Application of ILDR1 inhibitor in preparation of hepatocellular carcinoma drugs

Abstract

According to the application of the ILDR1 inhibitor in preparation of the hepatocellular carcinoma medicine, the ILDR1 inhibitor inhibits proliferation of hepatocellular carcinoma cells, and the ILDR1 is a new target for treating HCC and a biomarker related to diagnosis and prognosis. In-vitro experiments find that in an HCC cell line HCCLM3, down-regulation of the ILDR1 significantly inhibits the proliferation ability of tumors, down-regulation of the ILDR1 inhibits transcription of glutamine transaminase (GLS) which is a key enzyme for glutamine metabolism and enables down-regulation of the protein expression quantity of the GLS, and down-regulation of the ILDR1 may realize inhibition of the GLS by inhibiting an AKT signal channel 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 about 90% of all liver cancer cases. According to the latest statistics from the World Health Organization (WHO), HCC ranks fifth among all common cancers in the world and is the third leading cause of cancer-related death. This situation is particularly serious in my country, where the incidence of HCC ranks fourth among all tumors, but its mortality rate ranks second, seriously threatening the lives, health, and socioeconomic development of the Chinese people.

[0003] There are significant differences in the risk factors for HCC around the world, with hepatitis B virus (HBV) infection being dominant in Asia, hepatitis C virus (HCV) infection being more common in Japan, and non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and chronic alcoholism becoming 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 declined. However, the incidence of liver cancer associated with NAFLD and NASH is on the rise. NAFLD is considered to be a 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 interventions targeting metabolic pathways may become new treatment strategies.

[0004] Metabolic reprogramming of tumor cells is one of their prominent characteristics, a process that helps tumor cells adapt to rapid proliferation and high energy demands. In order to meet these demands, the metabolic pathways of tumor cells undergo significant changes, including enhanced sugar 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 development. 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 sugar metabolism, lipid metabolism, and amino acid metabolism, which not only re-meets their own energy needs, but also regulates gene expression and protein modification, ultimately promoting the proliferation and spread of tumor cells.

[0005] Glutamine plays multiple key roles in the process of tumor metabolic reprogramming. First, glutamine generates α-ketoglutarate (α-KG) through deamination and enters the tricarboxylic acid cycle (TCA cycle) to provide energy for tumor cells with high proliferation rates. Secondly, 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, glutathione (GSH) generated by glutamine metabolism plays a key role in maintaining cellular redox balance, enhancing the resistance of tumor cells to oxidative stress and chemotherapeutic drugs, and further promoting tumor survival and drug resistance. Glutaminase (GLS), as a key enzyme in glutamine metabolism, is responsible for converting glutamine to glutamate and participates in the TCA cycle and multiple biosynthetic pathways. GLS is crucial in glutamine metabolism in tumor cells. A large number of 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 tumor metabolic heterogeneity and drug resistance, GLS inhibitors have not yet widely benefited all cancer 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 participates in auditory signal transduction and potential immunoregulatory functions mainly 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 yet been fully elucidated. In the process of maintaining tight junctions between cells, ILDR1 may affect the growth and metabolic state of hepatocytes by regulating metabolic pathways, thereby playing a role in the occurrence and development of HCC. Based on this, the present invention provides the use of ILDR1 inhibitors in the preparation of hepatocellular carcinoma drugs. Summary of the invention

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

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

[0009] Furthermore, the ILDR1 inhibitor inhibited the proliferation ability of the liver cancer cell line HCCLM3 in vitro.

[0010] Furthermore, the ILDR1 inhibitor inhibited the protein expression of GLS in the liver cancer cell line HCCLM3.

[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 cell line through the PI3K-AKT-MYC signaling axis.

[0013] Beneficial effects:

[0014] The present invention shows that ILDR1 inhibitors inhibit the proliferation of liver cancer cells, and ILDR1 is a new target for the treatment of HCC and a biomarker related to diagnosis and prognosis. In vitro experiments show that in the HCCLM3 cell line, the downregulation of ILDR1 significantly inhibits the proliferation ability of tumors. Further in vitro experiments found that the downregulation of ILDR1 inhibits the transcription of glutamine metabolism key enzyme glutamine aminotransferase (GLS) and reduces its protein expression, and the downregulation of ILDR1 inhibits GLS by inhibiting 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 It is a graph detecting the proliferation rate of HCCLM3 cell line 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 picture of the immunoblotting result of detecting the expression change of the gene ILDR1 after the HCCLM3 cell line was treated with the GLS activity inhibitor for 48 hours;

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

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

[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 result of immunoblotting to detect the expression changes of genes ILDR1, AKT, p-AKT, MYC, and GLS after ILDR1 knockdown;

[0023] Fig. 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] Fig.10 Immunoblotting was used to validate the specificity of the ILDR1-PI3K-AKT-cMYC-GLS signaling axis. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0027] TCGA Liver hepatocellular carcinoma was downloaded from the UCSC Xena platform (https: / / xena.ucsc.edu) on March 22, 2024, and the expression matrix, gene names, and related survival data were organized using R language. Then, the high (top 75%) and low (bottom 25%) expression of ILDR1 in the LIHC dataset were grouped, and the actual number of months the patients survived was calculated by dividing the survival time of each group by 12. The survival function in R language was used to fit the above data to the survival curve.

[0028] The results are as follows Figure 1 As shown, Figure 1 The TCGA public database was used to explore the analysis of the gene ILDR1 on liver cancer survival. The survival time of liver cancer patients was analyzed according to the month as the time. As shown in the figure, the blue line (ILDR1 low expression group) and the yellow line (ILDR1 high expression group) were compared and found that the survival rate of patients in the ILDR1 low expression group was significantly higher than that in the ILDR1 high expression group. That is, low expression of ILDR1 can delay the death of HCC patients. Therefore, ILDR1 can be used as a prognostic indicator gene for the prognosis evaluation of primary liver cancer.

[0029] Example 2 Construction of transient transfection cell line

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

[0031] 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, the serum-free medium was replaced to starve the cells for 2 hours; (2) For each transfection sample, the complex was prepared according to the following steps: Use 250μl of serum-free DMEM medium to dilute the plasmid. At the same time, gently mix Lipofectamine 2000, 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, add the above mixture to the 6-well plate, incubate at 37℃ for 4 hours, and then replace the cell culture medium with complete cell culture medium. After incubating the cells for 24 hours, use immunoblotting to identify the protein expression of ILDR1.

[0032] Table 1 Small interfering RNA sequences

[0033]

[0034]

[0035] The operation method of the cell counting kit for detecting cell proliferation includes: first, cell plating, digesting and resuspending the stable cell line and the control cell line, 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) was used to detect cell proliferation, and the cells were inoculated in a 96-well plate with 2000 cells per well. After the cells were transfected, CCK-8 reagent was added at 4, 24, 48, and 72 hours of culture, respectively. After incubation in the incubator for 1 hour, the absorbance at 450nm was measured using an enzyme reader to evaluate cell proliferation.

[0036] Figure 2This was obtained by using the CCK8 experimental method to study the effect of 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 of the siCON group at 450nm was significantly higher than that of the siILDR1 group, which means that the proliferation of HCCLM3 cells in the siILDR1 group was significantly slowed down. Experiments have shown 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 TCGA database

[0038] The Tumor data in the TCGA-LIHC data were extracted and standardized, and then the expression values ​​of ILDR1 and GLS were subjected to Pearson correlation analysis. The expression of ILDR1 was significantly positively correlated with that of GLS, with a correlation coefficient of 0.42. The ggplot2 function in the R language was used to fit the curve of 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 in the above were subjected to Pearson correlation analysis, as shown in Figure 2. Figure 3 As shown, with the expression value of ILDR1 gene as the X-axis and the expression value of GLS gene as the Y-axis, 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 GLS, and the correlation coefficient was 0.42, indicating that the GLS gene and IDLR1 gene were highly correlated in liver cancer.

[0040] Example 4

[0041] 1. Treat HCCLM3 cells with Bis-2-(5-phenylacetylamino-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a GLS activity inhibitor. BPTES (Bis-2-(5-phenylacetylamino-1,3,4-thiadiazol-2-yl)ethyl sulfide) is a selective glutamate dehydrogenase (Glutaminase, GLS) inhibitor. BPTES can specifically bind to and inhibit the activity of GLS, reducing the conversion of glutamine to glutamate. After collecting cells, 2*10^5 cells were plated per well in a 6-well plate. After 12 hours, the cells adhered to the wall, and 2ul of 10mg / ml BPTES solution was added to each well. After waiting for 48 hours, protein samples were collected for immunoblotting experiments.

[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 (12,000rpm, 15min); (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) Process protein samples: After quantification, 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 electrophoretic separation (constant voltage 120V, 90 minutes); (6) Transfer and block: 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 detect it on the Odyssey fluorescence scanner.

[0043] Figure 4 This is a result of immunoblotting to detect the expression changes of the gene ILDR1 after 48 hours of using the GLS activity inhibitor on the HCCLM3 cell line. As shown in the figure, after 48 hours of using the GLS inhibitor, the thickness of the GLS band became thinner, indicating that the protein expression level of the GLS gene decreased, but the band of the ILDR1 gene did not change significantly, indicating that the protein expression level of the ILDR1 gene did not change much. The above shows that inhibiting the expression level of GLS has no effect on the expression of the ILDR1 gene, indicating that GLS cannot regulate ILDR1.

[0044] Example 5

[0045] Cell lentiviral infection 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 inoculated in a sterile 6-well culture plate. After the cells adhered to the wall, fresh culture medium was replaced and 1ul of polybrene (Polybrene) polycationic polymer with a concentration of 10mg / ml was added; (2) The infection dose (MOI=20) was determined according to the pre-screened virus titer, and the lentivirus with the corresponding MOI value was added to the culture dish and placed in the 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 stabilized after 2 weeks of culture, immunoblotting is used to test the 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 This is an immunoblot result diagram for detecting the expression changes of the genes GLS and ILDR1 after ILDR1 knockdown. As shown in the figure, the HCCLM3 cell line was infected with a lentivirus to knock down the ILDR1 gene, and the two knockdown methods of ILDR1 significantly reduced the thickness of the ILDR1 protein band, that is, significantly reduced the expression level of ILDR1. At the same time, the protein band of GLS in the two knockdown methods also became significantly thinner, indicating that the protein expression level of the GLS gene was significantly reduced. The above shows that inhibiting 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.

[0050] Example 6

[0051] Cell lentiviral infection and construction of stably overexpressed ILDR1 cell line: 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) Inoculate 2×10^5 HCCLM3 cells in a sterile 6-well culture plate. After the cells adhere to the wall, replace the culture medium with fresh medium and add 20ul, 1ug / ul Polybrene Plus; (2) Determine the infection dose (MOI=20) based on the pre-screened virus titer, add the lentivirus with the corresponding MOI value to the culture dish, and incubate it in an incubator for 12h; (3) Replace the culture medium with fresh medium after 12 hours, and observe the cell fluorescence under a fluorescence microscope after 24h; (4) After 48h of infection, add puromycin (2μg / mL) for screening. After the cells have been cultured and stabilized for 2 weeks, use immunoblotting to test the ILDR1 protein expression to ensure the transfection effect of the cells used in the experiment.

[0053] Figure 6 This is a picture of the immunoblotting results for detecting 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 cell model of ILDR1 overexpression, the thickness of the ILDR1 protein band became significantly thicker, that is, the expression level of ILDR1 was significantly increased. At the same time, the protein band of GLS in the two overexpression cell lines also became significantly thicker, indicating that the protein expression level of the GLS gene increased significantly. 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 for 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 gene differences between high and low expression of ILDR1 in the TCGA-LIHC database Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed on the differentially expressed genes. In the TCGA-LIHC database, the ILDR1 expression was grouped according to high (first 75%) and low (last 25%), and then gene difference analysis and KEGG enrichment analysis were performed. As shown in the figure, the X-axis represents the number of genes in the pathway in which the differentially expressed genes are enriched, the Y-axis represents the signal pathway in which the differentially expressed genes are enriched, and the color represents the adjusted P value of the above pathways, where the darker the color, the smaller the P value, and the greater the possibility of enrichment. According to the above, the differentially expressed genes between the high and low expression groups of ILDR1 are mainly enriched in the following pathways, neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, etc.

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

[0059] Example 8

[0060] Refer to the steps in Example 5 to obtain Figure 8 , Figure 8This is a result of immunoblotting to detect the expression changes of genes ILDR1, AKT, p-AKT, MYC, and GLS after ILDR1 knockdown. As shown in the figure, the HCCLM3 cell line was infected with lentivirus to knock down the ILDR1 gene, and the two knockdown methods of ILDR1 significantly reduced the thickness of the protein band of ILDR1, that is, significantly reduced the expression level of ILDR1. At the same time, the protein band of AKT did not change significantly in the two knockdown methods, indicating that the protein expression level of the AKT gene did not change significantly. At the same time, the protein band of p-AKT in the two knockdown methods became significantly thinner, indicating that the protein expression level of the p-AKT gene decreased significantly, that is, the level of phosphorylation of the gene AKT decreased significantly, that is, the degree of phosphorylation of the PI3K-AKT signaling pathway decreased significantly, and the degree of activation decreased significantly. At the same time, the protein band of MYC in the two knockdown methods also became significantly thinner, indicating that the protein expression level of the MYC gene decreased significantly. At the same time, the protein band of GLS in the two knockdown methods also became significantly thinner, indicating that the protein expression level of the GLS gene decreased significantly. The above shows 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 Fig. 9 , Fig. 9 This is the result of immunoblotting to detect the expression changes of genes ILDR1, AKT, p-AKT, MYC, and GLS after ILDR1 overexpression. Fig. 9As shown, the HCCLM3 cell line was infected with a lentivirus to overexpress the ILDR1 gene, wherein in the cell model of ILDR1 overexpression, the thickness of the ILDR1 protein band became significantly thicker, that is, ILDR1 overexpression significantly increased the expression level of ILDR1. At the same time, the protein band of AKT in ILDR1 overexpressing cells did not change significantly, indicating that the protein expression level of the AKT gene did not change significantly. At the same time, the protein band of p-AKT in ILDR1 overexpressing cells became significantly thicker, indicating that the protein expression level of the p-AKT gene increased significantly, that is, the level of phosphorylation of gene AKT increased significantly, that is, the PI3K-AKT signaling pathway was significantly phosphorylated, that is, the PI3K-AKT signaling pathway was significantly activated. At the same time, the protein band of MYC in ILDR1 overexpressing cells also became significantly thicker, indicating that the protein expression level of the MYC gene increased significantly. At the same time, the protein band of GLS in ILDR1 overexpressing cells also became significantly thicker, indicating that the protein expression level of the GLS gene increased significantly. The above shows 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] Combination Figure 8 and Fig. 9 This 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 AKT-specific inhibitor MK2006, which is a highly selective Akt1 / 2 / 3 inhibitor. MK-2206 is an allosteric inhibitor activated by the pleckstrin homology domain. MK-2206 inhibits the autophosphorylation of Akt at threonine 308 and serine 473. 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, wait for 48 hours, and collect protein samples for immunoblotting experiments.

[0066] MYC specific inhibitor 5-(4-ethylbenzylidene) rhodanine (10058-F4), 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, 10mg / ml MK-2206 solution to each well. After waiting for 48 hours, collect protein samples for immunoblotting experiments. Fig.10 .

[0067] Fig.10 To verify the specificity of the ILDR1-PI3K-AKT-cMYC-GLS signaling axis using immunoblotting experiments, AKT-specific inhibitor MK2006 (Cat. No.: Biyuntian SF2717) and MYC-specific inhibitor 10058-F4 (Cat. No.: Aibixin abs810789) were added to the ILDR1-overexpressing cell line. As shown in the figure, the protein expression of ILDR1, p-AKT, MYC and GLS in the ILDR1-overexpressing cell line increased significantly with the increase of ILDR1 gene expression, which is consistent with the results in the above figure, and once again proves the above conclusion. However, after adding the AKT-specific inhibitor MK2006 to the ILDR1-overexpressing cell line, the positive regulatory effect of ILDR1 on MYC and GLS protein expression disappeared, indicating that the regulation of ILDR1 on MYC and GLS depends 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 the regulation of ILDR1 on 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. Application of ILDR1 inhibitors in the preparation of hepatocellular carcinoma drugs.

2. Application of ILDR1 inhibitors in the preparation of drugs for early diagnosis, prognosis assessment or targeted therapy of primary liver cancer.

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 inhibited 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 inhibited the protein expression of GLS in the liver cancer cell line HCCLM3 cell line through the PI3K-AKT-MYC signaling axis.

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