynthesis and acyl-CoA metabolism, was significantly elevated in cells overexpressing SLC25A20. Figure 6 E), highlighting the robust expansion of the lipid-related cofactor repertoire. NADPH abundance was also increased in cells overexpressing slc25a20, suggesting that reduced equivalence availability may be enhanced ( Figure 6 E).

[0050] To visualize the effect on lipid storage, we performed BODIPY 493 / 503 staining, a fluorescent dye-based method for detecting intracellular neutral lipids and lipid droplets. Consistent with the lipid-promoting gene signature observed in our RNA-seq data, SLC25A20 overexpression increased neutral lipid accumulation in A549 cells. Figure 6 F). Conversely, compared to the corresponding control cells, SLC25A20 gene knockout reduced neutral lipid accumulation (F). Figure 6 F). These results indicate that SLC25A20 expression is associated with altered neutral lipid storage in LUAD cells. Overall, these findings suggest that SLC25A20 drives the lipidoproliferative program, possibly supporting the rapid expansion of LUAD cells by coordinating the availability of metabolic cofactors and the expression of synthetic enzymes.

[0051] Intracellular ATP levels were elevated in cells overexpressing slc25a20 and decreased in SLC25A20-KO cells. Figure 6 H, K). To further assess mitochondrial respiratory activity, we performed a Seahorse assay. The specific method is as follows: 3) Seahorse Oxygen Consumption Rate (OCR) Experiment: Cells were seeded at a density of 30,000 cells per well in Dulbecco's Modified Eagle Medium (DMEM) enriched with 10% fetal bovine serum (FBS) into Seahorse XF-96 cell culture microplates. After 12 h of incubation, the medium was replaced with Seahorse XF Base medium (pH 7.4), and 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine were added. The cells were then incubated at 37°C in a non-CO2 incubator for 1 h to allow pH and temperature equilibration. The basal OCR was recorded for three cycles and normalized to the total protein concentration per well using BCA assay. Data analysis was performed using Wave software (Agilent Technologies).

[0052] SLC25A20 overexpression increased basal and maximal oxygen uptake rates. Figure 6 I, J), while SLC25A20 knockout reduces OCR (I, J), while SLC25A20 knockout reduces OCR ( Figure 6 L, M), supporting the role of SLC25A20 in enhancing mitochondrial energy metabolism.

[0053] Since alterations in mitochondrial lipid metabolism also affect cellular redox status, we further measured total cellular ROS levels. ROS levels were decreased in SLC25A20-overexpressing cells, while increased in SLC25A20-KO cells. Figure 6 (G). These data indicate that SLC25A20 expression is associated with changes in cellular redox state. Combined with ATP (ATPAssay Kit, G4309-96T, Servicebio) measurements, ATP levels normalized to viable cell number, and OCR measurements, these findings suggest that SLC25A20 influences mitochondrial bioenergy output and redox homeostasis; however, total cellular ROS should be interpreted as a distinct functional reading rather than a direct result of ATP production.

[0054] In summary, these findings indicate that SLC25A20 overexpression alters the lipid-related metabolite profile, increases neutral lipid accumulation, enhances ATP and oxygen consumption, and reduces ROS levels; while SLC25A20 knockout reduces ATP and oxygen consumption and increases ROS.

[0055] SLC25A20-overexpressing cells and lung adenocarcinoma cells with high endogenous SLC25A20 levels were treated for 12 h with 10 μM (final concentration) of the fatty acid oxidation inhibitor (etomoxir HY-50202) and a control (e.g., an equal volume of DMSO). Intracellular ATP levels were first measured using a bioluminescent ATP assay. SLC25A20 overexpression increased intracellular ATP abundance, while etomoxicin significantly reduced ATP levels in SLC25A20-overexpressing A549 cells. Figure 7 A). These findings suggest that the enhanced bioenergetic activity driven by SLC25A20 depends at least in part on metabolism related to fatty acid oxidation. Consistent with this, etomoxib also inhibited ATP-related metabolic activity and cell growth in A549 cells, although the inhibition was more pronounced in an environment of slc25a20 overexpression.

[0056] Next, we used hippocampal extracellular flux analysis to examine mitochondrial respiratory activity. Etomoxicillin treatment significantly reduced oxygen consumption (OCR) in slc25a20-overexpressing A549 cells, and decreased basal respiration, ATP-related respiration, and maximal respiration. Figure 7 These results indicate that inhibition of cpt1-dependent FAO impairs the mitochondrial respiratory advantage derived from SLC25A20 overexpression. Simultaneously, etomoxicin reduced the OCR and respiratory parameters of parental A549 cells, further supporting the contribution of FAO-related mitochondrial respiration to the bioenergetics of LUAD cells under basal conditions (Figure S6).

[0057] Functionally, etomoxifen attenuated the slc25a20-driven malignant phenotype in A549 cells. In cells overexpressing slc25a20, etomoxifen reduced cell viability, colony formation, wound closure, and Matrigel invasion. Figure 7 f-1). BODIPY 493 / 503 staining further showed that etomoxifen reduced neutral lipid accumulation in Slc25a20-overexpressing cells, supporting the involvement of FAO-related lipid metabolic remodeling in this phenotype. Figure 7 (J). Similar inhibitory effects on proliferation, clonal growth, migration, invasion, and neutral lipid accumulation were also observed after A549 cells were treated for 12 h with a 10 μM fatty acid oxidation inhibitor (etomoxicillin) and a control (e.g., an equal volume of DMSO). Notably, etomoxicillin attenuated, but did not completely eliminate, the SLC25A20-related phenotype, suggesting that SLC25A20 may promote LUAD progression through a fAO-dependent metabolic mechanism as well as other metabolic mechanisms.

[0058] To further evaluate the effectiveness of FAO inhibition in an environment with high endogenous SLC25A20 levels, we treated H1975 cells with a 5 μM (final concentration) fatty acid oxidation inhibitor (etomoxicillin) and a control (equal volume of DMSO). In our cell line screening, H1975 cells showed high expression of endogenous SLC25A20. Similar to the results in A549 cells overexpressing SLC25A20, etomoxicillin reduced OCR, cell proliferation, colony formation, migration, invasion, and neutral lipid accumulation in H1975 cells (Figure S6). These results indicate that the inhibitory effect of FAO is not limited to the overexpression model but can also be observed in LUAD cells with relatively high endogenous SLC25A20 expression.

[0059] Given that etomoxicillin targets CPT1-dependent fatty acid entry into mitochondria, and that SLC25A20 mediates acylcarnitine / carnitine exchange across the mitochondrial inner membrane, these data should be interpreted as evidence of involvement in fatty acid oxidation-related metabolism, rather than proof that all SLC25A20-driven effects are entirely dependent on fatty acid oxidation. Combined with the reduced ROS accumulation observed with SLC25A20 overexpression, these results suggest that SLC25A20 supports the progression of LUAD by coordinating mitochondrial energy production, lipid metabolic remodeling, and redox homeostasis. Figure 8 ).

[0060] The results showed that fatty acid oxidation inhibition reduced oxygen consumption, ATP levels, cell proliferation, colony formation, migration, invasion, and lipid droplet accumulation. These results indicate that high SLC25A20 expression can be used to identify lung adenocarcinoma patients with active mitochondrial energy metabolism associated with fatty acid oxidation, and for treatment stratification, particularly for screening patients suitable for fatty acid oxidation inhibition, mitochondrial metabolism inhibition, or SLC25A20-related metabolic targeted interventions.

[0061] Example 4 Detection kit based on SLC25A20 expression level.

[0062] A kit for the diagnosis, prognostic assessment, risk assessment of malignant progression, and / or treatment stratification of lung adenocarcinoma, comprising reagents for detecting SLC25A20 mRNA and / or protein expression levels.

[0063] In one embodiment, the kit includes qRT-PCR primers or probes for detecting SLC25A20 mRNA, reverse transcription reagents, amplification reagents, internal reference gene detection primers or probes, a positive control, a negative control, and instructions. By detecting the expression level of SLC25A20 in the test sample relative to the internal reference gene and comparing it with a control or a preset threshold, the diagnosis, prognostic risk, or treatment stratification of lung adenocarcinoma can be determined.

[0064] In another embodiment, the kit includes an antibody, a secondary antibody, a chromogenic reagent or a fluorescent labeling reagent, a blocking buffer, a washing buffer, a positive control, a negative control, and instructions for use in detecting SLC25A20 protein. The expression level of SLC25A20 protein is detected by immunohistochemistry, immunofluorescence, Western blot, ELISA, or chemiluminescence, and the result is determined based on the expression intensity or scoring.

[0065] Example 5 A screening method for candidate therapies for lung adenocarcinoma based on SLC25A20.

[0066] Establish a lung adenocarcinoma cell model with high SLC25A20 expression or select endogenous lung adenocarcinoma cells with high SLC25A20 expression. Apply the test drug or treatment regimen to the cells and detect SLC25A20 expression levels, cell proliferation, colony formation, migration, invasion, apoptosis, ATP levels, oxygen consumption, lipid droplet accumulation, ROS levels, and / or fatty acid oxidation-related indicators.

[0067] When a test drug or treatment regimen can reduce the proliferation, colony formation, migration, invasion, ATP levels, oxygen consumption, lipid droplet accumulation, or tumorigenic capacity of SLC25A20-overexpressing lung adenocarcinoma cells, or increase ROS and / or apoptosis, it can be identified as a candidate anti-lung adenocarcinoma drug or treatment regimen. This method can be used to screen fatty acid oxidation inhibitors, mitochondrial metabolism inhibitors, SLC25A20-related metabolic pathway inhibitors, and their combination therapies.

[0068] To further evaluate the efficacy of SLC25A20 as a diagnostic biomarker for lung adenocarcinoma, this invention performed ROC curve analysis based on an independent validation cohort (containing 60 lung adenocarcinoma tissue samples and 60 paired adjacent normal tissue samples). Figure 9 As shown, the area under the ROC curve (AUC) for SLC25A20 expression level in distinguishing lung adenocarcinoma tissue from adjacent non-cancerous tissue was 0.957 (95% confidence interval: 0.904–1.000, P < 0.001), indicating its extremely high diagnostic accuracy. The optimal cut-off value was determined to be 0.424 based on the Youden Index. At this threshold, the diagnostic sensitivity was 95.7% and the specificity was 91.0%. When the SLC25A20 expression level in a subject's sample was ≥0.424, the subject could be identified as a lung adenocarcinoma patient or a high-risk individual for lung adenocarcinoma; when the SLC25A20 expression level was <0.424, the subject could be identified as a non-lung adenocarcinoma patient or a low-risk individual for lung adenocarcinoma. These results indicate that SLC25A20 has good diagnostic value for lung adenocarcinoma.

[0069] In this embodiment, the SLC25A20 expression level used for comparison with 0.424 is a relative expression level calculated using the following method: 1) Obtain the cycle threshold (Ct value) of SLC25A20 and the internal reference gene GAPDH in subject samples (lung adenocarcinoma tissue or adjacent normal tissue); 2) Calculate the ΔCt value for each sample using the formula: ΔCt = Ct(SLC25A20) - Ct(GAPDH); 3) Calculate the control ΔCt value for standardization (preferably using a set of known average ΔCt values ​​from non-cancerous or adjacent normal tissues as the control value); 4) Calculate the ΔΔCt value using the formula: ΔΔCt = ΔCt(subject sample) - ΔCt(control); 5) Calculate the relative expression level using the formula: Relative expression level = 2^(-ΔΔCt).

[0070] Finally, the relative expression level was compared with the optimal cutoff value of 0.424. If the relative expression level was ≥0.424, the subject was determined to have an increased risk of lung adenocarcinoma or be a lung adenocarcinoma patient.

[0071] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. Application of reagents for detecting SLC25A20 expression levels in the preparation of products for the diagnosis, prognostic assessment, risk assessment of malignant progression, and / or treatment stratification of lung adenocarcinoma.

2. The application according to claim 1, characterized in that, The SLC25A20 expression level includes the SLC25A20 mRNA expression level and / or the SLC25A20 protein expression level.

3. The application according to claim 1 or 2, characterized in that, The product is used to compare the expression level of SLC25A20 in subject samples with normal controls, adjacent normal tissue controls, healthy population controls, or a preset threshold. When the expression level of SLC25A20 is elevated, it indicates to the subject: (i) Increased risk of developing lung adenocarcinoma, poor prognosis of lung adenocarcinoma, and / or higher risk of malignant progression; and / or (ii) Suitable for treatment stratification assessment.

4. The application according to any one of claims 1-3, characterized in that, The samples were selected from lung adenocarcinoma tumor tissue, adjacent tissue, lung tissue biopsy samples, puncture samples, surgically resected samples, cytological samples, blood, serum, plasma, exosomes, circulating tumor cells, pleural effusion, sputum, or bronchoalveolar lavage fluid.

5. The application according to any one of claims 1-4, characterized in that, The reagents are selected from: (a) Primers, probes, microarray probes, sequencing adapters, digital PCR reagents, reverse transcription reagents, or amplification reagents used to detect SLC25A20 mRNA; and / or (b) Antibodies, antibody fragments, immunohistochemical reagents, immunofluorescence reagents, enzyme-linked immunosorbent assay (ELISA) reagents, chemiluminescence assay reagents, or mass spectrometry assay reagents used to detect SLC25A20 protein; The products mentioned include reagent kits, chips, detection systems, diagnostic models, prognostic assessment models, or treatment stratification models.

6. The application according to any one of claims 1-5, characterized in that, The prognostic assessment includes evaluating overall survival, progression-free survival, risk of recurrence, risk of metastasis, risk of tumor proliferation, risk of invasion, and / or risk of tumorigenic capacity.

7. The application according to any one of claims 1-6, characterized in that, The treatment stratification includes screening lung adenocarcinoma patients suitable for metabolic intervention therapy; the metabolic intervention therapy is selected from fatty acid oxidation inhibition therapy, mitochondrial oxidative phosphorylation inhibition therapy, lipid metabolism intervention, SLC25A20-related metabolic pathway intervention, chemotherapy combined with metabolic intervention, targeted therapy combined with metabolic intervention, or immunotherapy combined with metabolic intervention.

8. The application according to claim 7, characterized in that, The fatty acid oxidation inhibition therapy includes treatment that inhibits CPT1-dependent fatty acid entry into mitochondria, and / or treatment that inhibits SLC25A20-mediated acylcarnitine / carnitine exchange-related metabolic processes.

9. A kit for the diagnosis, prognostic assessment, risk assessment of malignant progression, and / or treatment stratification of lung adenocarcinoma, characterized in that, The kit includes reagents for detecting SLC25A20 expression levels, as well as at least one complementary component selected from internal control reagents, sample processing reagents, standards and instructions.

10. A method for screening candidate therapeutic drugs or treatment regimens for lung adenocarcinoma, characterized in that, Includes the following steps: To apply the test drug or treatment regimen to lung adenocarcinoma cells or samples that highly express SLC25A20; The assays were selected from one or more of the following indicators: SLC25A20 expression level, cell proliferation, colony formation, migration, invasion, apoptosis, ATP level, oxygen consumption rate, lipid droplet accumulation, ROS level, and fatty acid oxidation-related indicators. Drugs or treatment regimens that can reduce SLC25A20-related malignant or metabolic phenotypes compared to untreated controls are considered as candidate treatments.

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