Use of ildr1 inhibitors in the manufacture of a medicament for hepatocellular carcinoma

Abstract

The application provides application of an ILDR1 inhibitor in preparation of a hepatocellular carcinoma drug, the ILDR1 inhibitor in the application inhibits proliferation of liver cancer cells, ILDR1 is a new target for treating HCC and a biomarker related to diagnosis and prognosis, in vitro experiments find that after ILDR1 is down-regulated in an HCC cell line HCCLM3, the proliferation ability of tumors is significantly inhibited, in vitro experiments find that down-regulation of ILDR1 inhibits transcription of glutamine transaminase (GLS), a key enzyme in glutamine metabolism, and down-regulates the protein expression amount of GLS, and the down-regulation of ILDR1 may inhibit GLS by inhibiting an AKT signal path and a transcription factor MYC.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the use of an ILDR1 inhibitor in the preparation of a hepatocellular carcinoma drug. Background Art

[0002] Hepatocellular carcinoma (HCC) is a common primary liver cancer, accounting for approximately 90% of all liver cancer cases. According to the latest statistics from the World Health Organization (WHO), HCC ranks fifth among all common cancers worldwide and is the third leading cause of cancer-related death. This situation is particularly serious in my country, where HCC has the fourth highest incidence rate among all tumors and the second highest mortality rate, posing a serious threat to the lives, health, and socioeconomic development of the Chinese people.

[0003] There are significant differences in the risk factors for HCC worldwide. Hepatitis B virus (HBV) infection is predominant in Asia, hepatitis C virus (HCV) infection is more common in Japan, and non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and long-term alcohol abuse are the main pathogenic factors in Europe and North America. In recent years, with the large-scale promotion of HBV vaccination in my country, the incidence of HBV-related malignancies has decreased. However, the incidence of liver cancer related to NAFLD and NASH is on the rise. NAFLD is considered to be the manifestation of metabolic syndrome in the liver, involving metabolic disorders in multiple systems. These changes highlight the important role of metabolic disorders in the occurrence and development of HCC, suggesting that intervention targeting metabolic pathways may become a new treatment strategy.

[0004] Metabolic reprogramming of tumor cells is one of their most prominent characteristics. This process helps tumor cells adapt to rapid proliferation and high energy demands. To meet these demands, the metabolic pathways of tumor cells undergo significant changes, including enhanced glucose metabolism, fatty acid synthesis, and amino acid metabolism. Metabolic reprogramming is not only one of the hallmarks of cancer, but also a key driver of its occurrence and progression. In the tumor microenvironment, due to physicochemical conditions such as high pressure, low pH, and hypoxia, as well as the heterogeneity of tumor blood vessels, local tumor cells face limited metabolic resources, which accelerates the consumption of nutrients and the accumulation of metabolites. To meet this challenge, tumor cells regulate their own metabolic pathways, including glucose metabolism, lipid metabolism, and amino acid metabolism, not only to re-meet their own energy needs, but also to regulate gene expression and protein modification, ultimately promoting tumor cell proliferation and spread.

[0005] Glutamine plays multiple key roles in the metabolic reprogramming of tumors. First, glutamine is deaminated to produce alpha-ketoglutarate (a-KG), which enters the tricarboxylic acid cycle (TCA cycle) to provide energy for the high proliferation rate of tumor cells. Second, as an important nitrogen source, glutamine supports the synthesis of nucleotides, amino acids, and fatty acids, promoting the growth and proliferation of tumor cells. In addition, glutamine metabolism generates glutathione (GSH), which plays a key role in maintaining cellular redox balance, enhancing the resistance of tumor cells to oxidative stress and chemotherapy drugs, and further promoting tumor survival and drug resistance. Glutamine transaminase (Glutaminase, GLS) is a key enzyme in glutamine metabolism, responsible for converting glutamine to glutamate, which participates in the TCA cycle and various biosynthetic pathways. GLS is crucial in the glutamine metabolism of tumor cells. Numerous studies have shown that inhibiting the expression of GLS in tumor cells can significantly delay tumor progression and reduce the number of cancer stem cells. However, due to metabolic heterogeneity and drug resistance of tumors, GLS inhibitors have not been widely used in all tumor patients in clinical applications, and further research and optimization of treatment strategies are still needed.

[0006] ILDR1 (Immunoglobulin-like Domain Containing Receptor 1) is a key transmembrane protein that is involved in hearing signal transduction and potentially immune regulation by maintaining the integrity of tight junctions and regulating cell barrier function. Studies have shown that ILDR1 may regulate glutamine metabolism in hepatocytes, although its specific mechanism has not been fully elucidated. ILDR1 may affect the growth and metabolic state of hepatocytes by regulating metabolic pathways in the process of maintaining intercellular tight junctions, thereby playing a role in the occurrence and development of HCC. Based on this, the present application provides the use of an ILDR1 inhibitor in the preparation of a hepatocellular carcinoma drug. SUMMARY

[0007] The purpose of the present application is to provide the use of an ILDR1 inhibitor in the preparation of a hepatocellular carcinoma drug.

[0008] In one aspect, the present application provides the use of an ILDR1 inhibitor in the preparation of an early diagnosis, prognosis evaluation, or targeted therapy drug for primary liver cancer.

[0009] Further, the ILDR1 inhibitor inhibits the proliferation ability of the hepatocellular carcinoma cell line HCCLM3 cell line in vitro.

[0010] Further, the ILDR1 inhibitor inhibits the protein expression of GLS in the hepatocellular carcinoma cell line HCCLM3 cell line.

[0011] Furthermore, the ILDR1 inhibitor inhibited the activity of the PI3K-AKT signaling pathway in the liver cancer cell line HCCLM3.

[0012] Furthermore, the ILDR1 inhibitor inhibited the protein expression of GLS in the liver cancer cell line HCCLM3 through the PI3K-AKT-MYC signaling axis.

[0013] Beneficial effects:

[0014] The present invention demonstrates that ILDR1 inhibitors inhibit the proliferation of liver cancer cells, making ILDR1 a novel target for the treatment of HCC and a biomarker relevant to diagnosis and prognosis. In vitro experiments demonstrated that downregulation of ILDR1 significantly suppressed tumor proliferation in the HCCLM3 HCC cell line. Further in vitro experiments revealed that downregulation of ILDR1 inhibited the transcription of transglutaminase (GLS), a key enzyme in glutamine metabolism, and reduced its protein expression. This inhibition of GLS was achieved by suppressing the AKT signaling pathway and the transcription factor MYC. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is an analysis chart using the TCGA public database to explore the effect of gene ILDR1 on liver cancer survival;

[0016] Figure 2 This is a graph detecting the proliferation rate of HCCLM3 cell lines transfected with ILDR1 gene small interfering RNA;

[0017] Figure 3 This is a correlation analysis diagram between ILDR1 and GLS in the TCGA database;

[0018] Figure 4 This is a graph showing the immunoblotting results for detecting changes in ILDR1 gene expression after the HCCLM3 cell line was treated with a GLS activity inhibitor for 48 hours;

[0019] Figure 5 This is a graph of immunoblotting results for detecting changes in the expression of genes GLS and ILDR1 after ILDR1 knockdown;

[0020] Figure 6 This is a graph showing the immunoblotting results for detecting changes in the expression of genes GLS and ILDR1 after ILDR1 overexpression;

[0021] Figure 7 This is a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes in the TCGA-LIHC database;

[0022] Figure 8This is the immunoblotting result diagram for detecting the expression changes of genes ILDR1, AKT, p-AKT, MYC, and GLS after ILDR1 knockdown;

[0023] Figure 9 This is a graph of immunoblotting results for detecting changes in the expression of genes ILDR1, AKT, p-AKT, MYC, and GLS after overexpression of ILDR1;

[0024] Figure 10 Immunoblotting was used to validate the specificity of the ILDR1-PI3K-AKT-cMYC-GLS signaling axis. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0026] Example 1 Survival analysis of ILDR1 gene using TCGA database

[0027] The TCGA Liver hepatocellular carcinoma dataset was downloaded from the UCSC Xena platform (https: / / xena.ucsc.edu) on March 22, 2024. Expression matrices, gene names, and associated survival data were organized using the R language. Patients with high (top 75%) or low (bottom 25%) ILDR1 expression in the LIHC dataset were then grouped. The actual number of months of survival was calculated by dividing the survival time of each group by 12. Survival curves were fitted to these data using the survival function in the R language.

[0028] The results are as follows Figure 1 As shown, Figure 1 Using the TCGA public database, we investigated the role of the ILDR1 gene in HCC survival. Survival time in HCC patients was analyzed on a monthly basis. A comparison of the blue line (ILDR1 low expression group) and the yellow line (ILDR1 high expression group) in the figure reveals that the survival rate of patients in the ILDR1 low expression group was significantly higher than that in the ILDR1 high expression group. This suggests that low ILDR1 expression can delay death in HCC patients. Therefore, ILDR1 can serve as a prognostic indicator gene for the prognosis of primary HCC.

[0029] Example 2 Construction of transient transfection cell line

[0030] The siRNA transient transfection experiment used small interfering RNA (siRNA) with EGFP provided by Anhui General Biotechnology Co., Ltd. to transiently transfect HCCLM3 cells. The specific small interfering RNA sequence is shown in Table 1 below.

[0031] The specific operation steps are as follows: (1) 2×10^5 HCCLM3 cells were seeded into a sterile 6-well culture plate. After the cells adhered, the serum-free medium was replaced and the cells were starved for 2 hours. (2) For each transfection sample, the complex was prepared according to the following steps: Dilute the plasmid with 250μl serum-free DMEM medium. Gently mix Lipofectamine 2000 at the same time, and then take 4ul and dilute it in 250μl serum-free DMEM medium. Incubate at room temperature for 5 minutes. Then mix the diluted DNA and diluted Lipofectamine 2000 (total volume = 500μL). Gently mix and incubate at room temperature for 20 minutes. After mixing, the above mixture was added to the 6-well plate. After incubation at 37℃ for 4 hours, the cell culture medium was replaced with complete cell culture medium. After incubating the cells for 24 hours, the protein expression of ILDR1 was identified by immunoblotting.

[0032] Table 1 Small interfering RNA sequences

[0033]

[0034]

[0035] The cell counting kit is used to detect cell proliferation. The operation method includes: first, cell plating, digesting the stable cell line and the control cell line and then resuspending them, using a hemocytometer to count the cells, adjusting the number of cells, inoculating 2000 cells per well in a 96-well plate, and mixing the above cells using the 8-shaped shaking method. Cell counting kit-8 (CCK8) is used to detect cell proliferation. The cells are inoculated in a 96-well plate with 2000 cells per well. After the cells are transfected, CCK-8 reagent is added at 4, 24, 48, and 72 hours of culture, respectively. After incubation in the incubator for 1 hour, the absorbance at 450 nm is measured using a microplate reader to evaluate cell proliferation.

[0036] Figure 2This was achieved by using the CCK8 experimental method to investigate the effect of the ILDR1 gene on the cell proliferation rate of the liver cancer cell line HCCLM3. As shown in the comparison of siCON (black line) and siILDR1 (yellow line) in the above figure, after using small interfering RNA to affect the expression of ILDR1, the absorbance at 450nm of the siCON group was significantly higher than that of the siILDR1 group, indicating that the proliferation of HCCLM3 cells in the siILDR1 group was significantly slowed. The experiment showed that ILDR1 inhibition can significantly delay tumor cell proliferation. Therefore, ILDR1 inhibition can be used as a potential therapeutic target for primary liver cancer.

[0037] Example 3 Correlation analysis between genes in the TCGA database

[0038] Tumor data from the TCGA-LIHC data were extracted and standardized. Pearson correlation analysis was then performed on the expression values ​​of ILDR1 and GLS. The expression of ILDR1 was significantly positively correlated with that of GLS, with a correlation coefficient of 0.42. The ggplot2 function in R language was used to fit a curve showing the correlation between the expression of the two genes in the TCGA-LIHC database.

[0039] The data of tumor patients in the TCGA-LIHC data were extracted and standardized, and then the expression values ​​of ILDR1 and GLS were subjected to Pearson correlation analysis, as shown in Figure 2. Figure 3 As shown, the expression value of the ILDR1 gene was used as the X-axis and the expression value of the GLS gene was used as the Y-axis, and the expression values ​​of the two genes in each patient were subjected to Pearson correlation analysis. The above data showed that the expression of ILDR1 was significantly positively correlated with that of GLS, and the correlation coefficient was 0.42, indicating that the GLS gene and the IDLR1 gene were highly correlated in liver cancer.

[0040] Example 4

[0041] 1. Treat HCCLM3 cells with the GLS activity inhibitor Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES). BPTES (Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide) is a selective glutamate dehydrogenase (GLS) inhibitor. BPTES specifically binds to and inhibits GLS activity, reducing the conversion of glutamine to glutamate. After harvesting cells, plate 2 x 10^5 cells per well in a 6-well plate. After 12 hours of cell attachment, add 2 μl of 10 mg / ml BPTES solution to each well. Wait 48 hours before collecting protein samples for immunoblotting.

[0042] 2. The specific experimental steps of the immunoblotting experiment are as follows: (1) Cell lysis: After washing the cells in the culture dish, add NP40 cell lysis solution and gently shake on ice for 15-30 minutes; (2) Cell disruption: Use ultrasonic equipment to treat the cells and obtain the required cell lysis solution by high-speed centrifugation (12,000 rpm, 15 minutes); (3) BCA protein quantification: Add the sample to be tested and the protein standard to a 96-well plate, add BCA protein colorimetric solution, incubate in a 37°C incubator for 15-30 minutes, measure the absorbance and draw a standard curve to calculate the protein concentration of the sample; (4) Protein sample processing: After the quantification is completed, take a certain amount of protein, add 1 / 4 volume of 5×SDS, and incubate at 100°C Heat for 5 minutes to denature the protein, then cool the sample in an ice bath for 2 minutes to complete the preparation; (5) Perform protein electrophoresis: After preparing the SDS-PAGE gel, load the sample into a vertical electrophoresis tank and perform electrophoresis separation (constant voltage 120V, 90 minutes); (6) Transfer and blocking: After the electrophoresis is completed, transfer the gel to the NC membrane (add enough electrotransfer solution according to the prompt scale, transfer the membrane at a constant current of 0.23A for 90 minutes, and perform blocking treatment; (7) Antibody incubation and detection: After washing the NC membrane, incubate the primary antibody at 4°C overnight (or at room temperature for 2 hours). After the incubation is completed, wash the membrane and incubate the corresponding fluorescent-labeled secondary antibody; (8) Band detection: After the incubation is completed, wash the NC membrane and then detect it on the Odyssey fluorescence scanner.

[0043] Figure 4 This figure shows immunoblot results examining changes in ILDR1 expression in HCCLM3 cells after 48 hours of treatment with a GLS inhibitor. As shown, the GLS band became thinner after 48 hours of treatment, indicating decreased GLS protein expression. However, the ILDR1 band remained unchanged, indicating minimal changes in ILDR1 protein expression. This suggests that inhibiting GLS expression has no effect on ILDR1 expression, indicating that GLS cannot regulate ILDR1.

[0044] Example 5

[0045] Lentiviral infection of cells and construction of stable knockdown ILDR1 cell line: The shRNA stable transfection experiment used the lentiviral infection sequence of shRNA with EGFP provided by Anhui General Biotechnology Co., Ltd. The lentiviral infection sequence is shown in Table 2 below, and HCCLM3 cells were stably transfected. The specific operation steps are as follows: (1) 2×10^5 HCCLM3 cells were seeded in a sterile 6-well culture plate. After the cells adhered to the wall, fresh culture medium was replaced and 1ul of polybrene (polycationic polymer) at a concentration of 10mg / ml was added; (2) The infection dose (MOI=20) was determined based on the pre-screened virus titer, and the lentivirus with the corresponding MOI value was added to the culture dish and placed in an incubator for incubation for 12-16h; (3) After 12 hours, fresh culture medium was replaced. After 24 hours, the cell fluorescence can be observed using a fluorescence microscope; (4) 48 hours after infection, puromycin (2 μg / mL) is added for screening. After the cells are cultured and stabilized for 2 weeks, immunoblotting experiments are used to test ILDR1 protein expression to ensure the transfection effect of the cells used in the experiment.

[0046] Table 2 Lentivirus-constructed shRNA sequences

[0047]

[0048]

[0049] Figure 5 The following is an immunoblot analysis showing changes in GLS and ILDR1 expression following ILDR1 knockdown. As shown, HCCLM3 cells were infected with a lentiviral vector to knock down the ILDR1 gene. Both ILDR1 knockdown methods significantly reduced the thickness of the ILDR1 protein band, indicating a significant decrease in ILDR1 expression. Concurrently, the GLS protein band also became significantly thinner in both knockdown methods, indicating a significant decrease in GLS protein expression. This demonstrates that inhibiting ILDR1 expression significantly affects GLS expression, indicating that ILDR1 can significantly regulate GLS expression.

[0050] Example 6

[0051] Cell lentiviral infection and construction of stably overexpressed ILDR1 cell lines: The lentiviral infection experiment used the ILDR1 interference lentivirus and its control lentivirus (Control and AAV_ILDR1) provided by Shanghai Heyuan Biotechnology Co., Ltd.

[0052] The specific operation steps are as follows: (1) 2×10^5 HCCLM3 cells were inoculated into a sterile 6-well culture plate. After the cells adhered to the wall, fresh culture medium was replaced and 20ul, 1ug / ul Polybrene Plus was added; (2) The infection dose (MOI = 20) was determined based on the pre-screened virus titer, and the lentivirus with the corresponding MOI value was added to the culture dish and incubated in an incubator for 12 hours; (3) Fresh culture medium was replaced after 12 hours, and cell fluorescence could be observed using a fluorescence microscope after 24 hours; (4) Puromycin (2μg / mL) was added for screening 48 hours after infection. After the cells were cultured and stabilized for 2 weeks, the ILDR1 protein expression was tested using immunoblotting to ensure the transfection effect of the cells used in the experiment.

[0053] Figure 6 This is an immunoblot result diagram that detects the expression changes of the genes GLS and ILDR1 after ILDR1 overexpression. As shown in the figure, the HCCLM3 cell line was infected with a lentivirus to overexpress the ILDR1 gene. In the ILDR1 overexpression cell model, the thickness of the ILDR1 protein band became significantly thicker, which means that the expression level of ILDR1 was significantly increased. At the same time, the GLS protein band in the two overexpression cell lines also became significantly thicker, indicating that the protein expression level of the GLS gene was significantly increased. Figure 5 and Figure 6 This indicates that increasing the expression level of ILDR1 has a significant effect on the expression level of the GLS gene, indicating that the ILDR1 gene can significantly regulate the expression level of GLS.

[0054] In summary, ILDR1 is an important regulatory factor in regulating GLS protein expression.

[0055] Example 7

[0056] Differential analysis and enrichment analysis of the expression of ILDR1 gene in the TCGA-LIHC database: The study used the TCGA-LIHC database to divide the samples into high expression group (top 75%) and low expression group (bottom 25%) according to the expression level of ILDR1 gene. Subsequently, gene differential analysis and KEGG enrichment analysis were performed. KEGG (Kyoto Encyclopedia of Genes and Genomes) is a comprehensive biological database developed and maintained by Kyoto University in Japan, which aims to systematically integrate genomes, chemicals and their system function information. KEGG enrichment analysis is a bioinformatics method used to identify and explain the significant enrichment of specific gene sets in various biological pathways. Through this analysis, researchers can gain an in-depth understanding of the role of selected gene sets in biological processes, signal transduction pathways and their potential functions.

[0057] Figure 7 Analysis of differentially expressed genes in the TCGA-LIHC database revealed that differentially expressed genes were enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Groups were grouped by ILDR1 expression in the TCGA-LIHC database, with high (top 75%) and low (bottom 25%), followed by gene differential analysis and KEGG enrichment analysis. As shown in the figure, the X-axis represents the number of genes in the pathway in which the differentially expressed genes were enriched, and the Y-axis represents the signaling pathway in which the differentially expressed genes were enriched. The colors represent the adjusted P-values ​​for the pathways above, with darker colors indicating smaller P-values ​​and greater likelihood of enrichment. As shown above, differentially expressed genes between the ILDR1 high and low expression groups were mainly enriched in the following pathways: neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, and PI3K-Akt signaling pathway.

[0058] The PI3K-AKT signaling pathway can further regulate GLS expression by regulating the MYC gene, which is consistent with the theory that ILDR1 can regulate GLS. Therefore, the degree of activation of the PI3K-AKT signaling pathway and the expression of the MYC gene in ILDR1 overexpression and knockdown stable cell lines were investigated.

[0059] Example 8

[0060] Refer to the steps in Example 5 to obtain Figure 8 , Figure 8The following are immunoblot results examining changes in ILDR1, AKT, p-AKT, MYC, and GLS expression following ILDR1 knockdown. As shown, HCCLM3 cells were infected with lentivirus to knock down the ILDR1 gene. Both ILDR1 knockdown methods significantly reduced the thickness of the ILDR1 protein band, indicating a significant decrease in ILDR1 expression. Conversely, the AKT protein band remained unchanged in both knockdown methods, indicating no significant change in AKT protein expression. Furthermore, the p-AKT protein band became significantly thinner in both knockdown methods, indicating a significant decrease in p-AKT protein expression. This indicates a significant decrease in AKT phosphorylation, significantly reducing phosphorylation and activation of the PI3K-AKT signaling pathway. The MYC protein band also became significantly thinner in both knockdown methods, indicating a significant decrease in MYC protein expression. The GLS protein band also became significantly thinner in both knockdown methods, indicating a significant decrease in GLS protein expression. The above results show that inhibiting the expression level of ILDR1 has a significant effect on the expression levels of AKT, p-AKT, MYC, and GLS genes, indicating that the ILDR1 gene can significantly regulate the expression levels of AKT, p-AKT, MYC, and GLS.

[0061] Example 9

[0062] Refer to the steps in Example 6 to obtain Figure 9 , Figure 9 This is the immunoblotting result diagram for detecting the expression changes of genes ILDR1, AKT, p-AKT, MYC, and GLS after ILDR1 overexpression. Figure 9As shown, the HCCLM3 cell line was infected with a lentivirus to overexpress the ILDR1 gene. In the ILDR1-overexpressing cell model, the thickness of the ILDR1 protein band increased significantly, indicating that ILDR1 overexpression significantly increased ILDR1 expression. Meanwhile, the AKT protein band did not change significantly in the ILDR1-overexpressing cells, indicating that AKT protein expression levels did not change significantly. Furthermore, the p-AKT protein band in the ILDR1-overexpressing cells became significantly thicker, indicating a significant increase in p-AKT protein expression levels. This indicates a significant increase in AKT phosphorylation, indicating significant phosphorylation and activation of the PI3K-AKT signaling pathway. Furthermore, the MYC protein band in the ILDR1-overexpressing cells also became significantly thicker, indicating a significant increase in MYC protein expression levels. Furthermore, the GLS protein band in the ILDR1-overexpressing cells also became significantly thicker, indicating a significant increase in GLS protein expression levels. The above results show that upregulating the expression level of ILDR1 has a significant effect on the expression levels of AKT, p-AKT, MYC, and GLS genes, indicating that the ILDR1 gene can significantly regulate the expression levels of AKT, p-AKT, MYC, and GLS.

[0063] Combine Figure 8 and Figure 9 , which indicates that the ILDR1 gene can significantly regulate the expression levels of AKT, p-AKT, MYC, and GLS.

[0064] Example 10

[0065] Refer to the steps in Example 6 and treat cells with the AKT-specific inhibitor MK2006, a highly selective Akt1 / 2 / 3 inhibitor. MK-2206 is an allosteric inhibitor activated by the pleckstrin homology domain. MK-2206 inhibits autophosphorylation of Akt at threonine 308 and serine 473. First, collect cells and plate 2*10^5 cells per well in a 6-well plate. After 12 hours of cell attachment, add 2 μl of 10 mg / ml MK-2206 solution to each well. Wait 48 hours before collecting protein samples for immunoblotting.

[0066] MYC specific inhibitor 5-(4-ethylbenzylidene) rhodanine (10058-F4) can reduce the level of c-Myc protein in cells. First, collect cells, then plate 2*10^5 cells per well in a 6-well plate. After 12 hours, after the cells adhere to the wall, add 2ul of 10mg / ml MK-2206 solution to each well. After waiting for 48 hours, collect protein samples for immunoblotting. Figure 10 .

[0067] Figure 10 To validate the specificity of the ILDR1-PI3K-AKT-cMYC-GLS signaling axis using immunoblotting, the AKT-specific inhibitor MK2006 (Cat. No.: Biyuntian SF2717) and the MYC-specific inhibitor 10058-F4 (Cat. No.: Aibixin abs810789) were added to ILDR1-overexpressing cell lines. As shown in the figure, protein expression of ILDR1, p-AKT, MYC, and GLS increased significantly with increased ILDR1 gene expression in the ILDR1-overexpressing cell lines, consistent with the results in the previous figure and further supporting the aforementioned conclusions. However, treatment with the AKT-specific inhibitor MK2006 abolished the positive regulatory effect of ILDR1 on MYC and GLS protein expression in the ILDR1-overexpressing cell lines, indicating that ILDR1 regulation of MYC and GLS is dependent on the PI3K-AKT signaling pathway. Furthermore, after adding the MYC inhibitor 10058-F4 to the ILDR1-overexpressing cell line, the regulatory effect of ILDR1 on GLS protein expression also disappeared, suggesting that ILDR1's regulation of GLS is achieved through MYC.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Use of an ILDR1 inhibitor in the preparation of a drug for treating hepatocellular carcinoma, characterized in that: The ILDR1 inhibitor is an ILDR1 small interfering RNA, and the sequence of the ILDR1 small interfering RNA is: S1 Justice Chain: AGGCAAGAAUAGCAGGAAATT Antisense strand: UUUCCUGCUAUUCUUGCCUTT; S2 Justice Chain: GAGCAGAUCUCGUGAUAAATT Antisense strand: UUUAUCACGAGAUCUGCUCTT; S3 Justice Chain: CCAAUGGUGUCCUGGAGUATT Antisense strand: UACUCCAGGACACCAUUGGTT.

2. Use of an ILDR1 inhibitor in the preparation of a targeted therapeutic drug for treating primary liver cancer, characterized in that: The ILDR1 inhibitor is an ILDR1 small interfering RNA, and the sequence of the ILDR1 small interfering RNA is: S1 Justice Chain: AGGCAAGAAUAGCAGGAAATT Antisense strand: UUUCCUGCUAUUCUUGCCUTT; S2 Justice Chain: GAGCAGAUCUCGUGAUAAATT Antisense strand: UUUAUCACGAGAUCUGCUCTT; S3 Justice Chain: CCAAUGGUGUCCUGGAGUATT Antisense strand: UACUCCAGGACACCAUUGGTT.

3. The use according to any one of claims 1 or 2, characterized in that The ILDR1 inhibitor inhibits the proliferation ability of the liver cancer cell line HCCLM3 in vitro.

4. The use according to any one of claims 1 or 2, characterized in that The ILDR1 inhibitor suppressed the protein expression of GLS in the liver cancer cell line HCCLM3.

5. The use according to any one of claims 1 or 2, characterized in that The ILDR1 inhibitor inhibited the activity of the PI3K-AKT signaling pathway in the liver cancer cell line HCCLM3.

6. The use according to any one of claims 1 or 2, characterized in that The ILDR1 inhibitor suppressed GLS protein expression in the liver cancer cell line HCCLM3 through the PI3K-AKT-MYC signaling axis.

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