Use of glutaroyl-coa dehydrogenase in inducing ferroptosis of liver cancer cells

Abstract

The application provides application of glutaroyl-CoA dehydrogenase in inducing ferroptosis of liver cancer cells, and belongs to the technical field of biotechnology.The application provides application of glutaroyl-CoA dehydrogenase (GCDH) in preparing products for evaluating risk of liver cancer and evaluating prognosis risk of liver cancer, and the tumor stage and T grade can be estimated by detecting the expression amount of GCDH, and the survival effect of liver cancer patients is evaluated.The application provides application of the overexpression reagent of GCDH in preparing products for inhibiting proliferation of liver cancer cells and inducing ferroptosis of liver cancer cells, and the ferroptosis of liver cancer cells is promoted by overexpressing GCDH in cells, and the colony formation ability of liver cancer cells is reduced.Further, the application provides application of the overexpression reagent of GCDH combined with a CA9 inhibitor in preparing products for inhibiting proliferation of liver cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of glutaryl-CoA dehydrogenase in inducing ferroptosis in liver cancer cells. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the malignant tumors with the highest incidence and mortality rates worldwide. It has an insidious onset and rapid progression, with many patients diagnosed at an advanced stage, missing the optimal window for radical surgical treatment. Currently, clinical treatments for HCC mainly include surgical resection, radiotherapy, chemotherapy, and targeted therapy. However, traditional radiotherapy and chemotherapy suffer from poor specificity and strong toxic side effects. Targeted drugs, such as sorafenib and lenvatinib, can easily lead to drug resistance in tumor cells with long-term use, significantly reducing treatment efficacy and making long-term effective disease control difficult. Therefore, finding new therapeutic targets for HCC and developing novel and efficient treatment strategies have become crucial issues that urgently need to be addressed in the field of HCC research.

[0003] Ferroptosis is a programmed cell death mechanism distinct from apoptosis, autophagy, and necrosis. Its core characteristic is the abnormal accumulation of iron-dependent lipid peroxidation, ultimately leading to cell structural damage and death. Ferroptosis is closely related to iron metabolism imbalance, lipid metabolism disorder, and antioxidant system failure. Among these, the glutathione peroxidase 4 (GPX4)-mediated antioxidant pathway is a key step in inhibiting ferroptosis, while the cystine / glutamate antitransporter (System Xc) plays a crucial role. - The GPX4 complex (composed of SLC7A11 and SLC3A2) indirectly maintains GPX4 activity by regulating cystine uptake and glutathione (GSH) synthesis, thereby inhibiting lipid peroxidation and ferroptosis. However, recent studies have revealed that ferroptosis can also be regulated through various other pathways, leaving many aspects unresolved. Recent research has confirmed that ferroptosis is involved in the occurrence, development, and drug resistance of liver cancer, inducing ferroptosis in liver cancer cells, effectively killing tumor cells, especially showing significant inhibitory effects on drug-resistant liver cancer cells, providing a new research direction and potential strategy for liver cancer treatment.

[0004] Glutaryl-CoA dehydrogenase (GCDH) is a metabolic enzyme located in the mitochondrial matrix. Its classic physiological function is to participate in the catabolism of lysine, hydroxylysine, and tryptophan, catalyzing the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, thus maintaining normal metabolic balance in the body. Previous studies have shown that GCDH gene mutations lead to reduced or absent GCDH activity, causing type I glutamate hyperacidity and resulting in the accumulation of abnormal metabolites such as glutamate and 3-hydroxyglutamate in the body. This primarily damages the central nervous system, manifesting as macrocephaly and progressive abnormal muscle tone. To date, there are no reports in existing technologies regarding an association between GCDH and ferroptosis in liver cancer cells. Summary of the Invention

[0005] To address the problems existing in the prior art, the primary objective of this invention is to provide a detection reagent for glutaryl-CoA dehydrogenase for use in the preparation of products for assessing liver cancer risk and prognostic risk.

[0006] The second objective of this invention is to provide the application of an overexpression reagent of glutaryl-CoA dehydrogenase in the preparation of products that inhibit the proliferation of liver cancer cells and induce ferroptosis in liver cancer cells.

[0007] A third objective of this invention is to provide the application of an overexpression reagent of glutaryl-CoA dehydrogenase combined with a CA9 inhibitor in the preparation of a product that inhibits the proliferation of liver cancer cells.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of a reagent for detecting glutaryl-CoA dehydrogenase in the preparation of products for assessing liver cancer risk.

[0009] This invention provides the application of a reagent for detecting glutaryl-CoA dehydrogenase in the preparation of products for assessing the prognostic risk of liver cancer.

[0010] This invention provides the application of an overexpression reagent of glutaryl-CoA dehydrogenase in the preparation of products that inhibit the proliferation of liver cancer cells.

[0011] Preferably, overexpression of glutaryl-CoA dehydrogenase reduces the clonogenic ability of liver cancer cells.

[0012] This invention provides the application of an overexpression reagent of glutaryl-CoA dehydrogenase in the preparation of a product that induces ferroptosis in liver cancer cells.

[0013] Preferably, glutaryl-CoA dehydrogenase promotes the degradation of SLC7A11.

[0014] Preferably, glutaryl-CoA dehydrogenase downregulates ferroptosis inhibitor CA9.

[0015] This invention provides the application of an overexpression reagent of glutaryl-CoA dehydrogenase combined with a CA9 inhibitor in the preparation of a product that inhibits the proliferation of liver cancer cells.

[0016] Preferably, glutaryl-CoA dehydrogenase combined with CA9 inhibitors reduces the cell viability of liver cancer cells.

[0017] Preferably, the overexpression reagent for glutaryl-CoA dehydrogenase includes an overexpression vector for glutaryl-CoA dehydrogenase.

[0018] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention, by exploring the application of GCDH in inducing ferroptosis in hepatocellular carcinoma cells, not only fills a technological gap but also provides a novel, highly specific, efficient, low-toxicity, and easily convertible strategy for treating liver cancer. It effectively solves key technical challenges in existing liver cancer treatments and possesses significant clinical application value, social benefits, and economic benefits. Independent of traditional apoptosis and autophagy pathways, it can effectively kill liver cancer cells resistant to chemotherapy and targeted drugs, showing significant therapeutic potential, especially for advanced drug-resistant liver cancer. It can significantly improve the efficacy of liver cancer treatment and provide new treatment options for patients with advanced liver cancer. Attached Figure Description

[0019] Figure 1 Results of GCDH expression levels in hepatocellular carcinoma.

[0020] Figure 2 Decreased GCDH levels in HCC are associated with poor prognosis.

[0021] Figure 3 Results of GCDH inhibiting cell proliferation in vitro.

[0022] Figure 4 The result of GCDH overexpression inducing ferroptosis in HCC cells.

[0023] Figure 5 : expression profiles of ferroptosis-related proteins and genes.

[0024] Figure 6 GCDH inhibits ferroptosis in HCC by regulating CA9.

[0025] Figure 7 The interaction between GCDH and CA9 / SLC7A11 promotes ferroptosis in hepatocellular carcinoma cells. Detailed Implementation

[0026] This invention provides the application of a reagent for detecting glutaryl-CoA dehydrogenase (GCDH) in the preparation of products for assessing liver cancer risk. In liver cancer cells, both the protein and mRNA expression levels of GCDH are significantly reduced. Furthermore, GCDH expression levels are correlated with tumor stage and T grade; with increasing tumor stage and grade, GCDH expression levels show a decreasing trend.

[0027] This invention provides the application of GCDH detection reagents in the preparation of products for assessing the prognostic risk of hepatocellular carcinoma (HCC). GCDH has good diagnostic value in the development and prognosis of HCC. Decreased GCDH expression is associated with poorer survival in HCC patients, and a decrease in GCDH expression levels indicates a worse prognosis for HCC patients.

[0028] In this invention, the GCDH detection reagent includes reagents for detecting GCDH expression levels, including reagents for detecting GCDH protein expression levels or GCDH mRNA expression levels. As an optional embodiment, the GCDH protein expression level detection reagent includes a Western blot (WB) reagent; as an optional embodiment, the GCDH mRNA expression level detection reagent includes RNA-Seq sequencing-related reagents and RNA extraction and quantitative RT-PCR detection-related reagents. The GCDH primers in this invention are: upstream primer: 5'-ACTGAGATTACCCTGGGCCT-3' (SEQ ID No. 1); downstream primer: 5'-GTGAATGTCATGTGTACCTTCGT-3' (SEQ ID No. 2); the β-actin primers are: upstream primer: 5'-CTCCATCCTGGCCTCGCTGT-3' (SEQ ID No. 3); downstream primer: 5'-GCTGTCACCTTCACCGT TCC-3' (SEQ ID No. 4). The PCR reaction system consisted of: ddH2O 3.2µL, Primer-F (10µM) 0.4µL, Primer-R (10µM) 0.4µL, cDNA template 1µL, and 2×PowerUP SYBR GreenMaster Mix 5µL.

[0029] This invention provides the application of GCDH overexpression reagents in the preparation of products that inhibit the proliferation of liver cancer cells, wherein GCDH overexpression can reduce the clonogenic ability of liver cancer cells.

[0030] This invention provides the application of GCDH overexpression reagents in the preparation of products inducing ferroptosis in hepatocellular carcinoma cells. The GCDH overexpression induces a significant accumulation of lipid-derived ROS and increases the content of malondialdehyde (MDA), one of the final products of excessive lipid oxidation, accompanied by a significant depletion of GSH. GCDH induces ferroptosis in hepatocellular carcinoma cells by promoting SLC7A11 degradation and by downregulating the ferroptosis inhibitor CA9. GCDH regulates the expression of CA9 / SLC7A11, thereby modulating the subsequent ferroptosis susceptibility of hepatocellular carcinoma cells and promoting ferroptosis.

[0031] This invention provides the application of GCDH overexpression reagents combined with CA9 inhibitors in the preparation of products that inhibit the proliferation of hepatocellular carcinoma cells. This invention discovers that there is a physical interaction between GCDH and CA9 (through hydrogen bonds and salt bridges mediated by specific residues). Since CA9 participates in the regulation of pHi by producing bicarbonate, CA9 inhibitors (such as SLC-0111) can lead to pHi acidification. Therefore, GCDH overexpression combined with a CA9 inhibitor (such as SLC-0111) can induce acidification-induced cell death, thereby reducing the cell viability of hepatocellular carcinoma cells.

[0032] Preferably, the GCDH overexpression reagent of the present invention includes a GCDH overexpression vector. As an optional embodiment, the GCDH overexpression vector of the present invention is formed by inserting the cDNA sequence (NM_000159.4) of the GCDH gene into the PCDNA3.1(+) plasmid. Hind III and Xho The I site was prepared.

[0033] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0034] In the following examples, all data are expressed as Mean ± SEM. Graph Pad Prism 8.0 software was used for analysis. Significance differences were determined using unpaired t-tests, one-way ANOVA, or two-way ANOVA, followed by Turkey's multiple comparison test. A p-value < 0.05 was considered statistically significant.

[0035] Unless otherwise specified, the following embodiments are all conventional methods.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] Example 1 1. Experimental steps: (1) Clinical sample sources and analysis: Gene expression and clinical annotation data were downloaded from the TCGA database (including 50 normal tissues and 368 hepatocellular carcinoma (HCC) tumor tissues), the GSE14520 dataset (including 221 normal tissues and 225 HCC tumor tissues), and the ICGC database (including 202 normal tissues and 230 HCC tumor tissues), respectively. Immunohistochemical (IHC) detection data were obtained from the HPA database.

[0038] (2) Diagnostic value analysis and KM survival analysis: Receiver operating characteristic (ROC) curves were plotted using GraphPad Prism 8.0, and the diagnostic value of mitochondrial lipid metabolism genes in normal and cancerous tissues was evaluated based on the area under the curve (AUC). KM survival analysis plots were generated in R using the "survival" package.

[0039] (3) Construction of nomogram: Univariate and multivariate Cox regression analyses were used to incorporate GCDH expression and clinical data (including stage, TNM stage, sex, age, and tumor grade) into independent prognostic validation. Then, a nomogram model was constructed based on multivariate Cox regression analysis, and the performance of the nomogram was evaluated by plotting the area under the ROC curve and DCA under decision curve analysis.

[0040] (4) Protein-protein docking: The 3D structures of GCDH (PDB ID: 1SIQ) and CA9 (PDB ID: 6RQN) were retrieved from the Protein Database (PDB). Monomeric structures were submitted to HawkDock Server V2 (https: / / cadd.zju.edu.cn / hawkdock / ) for docking simulation and prediction of complex 3D structures. The generated models were analyzed using the PDBePISA online tool (https: / / www.ebi.ac.uk / pdbe / pisa / ) to characterize the interactive interface and visualize the data.

[0041] (5) Plasmid construction and transfection: The cDNA sequence of the GCDH gene (NM_000159.4) was inserted into the PCDNA3.1(+) plasmid. Hind III and Xho The plasmid was then transferred to the I site, followed by transformation of the ligation product into the DH5α strain. Single colony isolates were subsequently selected from LB agar plates containing penicillin. To verify successful plasmid construction, these colonies were subjected to colony PCR, restriction enzyme digestion, and sequencing. Cells were transfected in 6-well plates using Lipofectamine 3000 (Invitrogen, USA). Cells were collected 48 hours post-transfection for subsequent experiments. Cells transfected with the empty vector served as a control. Overexpression of the GCDH protein was verified by qRT-PCR and Western blot analysis.

[0042] (6) Cell line and culture conditions: Human hepatocyte cell line THLE-2, hepatocellular carcinoma cell lines HCCLM3, Huh7, and HepG2 were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin under 5% CO2 conditions.

[0043] (7) Proliferation and colony formation experiments: Cell viability was assessed using the Cell Counting Kit (Elabscience, Wuhan, China), strictly following the instructions. Cells were seeded into 96-well plates. The plates were incubated overnight in a CO2 incubator until approximately 70% confluence was achieved, at which point transient transfection was performed. Cell viability was measured at 24, 48, 72, and 92 hours post-transfection. 10 µL of CCK8 reagent and 90 µL of serum-free medium were added to each well, and the plates were incubated for 1 hour. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated according to the kit instructions.

[0044] (8) Cloning: Logarithmic growth phase cells were seeded at 2000 cells per well in 6-well plates. After transfection and culture for 14 days, cells were fixed with 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet for 15 min, rinsed, dried, and photographed. Cell clone counts were quantified using ImageJ software and plotted using GraphPad software. A p-value < 0.05 was considered statistically significant.

[0045] (9) ROS detection: Cells were transfected into 6-well or 96-well plates for 48 hours to induce GCDH overexpression. Cells were then incubated with DMEM containing 10 µM DCFH-DA at 37°C for 30 minutes. Fluorescence microscopy (EVOS® FL Auto, USA) and a microplate reader (BioTekGene5, USA) were used to acquire fluorescence images.

[0046] (10) Determination of reduced GSH / GSSH and MDA: Cell lysates were obtained using RIPA lysis buffer (Beyotime, China), and reduced glutathione levels were measured using a reduced glutathione assay kit (Beyotime, China). MDA was detected using a lipid peroxidation MDA assay kit (Beyotime, China).

[0047] (11) PHI measurement: Cells were seeded in 96-well plates (3000 cells per well), and after adhesion, transfected for 6 h. Then, SLC-0111 (a carbonic anhydrase IX / XII (CA9 / CA12) inhibitor) was added, and the cells were incubated for 48 h. pHi was measured using the BCECF AM fluorescence pH Assay Kit (Servicebio, China). After removing the growth medium, cells were loaded with 100 μl of BCF-AM assay working solution and incubated at 37°C for 30 min. The ratio was then measured using a microplate reader at EX / EM = 490 / 530 nm: EX / EM = 440 / 530 nm. A calibration curve was prepared using pH calibration buffer containing 10 μM nigrain (pH 6.0–8.0; 0.5 pH increments). An inverted 4PL nonlinear regression model was used to fit the calibration curve, and the experimental pHi values ​​were interpolated.

[0048] (12) RNA-Seq sequencing: Control and pCDNA-GCDH cells were used to identify differentially expressed mRNAs after GCDH overexpression. All three samples were washed twice with PBS, 1 mL of Trizol was added, and cells were collected into 1.5 mL centrifuge tubes. The cells were immediately flash-frozen in liquid nitrogen and sent to Sangon Biotech Co., Ltd. for mRNA sequencing. The processed transcriptome sequencing data were used for bioinformatics analysis, with |logFC|>1 and P<0.05 defined as significantly differentially expressed genes.

[0049] (13) RNA extraction and quantitative RT-PCR: Total RNA was extracted from cells using the RNA Easy Fast Tissue / Cell Kit (TIANGEN BIOTECH, Beijing, China) and reverse transcribed into cDNA using the RevertAid RT Kit (Thermo Scientific, USA). The cDNA was then analyzed using quantitative real-time PCR. GCDH primers: Upstream primer: 5'-ACTGAGATTACCCTGGGCCT-3' (SEQ ID No. 1); Downstream primer: 5'-GTGAATGTCATGTGTACCTTCGT-3' (SEQ ID No. 2); β-actin primers: Upstream primer: 5'-CTCCATCCTGGCCTCGCTGT-3' (SEQ ID No. 3); Downstream primer: 5'-GCTGTCACCTTCACCGT TCC-3' (SEQ ID No. 4). Reaction system: ddH2O 3.2µL, Primer-F (10µM) 0.4µL, Primer-R (10µM) 0.4µL, cDNA template 1µL, 2×PowerUP SYBR Green MasterMix 5µL. mRNA levels were normalized to β-actin (as internal reference) according to a 2... -ΔΔCT The method is calculated. Multiple independent samples are used (N≥3), and each sample is analyzed repeatedly.

[0050] (14) Immunoprecipitation: Cell precipitates were collected by centrifugation using RIPA lysis buffer. Following the instructions of the Protein G Agarose Gel (Beyotime, China) kit, the precipitated proteins were subjected to SDS-PAGE.

[0051] (15) Western blotting detection: Transfected cells were collected, RIPA lysis buffer was added, and the cells were incubated on ice for 30 minutes to lyse them. Protein concentration was determined using the BCA protein assay (Beyotime, China). Protein samples were separated by SDS-PAGE electrophoresis, transferred to a membrane for 1.5 h, and then blocked by incubation in TBST buffer with 5% skim milk for 2 h at room temperature. The membrane was incubated with GCDH antibody (1:500), NRF2 (1:500), GPX4 (1:500), and GAPDH antibody (1:5000) at 4°C for 15 h, washed three times with TBST, incubated with secondary antibody (1:5000) at room temperature for 2 h, washed three times with TBST, and then visualized using ECL chemiluminescence. Gray-scale analysis of the bands was performed using ImageJ software.

[0052] 2. Experimental Results: (1) GCDH is expressed at low levels in liver cancer: Samples from the database were analyzed, and the expression levels of GCDH in normal adult hepatic epithelial THLE-2 cells and HCCLM3, HepG2, and Huh7 hepatocellular carcinoma cells were detected using qRT-PCR and Western blotting. The results of GCDH expression in hepatocellular carcinoma (HCC) are as follows: Figure 1 As shown in the figure, (A) differential expression of GCDH in tumor tissues from the TCGA database. (B) differential expression of GCDH in tumor tissues from the GEO database. (C) differential expression of GCDH in tumor tissues from the ICGC database. (D) differential expression of GCDH in tumor tissues at the protein level using immunohistochemical data from the HPA database. (E) qRT-PCR detection of GCDH mRNA expression in normal adult liver epithelial THLE-2 cells and HCCLM3, HepG2, and Huh7 hepatocellular carcinoma cells. (F) Western blotting detection of GCDH protein expression in normal hepatocytes and hepatocellular carcinoma cells. (G) Statistical results of Western blotting.

[0053] The results showed that GCDH mRNA levels were downregulated in seven tumor types compared to normal tissues: BLCA (Bladder Urothelial Carcinoma), ESCA (Esophageal Carcinoma), LIHC (Liver Hepatocellular Carcinoma), LUAD (Lung Adenocarcinoma), OV (Ovarian Serous Cystadenocarcinoma), STAD (Stomach Adenocarcinoma), and THCA (Thyroid Carcinoma). Furthermore, GCDH expression was also downregulated in the GSE14520 and ICGC datasets. Immunohistochemical data from the HPA database further supported the downregulation of GCDH expression in HCC tissues at the protein level. Similarly, in various human HCC cell lines (HCCLM3, HepG2, and Huh7), the mRNA and protein expression levels of GCDH were significantly lower than those in normal adult hepatic epithelial THLE-2 cells. Furthermore, GCDH expression levels correlated with tumor stage and T grade, but decreased with increasing tumor stage and grade. In conclusion, these results indicate that GCDH is expressed at low levels in HCC.

[0054] (2) Decreased GCDH expression is associated with shorter survival in patients with hepatocellular carcinoma (HCC). Analysis of GCDH expression and clinical data in samples from the database revealed that decreased GCDH levels in HCC were associated with poor prognosis. Figure 2 As shown in the figure. (A) Receiver operating characteristic (ROC) curve analysis of GCDH in HCC patients. (B) Kaplan-Meier analysis comparing overall survival (OS) in patients with high and low GCDH expression. (C) Univariate Cox regression analysis of clinicopathological factors associated with OS in HCC. (D) Multivariate analysis of prognostic factors such as GCDH expression. (E) Nonograph calibration curves for GCDH expression and M stage in the TCGA-LIHC cohort. (F) Nonograph calibration curves, predictive probability (x-axis) versus observed frequency (y-axis). The x-axis represents the prognostic probability (0–100%), and the y-axis represents the observed OS (0–100%).

[0055] ROC analysis showed that GCDH had good diagnostic value for HCC (AUC = 0.8023) (A). Decreased GCDH expression was associated with worse survival in patients with hepatocellular carcinoma (B). In univariate analysis, decreased GCDH expression was significantly associated with poorer overall survival (HR = 1.989, 95% CI = 1.405–2.816, p < 0.001) (C). This association persisted after multivariate adjustment for tumor stage, T, and M (HR = 1.710, 95% CI = 1.179–2.841, p = 0.005), establishing GCDH as an independent prognostic biomarker (D). The nomogram incorporated GCDH expression and M stage to predict 5-year OS for HCC (C-index: 0.72) (E). Calibration curves showed good agreement between the predicted survival probability of the nomogram and the expected value of the ideal model (F). In conclusion, these results indicate that GCDH can serve as a prognostic biomarker for HCC.

[0056] (3) Overexpression of GCDH inhibits cell proliferation To investigate the role of GCDH in liver cancer, GCDH-overexpressing Huh7 and HepG2 cell lines were constructed by transient plasmid transfection, and RT-qPCR, WB, CCK-8 assay, and colony formation assay were performed for detection.

[0057] Results of GCDH inhibiting cell proliferation in vitro: Figure 3 As shown in the figure. (A) RT-qPCR detection of GCDH expression in Huh7 and HepG2 cells. (B) Western blot detection of GCDH expression in Huh7 and HepG2 cells. (C) CCK-8 assay detection of viability of Huh7 and HepG2 cells overexpressing GCDH. (D) Clonogenesis assay to detect the effect of GCDH overexpression on cell proliferation.

[0058] RT-qPCR and Western blot analysis confirmed successful overexpression of GCDH in HepG2 and Huh7 cell lines (A, B). CCK-8 assays showed that GCDH overexpression significantly inhibited the proliferation of Huh7 and HepG2 cells (C). Clonogenic assays further evaluated the colony-forming ability of transfected cells. GCDH overexpression significantly reduced the colony-forming ability of HepG2 and Huh7 cell lines (D). These results indicate that GCDH plays an important role in inhibiting the proliferation of HepG2 and Huh7 cells.

[0059] (4) GCDH overexpression induces ferroptosis in HCC cells To further elucidate the cell death patterns induced by GCDH overexpression and the molecular mechanisms promoting HCC cell death, multiple cell death inhibitors—Fer-1 (ferroptosis inhibitor), 3-MA (autophagy inhibitor), Nec-1 (necrosis inhibitor), and Z-VAD (apoptosis inhibitor)—were added to Huh7 and HepG2 cells, respectively, and their effects on proliferation were examined. pCDNA3.1-GCDH represents the experimental group overexpressing GCDH, and pCDNA3.1 represents the control group with an empty vector plasmid.

[0060] The results of GCDH overexpression inducing ferroptosis in HCC cells are as follows: Figure 4 As shown. (A) Effects of pCDNA3.1-GCDH and various cell death inhibitors, alone or in combination, on the proliferation of HCC cells Huh7 and HepG2. (B) Effects of pCDNA3.1-GCDH and various cell death inhibitors, alone or in combination, on the proliferation of HCC cells Huh7 and HepG2. (C) Intracellular ROS levels observed under a fluorescence microscope after Huh7 cells were treated with pCDNA3.1 and pCDNA3.1-GCDH plasmids for 48 h. (D) Quantitative detection of ROS levels using a fluorescence microplate reader. (E) Relative MDA content in Huh7 cells. (F) Relative GSH content in Huh7 cells. p<0.05, p<0.01, p<0.001, p<0.0001.

[0061] The results showed that the inhibitory effect of GCDH overexpression in HCC cells was mainly attenuated by ferroptosis inhibition (Fer-1) (Figures A and B). Although apoptosis inhibitors could slightly attenuate the inhibition of proliferation, neither autophagy nor necroptosis inhibition could attenuate the inhibition of proliferation. Furthermore, clonogenic assays showed consistent results (C). Ferroptosis is an iron-dependent phospholipid peroxidation-mediated pathway regulating cell death. Figure 4 As shown, compared with the control group, GCDH overexpression induced a significant accumulation of lipid-derived ROS (D, E) and increased one of the final products of excessive lipid oxidation, malondialdehyde (MDA) (F), accompanied by a significant depletion of GSH (G). In summary, the results confirm that GCDH overexpression can induce ferroptosis in HCC cells.

[0062] (5) GCDH induces ferroptosis by promoting the degradation of SLC7A11. To further elucidate the molecular mechanism by which GCDH overexpression promotes ferroptosis in HCC cells, the expression of ferroptosis-related factors GPX4, SLC7A11, and NRF2 in Huh7 cells (a Huh7 cell line overexpressing GCDH was constructed by transient plasmid transfection) was examined.

[0063] Ferrocyte-related protein and gene expression profiles, such as Figure 5 As shown in the figure. (A) Protein levels of NRF2, GPX4, and SLC7A11 in Huh7 cells after 48 h of treatment. (B) Protein expression statistics. (C) Expression levels of NRF2, GPX4, and SLC7A11 in TCGA-LIHC data. Error bars represent mean ± SD. ns, not significant; P<0.05, P<0.01, P<0.001, P<0.0001.

[0064] Western blot (WB) results showed significant differences in GCDH and SLC7A11 protein expression (A, B). Analysis of TCGA data revealed no significant difference in NRF2 mRNA levels between samples with low / high GCDH expression, but significant differences were observed in the mRNA levels of GPX4 and SLC7A11 (C). These results suggest that GCDH may function in hepatocellular carcinoma cells through SLC7A11, inducing ferroptosis by promoting SLC7A11 degradation.

[0065] (6) GCDH regulates ferroptosis through carbonic anhydrase IX (CA9) / SLC7A11. To further investigate the specific mechanism by which GCDH regulates ferroptosis in hepatocellular carcinoma cells, RNA-seq analysis was performed on Huh7 cells overexpressing GCDH, with Huh7 cells transfected with an empty vector serving as the control. The results are as follows: Figure 6 As shown in the figure, (A) Differential gene expression profile in Huh7 cells overexpressing GCDH (pCDNA3.1-GCDH vs control) | 195 DEGs (p<0.05, |log2FC|>1). (B) KEGG pathway enrichment analysis of differentially expressed genes in Huh7 cells (p<0.05). (C) GCDH-regulated genes enriched in nitrogen metabolism and mineral uptake pathways. (D) GO enrichment analysis of 195 GCDH-regulated genes (p<0.05). (E) Intersection of GCDH-regulated genes with known ferroptosis regulatory genes (FerrDb v2.0). (F) Heatmap analysis of ferroptosis-related protein expression in GCDH-overexpressing / control Huh7 cells. (G) Expression of CA9 and DUOX2 in hepatocellular carcinoma tissue samples from the TCGA database (normal=50, tumor=368). (H) Western blot analysis of CA9 and DUOX2 protein expression in Huh7 cells 48 h after transfection with pCDNA3.1-GCDH, with GAPDH as an internal control. (I) Statistical analysis of protein expression rates. (J) Western blot analysis of CA9 and SLC7A11 expression in Huh7 cells 48 h after transfection with pCDNA3.1-GCDH, with GAPDH as an internal control. (K) Statistical analysis of protein expression ratios. Error bars represent mean ± SD. ns, not significant; P<0.05, P<0.01, P<0.001, P<0.0001.

[0066] Volcano plots showed differential expression of 195 genes, with 125 upregulated and 70 downregulated (A). KEGG pathway enrichment analysis revealed that nitrogen metabolism was one of the most significantly enriched pathways in the pCDNA3.1-GCDH group compared to the control group (B, C). Gene ontology function enrichment showed a significant correlation between GCDH and carbonate dehydratase activity (D).

[0067] Intersection analysis of ferroptosis-related regulatory genes (including driver and repressor factors) in the FerrDb database with differentially expressed genes related to GCDH revealed that under GCDH overexpression conditions, the ferroptosis repressor CA9 (p<0.001) was significantly downregulated, while the ferroptosis driver DUOX2 (p<0.05) was significantly upregulated (F). Consistent with this, Western blotting detected decreased CA9 and increased DUOX2 protein expression in GCDH-transfected hepatocellular carcinoma cells (H, I). However, TCGA data analysis showed no significant association between DUOX2 expression and the low / high GCDH expression groups, while CA9 expression was significantly correlated with the low / high GCDH expression groups (G). Furthermore, in GCDH-overexpressing cells, CA9 was linked to nitrogen metabolism and carbonate dehydrating enzyme activity via KEGG pathway analysis and molecular functional annotation (E). These findings suggest that GCDH overexpression primarily functions through CA9. In addition, compared to the pCDNA3.1-GCDH group, Fer-1 treatment attenuated the protein levels of CA9 and SLC7A11 (J, K). The above findings demonstrate that GCDH regulates the expression of CA9 / SLC7A11, thereby modulating the subsequent ferroptosis susceptibility of liver cancer cells.

[0068] (7) GCDH binds to CA9 to regulate intracellular pHi and induce ferroptosis. To further investigate the potential interaction between GCDH and CA9, a protein-protein docking approach was used to predict their binding affinity.

[0069] The interaction between GCDH and CA9 promotes ferroptosis in liver cancer cells. Figure 7 As shown. (A) Three-dimensional model of the GCDH-CA9 protein complex and prediction of its binding free energy. (B) Hydrogen bond formation at the GCDH-CA9 interaction interface. (C) Hydrogen bond length at the GCDH-CA9 interaction interface. (D) Salt bridge at the GCDH-CA9 interaction interface. (E) Immunoprecipitation (Co-Ip) of GCDH and Ca9 in Huh7 cells. (F) pHi measurement of Huh7 cells 48 hours after the above treatment. Treatment concentrations of CA9 inhibitor SLC-0111 (0, 25, 50, 100, 200 M). (G) pHi measurement of Huh7 cells 48 hours after the above treatment. Treatment concentration of CA9 inhibitor SLC-0111 (100 μM). (H) Cell viability after treatment of GCDH-overexpressing cells with CA9 inhibitor SLC-0111 (100 μM).

[0070] The results showed that the binding free energy of the GCDH-CA9 complex was -5.6 kcal / mol, indicating a thermodynamically favorable and stable interaction (A). Key residues involved in hydrogen bonding included Lys-315, Lys-331, Pro-7, Glu-12, Glu-296, Gln-306, Gln-313, and Ile-382 of GCDH, and Arg-106, Asp-7, Arg-20, Gly-244, Arg-12, Thr-239, His-105, and Arg-231 of CA9 (B). The corresponding hydrogen bond distances were 3.58 Å, 2.60 Å, 3.27 Å, 2.63 Å, 2.22 Å, 3.28 Å, 3.25 Å, and 3.26 Å, respectively (C). Furthermore, salt bridges are formed between Lys-331 and Glu-296 of GCDH and Asp-7 and Arg-12 of CA9 (D). This was experimentally verified using Co-IP, confirming the physical interaction between GCDH and CA9 in vivo (E). Since CA9 participates in pHi regulation by producing bicarbonate, the pHi of Huh7 cells treated with SLC-0111 confirmed that CA9 inhibitors induced pHi acidification (F). Subsequently, treatment of GCDH-overexpressing Huh7 cells with SLC-0111 showed that the pHi level achieved in the GCDH-overexpression group was comparable to that induced by CA9 inhibitors. Moreover, combined treatment with GCDH overexpression and SLC-0111 resulted in a more significant decrease in cell viability (G, H). In summary, GCDH and CA9 interact through specific residue-mediated hydrogen bonds and salt bridges, and CA9-mediated intracellular pHi regulation plays a crucial role in GCDH overexpression-induced ferroptosis in hepatocellular carcinoma cells, demonstrating a coordinated mechanism regulating ferroptosis-related cellular functions.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of glutaric acid monoacyl-CoA dehydrogenase detection reagent in the preparation of products for assessing liver cancer risk.

2. Application of glutaric acid monoacyl-CoA dehydrogenase detection reagent in the preparation of products for assessing the prognostic risk of liver cancer.

3. Application of glutaryl-CoA dehydrogenase overexpression reagent in the preparation of products that inhibit the proliferation of liver cancer cells.

4. The application according to claim 3, characterized in that, Overexpression of glutaryl-CoA dehydrogenase reduces the clonal formation ability of liver cancer cells.

5. Application of glutaryl-CoA dehydrogenase overexpression reagent in the preparation of products that induce ferroptosis in hepatocellular carcinoma cells.

6. The application according to claim 5, characterized in that, Glutamic acid monoacyl-CoA dehydrogenase promotes the degradation of SLC7A11.

7. The application according to claim 5, characterized in that, Glutamic acid monoacyl-CoA dehydrogenase downregulates ferroptosis inhibitor CA9.

8. Application of glutaryl-CoA dehydrogenase overexpression reagent combined with CA9 inhibitor in the preparation of products that inhibit the proliferation of liver cancer cells.

9. The application according to claim 8, characterized in that, Glutaryl-CoA dehydrogenase combined with CA9 inhibitors reduces the cell viability of liver cancer cells.

10. The application according to any one of claims 3 to 9, characterized in that, The overexpression reagent for glutaryl-CoA dehydrogenase includes an overexpression vector for glutaryl-CoA dehydrogenase.

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