Use of plekhh1 and inhibitors thereof in the preparation of a medicament or kit for inducing ferroptosis

Abstract

This invention relates to the application of PLEKHH1 and its inhibitors in the preparation of drugs or kits that induce ferroptosis, belonging to the field of tumor treatment technology. This invention discovers that knocking down the PLEKHH1 gene in liver cancer cells can induce ferroptosis. Therefore, this invention uses the PLEKHH1 gene as a novel target for cancer cells to resist ferroptosis. By knocking down this gene, it eliminates ferroptosis escape from cancer cells, induces ferroptosis, and thus exerts a therapeutic effect on tumors.

Description

Technical Field

[0001] This invention relates to the field of tumor treatment technology, and more particularly to the application of PLEKHH1 and its inhibitors in the preparation of drugs or kits that induce ferroptosis. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common and deadliest malignant tumors worldwide. The high recurrence rate and treatment resistance of HCC highlight the urgency of discovering novel molecular targets and developing effective intervention strategies. Ferroptosis, a novel form of regulatory cell death characterized by iron-dependent lipid peroxidation, has shown great potential in cancer treatment due to its ability to effectively eliminate cancer cells resistant to conventional drugs. HCC cells possess unique metabolic reprogramming and a strong ability to escape ferroptosis, but the specific molecular mechanisms by which they resist ferroptosis are not yet fully understood.

[0003] Current treatments for hepatocellular carcinoma (HCC) have certain limitations: 1) Traditional treatments are ineffective and lack specificity: Existing HCC treatments have limited efficacy and poor prognosis, and lack effective strategies to specifically reverse the endogenous resistance mechanisms of cancer cells; 2) Lack of effective ferroptosis induction targets and mechanisms: Current technologies have not yet clarified the key protective mechanisms and core molecular switches against ferroptosis in HCC cells, making it difficult to develop highly specific and efficient ferroptosis inducers; 3) Unclear molecular pathways: The molecular pathways by which HCC cells resist ferroptosis by regulating lipid metabolism are still unclear, which restricts the precise development of targeted drugs.

[0004] Therefore, seeking molecular targets and drugs that can effectively and specifically overcome the endogenous resistance to ferroptosis in HCC cells has become a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of PLEKHH1 and its inhibitors in the preparation of drugs or kits that induce ferroptosis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the use of PLEKHH1 inhibitors in the preparation of drugs or kits that induce ferroptosis in cancer cells.

[0007] Secondly, the present invention provides the use of PLEKHH1 inhibitors in the preparation of drugs or kits that disrupt the lipid peroxidation balance of cancer cells.

[0008] Furthermore, the cancer cells are liver cancer cells.

[0009] This invention reveals that knocking down the PLEKHH1 gene in liver cancer cells leads to a significant increase in triglyceride (TAG) levels and marked lipid droplet accumulation. It also reduces the levels of malondialdehyde (MDA), a lipid peroxide end product, and Fe. 2+ The level increased significantly, indicating that knocking down the PLEKHH1 gene affected Fe. 2+ Metabolism disrupts the cellular lipid peroxidation balance, thereby inducing ferroptosis. Therefore, this invention uses the PLEKHH1 gene as a novel target for cancer cells to resist ferroptosis. By knocking down this gene, ferroptosis escape from cancer cells is eliminated, inducing ferroptosis. Thus, PLEKHH1 inhibitors can induce ferroptosis in cancer cells and disrupt their lipid peroxidation balance.

[0010] Thirdly, the present invention provides the use of PLEKHH1 inhibitors in the preparation of drugs or kits that promote M1 polarization of macrophages in the tumor microenvironment and / or inhibit M2 polarization of macrophages in the tumor microenvironment.

[0011] Furthermore, the tumor is liver cancer.

[0012] This invention found that the M1 / M2 ratio in liver cancer cells of the PLEKHH1 knockdown group was significantly higher than that of the control group. Furthermore, in the conditioned medium (tumor microenvironment) of the knockdown group, the markers of M2 macrophages, ARG1, IL-10, and TGF-β, were significantly decreased, while the markers of M1 macrophages, INOS and IL-12, were significantly increased. This indicates that PLEKHH1 knockdown significantly promotes M1 macrophage polarization in the tumor microenvironment and inhibits M2 macrophage polarization. Therefore, PLEKHH1 inhibitors have the effect of promoting M1 macrophage polarization and / or inhibiting M2 macrophage polarization in the tumor microenvironment.

[0013] Fourthly, this invention provides the application of PLEKHH1 inhibitors in the preparation of drugs or kits that mediate the regulation of PPARγ expression through the FKBP5-PPARγ signaling pathway.

[0014] As a preferred embodiment of the fourth aspect, the regulation of the FKBP5-PPARγ signaling pathway is to reduce the expression level of PPARγ gene mRNA or PPARγ protein and block PPARγ nuclear translocation.

[0015] This invention investigated the molecular mechanism by which the PLEKHH1 gene regulates ferroptosis in cancer cells, finding that knocking down the PLEKHH1 gene in liver cancer cells resulted in a downregulation of PPARγ expression. Protein interaction database analysis revealed a potential interaction between PLEKHH1 and FKBP5; molecular docking prediction analysis indicated a strong interaction between FKBP5 and PPARγ. Therefore, this invention demonstrates that PLEKHH1 regulates the FKBP5-PPARγ signaling axis by mediating FKBP5, and that the PLEKHH1 gene influences lipid metabolism by regulating the FKBP5-PPARγ signaling axis, thereby regulating ferroptosis. Thus, PLEKHH1 inhibitors can regulate the FKBP5-PPARγ signaling pathway, resulting in reduced PPARγ mRNA or protein expression levels and blockade of PPARγ nuclear translocation.

[0016] Fifthly, the present invention provides the application of PLEKHH1 inhibitors in the preparation of antitumor drugs.

[0017] This invention demonstrates through cell experiments that knocking down the PLEKHH1 gene in HepG2 and Hep3B liver cancer cells effectively inhibits the traditional malignant tumor behaviors of liver cancer cells, including proliferation, migration, and invasion. Therefore, PLEKHH1 inhibitors possess anti-tumor efficacy and can be formulated into anti-tumor drugs.

[0018] As a preferred embodiment of the first, second, third, fourth, and fifth aspects, the PLEKHH1 inhibitor is a nucleotide sequence of siRNA targeting the PLEKHH1 gene as shown in any one of SEQ ID NO:1-6.

[0019] This invention designs three candidate siRNA sequences (PLEKHH1 si1, PLEKHH1 si2, and PLEKHH1 si3) with optimal interference efficiency targeting different exon regions of the PLEKHH1 gene. The sequence of the sense strand of PLEKHH1 si1 is shown in SEQ ID NO:1; the sequence of the antisense strand of PLEKHH1 si1 is shown in SEQ ID NO:2; the sequence of the sense strand of PLEKHH1 si2 is shown in SEQ ID NO:3; the sequence of the antisense strand of PLEKHH1 si2 is shown in SEQ ID NO:4; the sequence of the sense strand of PLEKHH1 si3 is shown in SEQ ID NO:5; and the sequence of the antisense strand of PLEKHH1 si3 is shown in SEQ ID NO:6.

[0020] Experimental results showed that PLEKHH1 si3 exhibited the most significant inhibitory efficiency, inhibiting PLEKHH1 mRNA expression by over 70%. Therefore, PLEKHH1 si3 is the optimal knockdown sequence for this invention.

[0021] In a sixth aspect, the present invention provides an antitumor drug, wherein the drug uses a PLEKHH1 inhibitor as its active ingredient.

[0022] As a preferred embodiment of the sixth aspect, the PLEKHH1 inhibitor is an siRNA targeting the PLEKHH1 gene, and the nucleotide sequence of the siRNA is shown in any one of SEQ ID NO:1-6.

[0023] As a preferred embodiment of the sixth aspect, the medicament further includes pharmaceutically acceptable excipients.

[0024] As a preferred embodiment of the sixth aspect, medically acceptable excipients include fillers, wetting agents, disintegrants, lubricants, solubilizers, solvents, and emulsifiers.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention is the first to discover and confirm that PLEKHH1 is a key factor for liver cancer cells to resist ferroptosis, providing a new molecular target for treating liver cancer by inducing ferroptosis.

[0026] 2. This invention reveals the complete pathway by which PLEKHH1 affects lipid metabolism and ferroptosis by regulating the FKBP5-PPARγ signaling axis, thus clarifying the development direction for new anti-tumor drugs.

[0027] 3. This invention also found that PLEKHH1 mediates the FKBP5-PPARγ signaling axis to regulate microenvironment macrophage polarization (promoting M1 polarization and / or inhibiting M2 polarization).

[0028] 4. This invention reveals for the first time that PLEKHH1 inhibitors not only inhibit the traditional malignant behaviors of cancer cells, such as proliferation, migration, and invasion, but also induce lipid peroxidation (ferroptosis) to directly kill cancer cells, induce tumor cells to release large amounts of IL-6 and TNF-α, and promote macrophage M1 polarization, thus exerting pro-inflammatory effects. Therefore, PLEKHH1 inhibitors have multiple anti-tumor effects. Attached Figure Description

[0029] Figure 1To investigate the expression of the PLEKHH1 gene in hepatocellular carcinoma (HCC) tissues and its role in tumor progression, the following diagrams are presented: (Figure A shows that the expression level of PLEKHH1 in HCC tissues (n=374) was significantly higher than that in adjacent normal tissues (n=50, ***p<0.001) based on the TCGA database analysis; Figure B shows that the expression level of PLEKHH1 in different grades of HCC tissues (G1-G4) was significantly different from that in normal tissues (***p<0.001); Figure C shows the Kaplan-Meier survival curves.) Figure 2 RT-qPCR RNA electrophoresis images of PLEKHH1si1, PLEKHH1si2, and PLEKHH1si3; Figure 3 Figure 1 shows the RT-qPCR amplification and melting curves (Figure A is the amplification curve of PLEKHH1; Figure B is the melting curve of PLEKHH1; Figure C is the amplification curve of GAPDH; Figure D is the melting curve of GAPDH). Figure 4 Schematic diagrams showing the knockdown efficiency of different PLEKHH1 siRNAs and their effects on the proliferation of different hepatocellular carcinoma cell lines (Figure A: Western blot analysis of PLEKHH1 protein expression levels in Hep3B cells transfected with PLEKHH1si1, PLEKHH1si2, and PLEKHH1si3; Figure B: Statistical graph of PLEKHH1 protein expression levels; Figure C: Western blot analysis of PLEKHH1 protein expression levels in Hep3B and HepG2 cells transfected with PLEKHH1si3; Figure D: Statistical graph of PLEKHH1 protein expression levels; Figure E: RT-qPCR analysis of mRNA expression levels in Hep3B cells transfected with PLEKHH1si1, PLEKHH1si2, and PLEKHH1si3; Figure F: ) Figure G shows the results of the CCK-8 assay after PLEKHH1 knockdown in Hep3B cells; Figure G is a schematic diagram of the results of the CCK-8 assay after PLEKHH1 knockdown in HepG2 cells. Figure 5 Schematic diagram of the inhibitory effect of PLEKHH1 knockdown on the migration and invasion of liver cancer cells (Figure A shows representative images of the migration ability of Hep3B and HepG2 cells from Day 0 to Day 3 after PLEKHH1 knockdown as detected by scratch assay; Figure B shows the quantitative statistical graph of scratch healing rate of Hep3B cells; Figure C shows the quantitative statistical graph of scratch healing rate of HepG2 cells; Figure D shows the effect of PLEKHH1 knockdown on the ability of liver cancer cells to penetrate matrix gel as detected by Transwell invasion assay, showing a representative field of view of crystal violet staining; Figure E shows the quantitative analysis of the number of cells that have penetrated the membrane). Figure 6Figure A shows the clinical characteristics of PLEKHH1 and its correlation with lipid metabolism in HCC (Figure A is a schematic diagram of the GEO dataset analysis results; Figure B is a schematic diagram of the GSVA analysis results). Figure 7 This diagram illustrates how PLEKHH1 deficiency can induce lipid peroxidation (ferroptosis) in liver cancer cells; (Figure A shows the results of lipid droplet staining in the knockdown group of Hep3B cells; Figure B shows the results of C11-BODIPY staining and quantitative fluorescence; Figure C shows the results of biochemical detection of total iron concentration, MDA, and TAG content in Hep3B cells). Figure 8 This diagram illustrates how PLEKHH1 deficiency can induce pro-inflammatory polarization in macrophages within conditioned medium (tumor microenvironment). (Figure A shows the content of M1 and M2 macrophages in Hep3B cell conditioned medium detected by flow cytometry; Figure B shows the M1 / M2 ratio; Figure C shows the levels of INOS and IL-12 markers for M1 macrophages and ARG1, IL-10, and TGF-β markers for M2 macrophages in Hep3B cell conditioned medium detected by Q-PCR; Figure D shows the results of ELISA detection of IL-6 and TNF-α levels in Hep3B cells.) Figure 9 Figure 1 shows the abnormal accumulation of FKBP5 protein and the induction of PPARγ nuclear translocation disorder caused by PLEKHH1 deficiency (Figure A is the interaction network diagram of PLEKHH1 with FKBP5 and CUL4A, etc. shown in the BioGRID database; Figure B is a schematic diagram of the high affinity binding of FKBP5 and PPARγ shown by molecular docking 3D structure prediction (Z-score=1421.979); Figure C is a schematic diagram of Western blotting and nucleocytoplasmic separation experiments; Figure D is a schematic diagram of Q-PCR detection of PPARγ mRNA level; Figure E is a confocal image of immunofluorescence staining; Figure F is a quantitative analysis diagram of PPARγ nucleocytoplasmic ratio; Figure G is a comparison diagram of overall fluorescence intensity). Detailed Implementation

[0030] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0031] The PLEKHH1 gene is a protein 1 (Pleckstrin Homology Domain-Containing Family H Member 1) encoding a PH domain and a plekstrin homology domain, located on human chromosome 17q23.1. The encoded protein belongs to a family of signal transduction-related proteins, and its core functions are closely related to intracellular cytoskeleton remodeling, membrane transport, and signaling pathway regulation.

[0032] The FKBP5 gene encodes FK506-binding protein 5, a member of the immunoaffinity protein family, located on human chromosome 6p21.31. The encoded protein is a molecular chaperone protein with peptidyl prolyl cis-trans isomerase (PPIase) activity, playing a central role in physiological and pathological processes such as glucocorticoid receptor signaling regulation, stress response modulation of drug action mechanisms, and susceptibility to stress-related diseases.

[0033] The PPARγ gene encodes the peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily located on human chromosome 3p25. The encoded protein is a ligand-dependent transcription factor that plays a central role in physiological processes such as adipocyte differentiation, regulation of glucose and lipid metabolism, and suppression of inflammatory responses.

[0034] Example 1 investigates the expression of the PLEKHH1 gene in hepatocellular carcinoma tissues and its role in tumor progression. 1. Online database analysis: We analyzed the expression level of the PLEKHH1 gene in adjacent normal tissue and hepatocellular carcinoma tissue using RNA-seq data from the TCGA (The Cancer Genome Atlas) database.

[0035] The results are as follows Figure 1 As shown, compared with adjacent normal tissue, the expression of PLEKHH1 gene in hepatocellular carcinoma tissue was significantly upregulated (Nomal, n=50; Tumor, n=374; ***p<0.001; Figure 1 A). Furthermore, high expression of PLEKHH1 is associated with tumor grade ( Figure 1 B), with the highest expression in G3 tissue. Kaplan-Meier survival curve analysis showed that patients with high PLEKHH1 gene expression had a significantly shorter median survival (n=185; ***p<0.001; Figure 1 C). Univariate Cox regression analysis (HR=1.53, 95%CI=1.17-2.01, p<0.001) and multivariate Cox regression analysis (HR=1.55, 95%CI=1.16-2.08, p<0.001) revealed that PLEKHH1 was a significantly high-risk factor, suggesting that PLEKHH1 is an independent adverse prognostic factor. The results of univariate and multivariate Cox regression analyses are shown in Tables 1 and 2.

[0036] Table 1 Results of univariate Cox regression analysis Table 2 Results of multivariate Cox regression analysis 2. Cell experiments: (1) Design and synthesize siRNA sequences: The full-length transcript sequence of human PLEKHH1 was obtained through NCBI. Three candidate siRNA sequences with optimal interference efficiency (PLEKHH1 si1, PLEKHH1si2, PLEKHH1si3) were designed for different exon regions of the PLEKHH1 gene and the siRNA sequences were synthesized. The siRNA sequences (SEQ ID NO:1-6) are detailed in Table 3.

[0037] (2) Cell transfection with siRNA sequence (RNA): The Hep3B cell line was passaged one day before transfection to achieve a confluence of 30%–50%. b. Transfection was performed using Lipofectamine™ RNAiMAX (invitrogen, Cat. No. 13778075) transfection reagent, with a working concentration of 50 nM siRNA, and Opti-MEM (invitrogen, Cat. No. 31985070) medium.

[0038] c. Use a 24-well plate with 500 μl of culture medium. Dissolve 1.25 μl of siRNA stock solution (20 μM) in 100 μl of Opti-MEM medium to obtain solution A; dissolve 1 μl of Lipofectamine™ RNAiMAX in Opti-MEM medium to obtain solution B. Mix solutions B together; then mix solutions A and B, let stand for 20 minutes, and add to the cells for transient transfection. After incubation for 4 hours, replace with cell growth medium.

[0039] (3) Detection of transfected cells: Transfection efficiency was detected by RT-qPCR and Western Blot, as well as the proliferation, migration and invasion capabilities of knockdown cell lines. The RT-qPCR primer sequences (SEQ ID NO:7-10) are shown in Table 4.

[0040] ①RT-qPCR RNA electrophoresis identification results are as follows Figure 2 As shown, the amplification curve and melting curve of RT-qPCR are as follows: Figure 3 As shown, the results indicate that the RNA sample is of good quality and intact, the PCR amplification efficiency is high and the primers are highly specific, with no non-specific amplification.

[0041] ② Results of knockdown efficiency for different siRNA sequences are as follows Figure 4 As shown, the results indicate that after transfecting Hep3B cells with three different siRNA sequences (PLEKHH1 si1, PLEKHH1 si2, and PLEKHH1 si3), PLEKHH1 si3 showed the most significant inhibitory efficiency. Figure 4 (A, 4B, 4E), which inhibited PLEKHH1 mRNA expression by more than 70%. Therefore, PLEKHH1 si3 was selected as the optimal knockdown sequence for subsequent experiments.

[0042] Subsequently, we further validated the knockdown effect of PLEKHH1 si3 in two hepatocellular carcinoma cell lines, Hep3B and HepG2. Western blot results confirmed that the PLEKHH1 protein level in both cell lines was significantly lower than that in the control group (NC) after transfection with PLEKHH1 si3. Figure 4 C, 4D).

[0043] Table 3 Three PLEKHH1 siRNA sequences Table 4. Q-PCR Primer Sequences ③ Cell phenotype detection: Based on the HepG2 and Hep3B cell lines with PLEKHH1 knockdown described above, the proliferation ability of the knockdown cell lines was detected by CCK-8 assay, the migration ability of the knockdown cell lines was detected by cell scratch assay, and the invasion ability of the knockdown cell lines was detected by Transwell assay.

[0044] The results of the CCK-8 experiment are as follows: Figure 4 As shown in FG, compared with the control group, knockdown of PLEKHH1 significantly inhibited Hep3B ( Figure 4 F) and HepG2 cells (F) Figure 4 The inhibitory effect on the proliferation rate of hepatocellular carcinoma cells (G) was significantly reduced at 48 and 72 hours, exhibiting a time-dependent effect. These data suggest that PLEKHH1 plays a crucial role in maintaining the malignant proliferative capacity of liver cancer cells.

[0045] The scratch test results are as follows Figure 5 As shown in AC, compared with the control group, the scratch healing rate of Hep3B and HepG2 cells after PLEKHH1 knockdown was significantly reduced, indicating that their horizontal migration ability was impaired.

[0046] Transwell invasion test results are as follows: Figure 5As shown in the DE, knocking down PLEKHH1 significantly reduced the number of Hep3B and HepG2 cells that penetrated the matrix gel, confirming that their longitudinal invasion ability was suppressed.

[0047] These results indicate that knocking down the PLEKHH1 gene can significantly inhibit the traditional malignant behaviors of liver cancer cells: proliferation, migration, and invasion, suggesting that PLEKHH1 inhibitors have multiple anti-tumor effects.

[0048] Example 2 investigates the role of PLEKHH1 in lipid peroxidation of liver cancer cells and macrophage polarization in the tumor microenvironment. Our analysis using the GEO dataset confirmed a significant negative correlation between PLEKHH1 expression and fatty acid oxidation-related genes (such as CPT2) (p<0.01). Figure 6 A, red indicates negative correlation, blue indicates positive correlation). Our GSVA analysis revealed that lipid pathways such as fatty acid metabolism were significantly enriched in the PLEKHH1 low-expression group (A, red indicates negative correlation, blue indicates positive correlation). Figure 6 B).

[0049] Based on the above PLEKHH1 knockdown cell line Hep3B, it was found that significant lipid accumulation occurred in Hep3B cells after PLEKHH1 knockdown. Figure 7 A). C11-BODIPY staining and quantitative fluorescence detection showed a significant expansion of the total lipid pool and intense lipid peroxidation in the knockdown group. Figure 7 B). Simultaneously, biochemical assays showed that PLEKHH1 knockdown significantly increased total cellular iron concentration (p<0.01) and the levels of lipid peroxidation markers malondialdehyde (MDA) and triglycerides (TAG) (p<0.001). Figure 7 C).

[0050] Flow cytometry analysis revealed that the M1 / M2 ratio in the knockdown group was significantly higher than that in the control group. Figure 8 AB); Q-PCR results showed that, compared with the control group, the markers ARG1, IL-10, and TGF-β of M2 macrophages were significantly decreased in the knockdown group conditioned medium (i.e., the tumor microenvironment), while the markers INOS and IL-12 of M1 macrophages were significantly increased. This indicates that PLEKHH1 knockdown significantly promotes the M1 polarization of macrophages in the tumor microenvironment and inhibits their M2 polarization. Figure 8 C). Furthermore, ELISA testing confirmed that the knockdown group released large amounts of IL-6 and TNF-α (C). Figure 8 D). This result is consistent with Figure 6 As predicted, knocking down PLEKHH1 can effectively reverse the "cold tumor" microenvironment in liver cancer.

[0051] In summary, these findings indicate that PLEKHH1 deficiency leads to abnormal lipid accumulation in liver cancer cells, accompanied by increased iron overload and lipid peroxidation damage, triggering lipid peroxidation (ferroptosis) in liver cancer cells. Simultaneously, PLEKHH1 deficiency also promotes the release of large amounts of pro-inflammatory factors from tumor cells and the transformation of macrophages into a pro-inflammatory phenotype, thereby exerting a tumor-killing effect. These key changes suggest that PLEKHH1 knockdown promotes ferroptosis and anti-tumor effects in Hep3B cells.

[0052] Example 3 investigates the molecular mechanism by which PLEKHH1 mediates FKBP5 regulation of the PPARγ signaling axis. The potential protein interaction network of PLEKHH1 (including FKBP5 and CUL4A, etc.) was confirmed using the BioGRID protein interaction database. Figure 9 A). 3D structure prediction analysis using molecular docking showed a high affinity binding between PPARγ and FKBP5 (Z-score = 1421.979). Figure 9 B).

[0053] Western blotting and nucleo-cytoplasmic separation experiments confirmed that knockdown of PLEKHH1 significantly upregulated FKBP5 protein and caused significant cytoplasmic retention of PPARγ. Figure 9 C); Q-PCR detection confirmed that PPARγ mRNA expression was significantly downregulated in the knockdown group cells ( Figure 9 D). Immunofluorescence staining confocal images showed impaired nuclear translocation of PPARγ in the knockdown group. Figure 9 E), quantitative analysis of immunofluorescence staining images confirmed a significant decrease in the PPARγ nucleocytoplasmic ratio in the knockdown group (E), Figure 9 F) and overall fluorescence intensity decreased ( Figure 9 G).

[0054] In summary, the experimental results indicate that the PLEKHH1 gene mediates the nuclear localization and expression of PPARγ through FKBP5. Knockout of the PLEKHH1 gene leads to impaired nuclear translocation of PPARγ (cytoplasmic retention) and transcriptional dysfunction (decreased mRNA and protein levels), thereby inducing lipid peroxidation (ferroptosis) in tumor cells and promoting the release of large amounts of IL-6 and TNF-α, which in turn promotes macrophage M1 polarization and exerts a pro-inflammatory effect. This invention reveals for the first time a novel mechanism by which PLEKHH1 regulates pro-inflammatory effects such as ferroptosis in liver cancer cells and abnormal macrophage polarization. PLEKHH1 can serve as an effective target for the diagnosis and treatment of liver cancer. Therefore, the PLEKHH1 inhibitor and its accompanying kit provided by this invention have therapeutic efficacy for tumors.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of PLEKHH1 inhibitors in the preparation of drugs or kits that induce ferroptosis in cancer cells.

2. Application of PLEKHH1 inhibitors in the preparation of drugs or kits that disrupt the lipid peroxidation balance of cancer cells.

3. Application of PLEKHH1 inhibitors in the preparation of drugs or kits that promote macrophage M1 polarization and / or inhibit macrophage M2 polarization in the tumor microenvironment.

4. Application of PLEKHH1 inhibitors in the preparation of drugs or kits that mediate the regulation of PPARγ expression through the FKBP5-PPARγ signaling pathway.

5. The application as described in claim 4, characterized in that, The regulation of the FKBP5-PPARγ signaling pathway involves reducing the expression level of PPARγ gene mRNA or PPARγ protein, thereby blocking PPARγ nuclear translocation.

6. Application of PLEKHH1 inhibitors in the preparation of antitumor drugs.

7. The application of the PLEKHH1 inhibitor according to any one of claims 1 to 6, characterized in that, The PLEKHH1 inhibitor is a siRNA that targets the PLEKHH1 gene.

8. The application of the PLEKHH1 inhibitor as described in claim 7, characterized in that, The nucleotide sequence of the siRNA targeting the PLEKHH1 gene is shown in any one of SEQ ID NO:1-6.

9. An antitumor drug, characterized in that, The drug uses a PLEKHH1 inhibitor as its active ingredient.

10. The antitumor drug as described in claim 9, characterized in that, The PLEKHH1 inhibitor is a siRNA that targets the PLEKHH1 gene, and the nucleotide sequence of the siRNA is shown in any one of SEQ ID NO:1-6.

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