SLC25A10 promotes drug resistance to hepatocellular carcinoma

Abstract

The present application relates to SLC25A10 leading to drug resistance of liver cancer cells, specifically, liver cancer cells are in a hypoxic environment, and hypoxia enhances the expression of SLC25A10 and triggers the splicing variation of SLC25A10 from isomer 1 to isomer 3. Isomer 3 of SLC25A10 enters the nucleus by combining with nuclear transport protein IPO7, and combines with transcription factor CEBPB, up-regulates the expression of BCL2A1, thereby enhancing the drug resistance of HCC cells to etoposide. According to the mechanism, a screening method and kit for developing drugs for treating drug resistance of liver cancer are developed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to SLC25A10 promoting drug resistance to liver cancer. BACKGROUND

[0002] Liver cancer, especially hepatocellular carcinoma (HCC), is one of the malignant tumors with high mortality and rising incidence worldwide. Chemotherapy, targeted therapy and immunotherapy are the main treatment methods for liver cancer at present. Radiotherapy and chemotherapy can kill cancer cells, but also cause harm to normal cells and trigger a series of side effects. Moreover, liver cancer cells are prone to drug resistance to various chemotherapy drugs and targeted therapy drugs, which greatly limits the treatment effect.

[0003] In recent years, with the progress of molecular biology and drug research and development technology, targeted therapy and immunotherapy have become a new hotspot in the treatment of liver cancer. Targeted therapy drugs act specifically on specific molecular markers on cancer cells, improving the treatment effect and reducing side effects. However, the multi-drug resistance (MDR) phenomenon of liver cancer is still one of the main difficulties faced by clinical treatment. Immunotherapy kills cancer cells by activating the patient's own immune system. Although immunotherapy brings new hope to patients with advanced liver cancer, its efficacy is still limited and may cause immune-related adverse reactions.

[0004] Therefore, there is an urgent need in the art for a screening method for drugs treating liver cancer drug resistance. SUMMARY

[0005] The present application relates to the field of biological medicine, and particularly relates to SLC25A10 promoting drug resistance to liver cancer.

[0006] The first aspect of the present application provides a method for screening candidate compounds for treating liver cancer, comprising the steps of:

[0007] (s1) testing group, culturing liver cancer cells in a culture system containing a test compound, and measuring data selected from the following group for the test group:

[0008] (a) the number of first complexes formed by SLC25A10 isoform 3 and IPO7 A1; and / or

[0009] (b) the number of second complexes formed by SLC25A10 isoform 3 and CEBPB B1;

[0010] and in the control group, culturing liver cancer cells in a culture system with the same conditions and without the test compound, and measuring data selected from the following group for the control group:

[0011] (c) the number of first complexes formed by SLC25A10 isoform 3 and IPO7 A0; and / or

[0012] (d) the number of the second complex B0 formed by SLC25A10 isoform 3 and CEBPB; and

[0013] (s2) comparing the number of the first complex and / or the second complex in the test group and the control group,

[0014] wherein if the number of the first complex Al in the test group is significantly lower than the number of the first complex A0 in the control group, and / or the number of the second complex Bl in the test group is significantly lower than the number of the second complex B0 in the control group, it suggests that the test compound is a candidate compound for treating liver cancer.

[0015] In another preferred embodiment, the first complex is detected by a first detection reagent set, wherein the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a second antibody against IPO7.

[0016] In another preferred embodiment, the first antibody and the second antibody each independently carries a detectable label, and / or is coupled to a magnetic bead.

[0017] In another preferred embodiment, the second complex is detected by a second detection reagent set, wherein the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a third antibody against CEBPB.

[0018] In another preferred embodiment, the first antibody and the third antibody each independently carries a detectable label, and / or is coupled to a magnetic bead.

[0019] In another preferred embodiment, the first antibody, the second antibody, and the third antibody each independently carries a detectable label, and / or is coupled to a magnetic bead.

[0020] In another preferred embodiment, the detection of the first complex and / or the detection of the second complex employs a method selected from the group consisting of ELISA, immunoprecipitation, or a combination thereof.

[0021] In another preferred embodiment, the test compound is selected from the group consisting of an antibody or a binding fragment thereof, a small molecule compound, a nucleic acid, a PROTEC compound.

[0022] In another preferred embodiment, the liver cancer cells in the test group and the control group are drug-resistant liver cancer cells.

[0023] In another preferred embodiment, the drug-resistant liver cancer cells are resistant to a drug selected from the group consisting of etoposide, fluorouracil, capecitabine, cisplatin, oxaliplatin, or a combination thereof.

[0024] In another preferred embodiment, the method further comprises a step (s3a):

[0025] In the drug resistance test group, the candidate compound is added to the culture system of the liver cancer cells, and the number and / or growth of the liver cancer cells are observed;

[0026] In the drug resistance control group, no test compound is added to the culture system of the liver cancer cells, and the number and / or growth of the liver cancer cells are observed;

[0027] wherein if the number or growth rate of the liver cancer cells in the test group is less than that in the control group, it indicates that the candidate compound can reverse the drug resistance of the drug-resistant liver cancer cells and / or sensitize the drug-resistant liver cancer cells.

[0028] In another preferred embodiment, the method further comprises a step (s3b) of further administering the candidate compound to a non-human mammal model to determine the therapeutic effect of the candidate compound on liver cancer in the non-human mammal.

[0029] In another preferred embodiment, the non-human mammal is a mammal inoculated with liver cancer cells.

[0030] In another preferred embodiment, the liver cancer cells are drug-resistant liver cancer cells.

[0031] In another preferred embodiment, "significantly lower" means A1 / A0≤1 / 2, preferably ≤1 / 3, and more preferably ≤1 / 4.

[0032] In another preferred embodiment, "significantly lower" means B1 / B0≤1 / 2, preferably ≤1 / 3, and more preferably ≤1 / 4.

[0033] In another preferred embodiment, the amino acid sequence of SLC25A10 isoform 3 is shown in SEQ ID NO: 18.

[0034] In another preferred embodiment, the cells include liver cancer cells.

[0035] In another preferred embodiment, the cells are drug-resistant liver cancer cells.

[0036] In another preferred embodiment, the cells are in vitro cultured cells.

[0037] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0038] In a second aspect of the present application, a reagent combination for screening candidate compounds for treating liver cancer is provided, comprising:

[0039] (1) a first detection reagent set for detecting a first complex, wherein the first complex is a complex of SLC25A10 isoform 3 and IPO7; and

[0040] (2) a second detection reagent set for detecting a second complex, the second complex being a complex of SLC25A10 isoform 3 and CEBPB.

[0041] In another preferred embodiment, the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a second antibody against IPO7;

[0042] The second detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a third antibody against CEBPB.

[0043] In another preferred embodiment, the first antibody, the second antibody, and the third antibody are each independently labeled with a detectable label, and / or coupled to a magnetic bead.

[0044] In another preferred embodiment, the reagent combination is used for screening candidate compounds for treating liver cancer.

[0045] In another preferred embodiment, the reagent combination is further used for screening candidate compounds for reversing drug resistance of drug-resistant liver cancer cells and / or sensitizing drug-resistant liver cancer cells.

[0046] In another preferred embodiment, the drug-resistant liver cancer cells are resistant to a drug selected from the group consisting of etoposide, fluorouracil, capecitabine, cisplatin, oxaliplatin, or a combination thereof.

[0047] In a third aspect, the present application provides a kit, the kit comprising:

[0048] (f1) a first container and a first detection reagent set for detecting a first complex in the first container, the first complex being a complex of SLC25A10 isoform 3 and IPO7; and

[0049] (f2) a second container and a second detection reagent set for detecting a second complex in the second container, the second complex being a complex of SLC25A10 isoform 3 and CEBPB.

[0050] In another preferred embodiment, the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a second antibody against IPO7;

[0051] The second detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a third antibody against CEBPB.

[0052] In another preferred embodiment, the kit further comprises a third container and drug-resistant liver cancer cells in the third container.

[0053] In another preferred embodiment, the kit is used for screening candidate compounds for treating liver cancer.

[0054] In another preferred embodiment, the kit is further used for screening candidate compounds for reversing drug resistance and / or sensitizing drug-resistant liver cancer cells.

[0055] In another preferred embodiment, the drug-resistant liver cancer cells are resistant to a drug selected from the group consisting of etoposide, fluorouracil, capecitabine, cisplatin, oxaliplatin, or a combination thereof.

[0056] It should be understood that, within the scope of the present application, all the technical features described above and the technical features described in detail hereinafter (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 SLC25A10 is closely related to HCC. A: Heatmap of expression profiles of three HCC cell lines under hypoxia or normoxia conditions. RNA sequencing was performed in Huh7, HepG2 and MHCC-97H cell lines. B: Volcano plot of differentially expressed genes in Huh7 cells. C: Immunoblot analysis of SLC25A10 expression in representative clinical samples. D: Immunohistochemical detection of SLC25A10 expression in HCC samples. E: Detection of SLC25A10 expression in HCC cells. F: Analysis of SLC25A10 expression in various tumor types and their corresponding normal tissues based on TCGA database. G: Analysis of SLC25A10 gene mutation frequency based on TCGA database. H: Kaplan-Meier (KM) curve analysis of patient survival (total of 476 cases).

[0058] Figure 2Huh7 cells under hypoxic conditions. B: Analysis of splicing of SLC25A10 expression under hypoxic conditions. C: Detection of SLC25A10 isoform 3 expression under hypoxic conditions by Q-PCR. D: Detection of SLC25A10 in cell fractionation. E: Immunofluorescence confocal imaging of SLC25A10 (magnification x 400). F: Immunohistochemical analysis of SLC25A10 in clinical samples (magnification x 100). G: Detection of mutant SLC25A10 in cell fractionation. The nuclear localization sequence PPWPWPP in SLC25A10 isoform 3 was mutated to PPAPAPP. H: Immunofluorescence confocal imaging of SLC25A10 in Huh7 cells expressing mutant SLC25A10 isoform 3. I: Silver staining of Huh7 cell lysate after immunoprecipitation with SLC25A10. J: Identification of candidate proteins after immunoprecipitation of Huh7 cell lysate with SLC25A10, candidate proteins were selected by mass spectrometry. K: Immunofluorescence confocal imaging of SLC25A10 in IPO7 knockdown Huh7 cells. L: Detection of SLC25A10 in cell fractionation in IPO7 knockdown Huh7 cells.

[0059] Figure 3 SLC25A10 isoform 3 reduces etoposide-induced apoptosis in HCC cells. A: Gene ontology (GO) analysis of differentially expressed genes in Huh7 cells. B: Growth analysis of HepG2 cells with knockdown of SLC25A10 isoform 3. 10 4 cells were seeded at the start of the experiment. C: Growth analysis of Huh7 cells with overexpression of SLC25A10 isoform 3. 10 4 cells were seeded at the start of the experiment. D: Colony formation experiment of HepG2 cells with knockdown of SLC25A10 isoform 3. 10 3 cells were seeded at the start of the experiment. E: Colony formation experiment of Huh7 cells with overexpression of SLC25A10 isoform 3. 10 3 cells were seeded at the start of the experiment. F: Detection of apoptotic cells in HepG2 cells with knockdown of SLC25A10 isoform 3 by flow cytometry. G: Detection of apoptotic cells in Huh7 cells with overexpression of SLC25A10 isoform 3 by flow cytometry. H: Detection of apoptotic signals in HepG2 cells with knockdown of SLC25A10 isoform 3. I: Detection of apoptotic signals in Huh7 cells with overexpression of SLC25A10 isoform 3.

[0060] Figure 4SLC25A10 isoform 3 upregulates BCL2A1 expression. A: Analysis of apoptosis-related protein expression in SLC25A10-affected HCC cells. B: Verification of BCL2A1 expression in HCC cells overexpressing or knockdown SLC25A10 isoform 3 by Q-PCR. C: Verification of BCL2A1 expression in HCC cells overexpressing or knockdown SLC25A10 by immunoblotting analysis. D: Effect of BCL2A1 knockdown on colony formation of HCC cells overexpressing or not overexpressing SLC25A10 isoform 3. E: Effect of BCL2A1 knockdown on HCC cell apoptosis by flow cytometry analysis. F: Effect of SLC25A10 transport inhibitor BMA on isoform 3 upregulated BCL2A1 expression, the concentration of BMA is 8 mM. G: Effect of BMA on ATP / ADP ratio. H: Effect of BMA on HCC cell apoptosis by flow cytometry, the concentration of etoposide is 15 mM.

[0061] Figure 5 SLC25A10 isoform 3 binds to transcription factor CEBPB to regulate BCL2A1 expression. A: Overlap of SLC25A10 binding proteins with transcription factors. SLC25A10 binding proteins were selected by mass spectrometry. B: Immunoblotting analysis of potential transcription factors binding to SLC25A10, immunoblotting was performed after co-immunoprecipitation. C: Immunoblotting analysis of potential transcription factors binding to SLC25A10 in Huh7 cells under hypoxic conditions. D: ChIP-seq analysis of CEBPB on BCL2A1 gene locus. E: Immunoblotting analysis of BCL2A1 in Huh7 cells knockdown CEBPB. F: Effect of CEBPB knockdown on isoform 3-regulated apoptosis by colony formation assay. G: Effect of CEBPB knockdown on isoform 3-regulated apoptosis by flow cytometry. H: Effect of CEBPB knockdown on isoform 3-regulated apoptosis by TUNEL assay.

[0062] Figure 6 Disrupting SLC25A10 binding to IPO7 enhances sensitivity to etoposide in mouse models. A: Detection of growth of Huh7 cells overexpressing SLC25A10 isoform 3 xenografts in PCX mouse models, with or without etoposide treatment. The number of cells injected each time is 5 x 10 6, the dose of etoposide is 20 mg / kg. B: Immunoblot analysis of BCL2A1 in representative xenografts, with or without overexpression of isoform 3. C: Detection of growth of Huh7 cell xenografts with knockdown of BCL2A1 in PCX mouse models and treated with etoposide. D: Detection of growth of Huh7 cell xenografts with knockdown of CEBPB in PCX mouse models and treated with etoposide. E: Detection of growth of Huh7 cell xenografts with overexpression of mutant isoform 3 in PCX mouse models and treated with etoposide.

[0063] Figure 7 Fig. 1 shows the detection of SLC25A10 knockdown efficiency in HepG2 cells and upregulation compared to other HCC cells; Fig. 1B shows the detection of SLC25A10 iso3 overexpression efficiency in Huh7 cells.

[0064] Figure 8 Fig. 2 shows the effect of SLC25A10 isoform 3 or ΔISO3 on the growth of Huh7 cells (representative images of colony formation).

[0065] Figure 9 Fig. 3A shows the effect of knockdown of SAFB2 on Huh7 cells evaluated by flow cytometry analysis; Fig. 3B shows the effect of knockdown of SAFB2 on Huh7 cells detected by colony formation.

[0066] Figure 10 Fig. 4 shows representative tumor photos of each group in the examples. DETAILED DESCRIPTION

[0067] The present inventors, through extensive and in-depth research, through a large number of experiments and screening, provide the application of SLC25A10 and SLC25A10 isoform 3 as a target in the screening of drugs for treating liver cancer. The present inventors have for the first time accidentally discovered that SLC25A10 promotes the resistance of HCC to etoposide in vitro and in mouse models. Specifically, hypoxia enhances the expression of SLC25A10 and triggers the splicing variation of SLC25A10 from isoform 1 to isoform 3. Isoform 3 of SLC25A10 enters the nucleus by binding to nuclear transport protein IPO7 and binds to transcription factor CEBPB, upregulating the expression of BCL2A1, thereby enhancing the resistance of HCC cells to etoposide. On this basis, the present application is completed.

[0068] TERMS

[0069] For the purposes of the present invention, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in the present disclosure have the meanings that are commonly understood by one of ordinary skill in the art in the field of the present invention. Before the present invention is described, it is to be understood that the invention is not limited to the particular methodologies and experimental conditions described, as such methodologies and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0070] As used herein the term "comprising" or grammatical variants thereof is to be taken as specifying including but not limited to, that is, it indicates the presence of stated features but not to the exclusion of others.

[0071] The term "about" can mean values or components that are within an acceptable error range for the specific value or component determined by one of ordinary skill in the art, which will vary from one context to another. It will depend for example on the limit of detection of a measuring technique, or on the degree of accuracy required for a given application.

[0072] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be understood to include the interpretation of values that fall within that range, including the values themselves, and, where appropriate, sub-ranges falling within that range.

[0073] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0074] SLC25A10

[0075] Solute carrier family (SLC) as the main carrier of nutrient uptake and transport, is a large class of transport proteins in cells, which plays an important role in tumor occurrence and development.

[0076] SLC25A10 is a transport protein located on the inner membrane of mitochondria, also known as mitochondrial dicarboxylate carrier (DIC), which mainly transports dicarboxylate such as malate and succinate from the inside of mitochondria to the outside of mitochondria, exchanges phosphate, sulfate and thiosulfate, so as to provide substrate for gluconeogenesis and urea synthesis process, and then maintain the distribution and homeostasis of intermediates in the process of TCA cycle in mitochondria.

[0077] IPO7

[0078] Transporter IPO7, also known as importin 7 or RANBP7, is a protein that plays a crucial role in cells. The IPO7 gene is located in the 11p15.4 region of human chromosome, with multiple splice variants and orthologues. IPO7 protein is one of the importin-beta-like transport receptors, with specific structural and functional domains that are essential for its involvement in nuclear transport processes.

[0079] As an importin-beta-like transport receptor, IPO7 is involved in the nuclear import process of proteins with classic nuclear localization signals. It co-mediates the nuclear transport of proteins with the importin-alpha / beta complex and GTPase Ran, which is one of the important pathways for the transport of intracellular substances. IPO7 has a Ran-dependent transport cycle, which can quickly and bidirectionally cross the nuclear membrane, thus achieving the nuclear import and export of proteins.

[0080] CEBPB

[0081] Transcription factor CEBPB, also known as C / EBPβ or NF-IL6 (Interleukin-6 nuclear transcription factor), is an important member of the CAAT region / enhancer binding protein (CEBP) family, mainly located in the nucleus. CEBPB belongs to the bZIP protein family, with a highly conserved b-ZIP domain at the C-terminus and a proximal basic amino acid region, which together mediate protein dimerization and specific binding of transcription factors to DNA sequences. The N-terminal domain has relatively low conservation, but contains short motifs that interact with transcriptional co-factors to trigger gene transcription.

[0082] CEBPB is abnormally expressed in various tumors and can serve as a target for tumor treatment. For example, in glioblastoma, CEBPB specifically drives the formation of M2-type tumor-associated macrophages to promote the growth of malignant tumors. In addition, CEBPB also up-regulates the expression of LINC01133, which induces cancer cells to acquire ferroptosis resistance by enhancing the mRNA stability of FSP1.

[0083] Method for screening drugs for treating liver cancer resistance

[0084] The present inventors surprisingly found that SLC25A10 promotes drug resistance of liver cancer, specifically, liver cancer cells are in a hypoxic environment, and hypoxia enhances the expression of SLC25A10 and triggers the splicing variation of SLC25A10 from isoform 1 to isoform 3. Isoform 3 of SLC25A10 enters the nucleus by binding with nucleoporin IPO7, and binds with transcription factor CEBPB, up-regulates the expression of BCL2A1, thereby enhancing the drug resistance of HCC cells to etoposide.

[0085] The screening method of candidate compounds for treating liver cancer of the present application comprises the steps of:

[0086] (s1) in the test group, culturing liver cancer cells in a culture system containing a test compound, and determining data of the test group selected from the group consisting of:

[0087] (a) the number of a first complex formed by SLC25A10 isoform 3 and IPO7 A1; and / or

[0088] (b) the number of a second complex formed by SLC25A10 isoform 3 and CEBPB B1;

[0089] and in the control group, culturing liver cancer cells in a culture system under the same conditions except that the culture system does not contain the test compound, and determining data of the control group selected from the group consisting of:

[0090] (c) the number of a first complex formed by SLC25A10 isoform 3 and IPO7 A0; and / or

[0091] (d) the number of a second complex formed by SLC25A10 isoform 3 and CEBPB B0; and

[0092] (s2) comparing the number of the first complex and / or the second complex in the test group and the control group,

[0093] wherein if the number of the first complex A1 in the test group is significantly lower than the number of the first complex A0 in the control group, and / or the number of the second complex B1 in the test group is significantly lower than the number of the second complex B0 in the control group, it indicates that the test compound is a candidate compound for treating liver cancer.

[0094] In a preferred embodiment, the liver cancer cells in the test group and the control group are drug-resistant liver cancer cells. In a preferred embodiment, the drug-resistant liver cancer cells are resistant to a drug selected from the group consisting of etoposide, fluorouracil, capecitabine, cisplatin, oxaliplatin, or a combination thereof.

[0095] In a preferred embodiment, the first complex is detected by a first detection reagent set, wherein the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a second antibody against IPO7.

[0096] The second complex is detected by a second detection reagent set, wherein the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a third antibody against CEBPB.

[0097] In a preferred embodiment, the first complex is detected by a first detection reagent set, wherein the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a second antibody against IPO7. In a preferred embodiment, the second complex is detected by a second detection reagent set, wherein the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a third antibody against CEBPB. In a preferred embodiment, the first antibody, the second antibody, and the third antibody are each independently labeled with a detectable label and / or coupled to a magnetic bead.

[0098] Screening kit for candidate compounds for treating liver cancer

[0099] The kit of the present application for screening candidate compounds for treating liver cancer comprises:

[0100] (f1) a first container and a first detection reagent set for detecting a first complex in the first container, the first complex being a SLC25A10 isoform 3 and IPO7 complex; and

[0101] (f2) a second container and a second detection reagent set for detecting a second complex in the second container, the second complex being a SLC25A10 isoform 3 and CEBPB complex.

[0102] In a preferred embodiment, the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a second antibody against IPO7.

[0103] The second detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a third antibody against CEBPB.

[0104] Generally, the kit further comprises an instruction manual or the like. The instruction manual records include the following:

[0105] (s1) in the test group, culturing liver cancer cells in a culture system containing a test compound, and measuring data of the test group selected from the group consisting of:

[0106] (a) the amount of a first complex formed by SLC25A10 isoform 3 and IPO7, A1; and / or

[0107] (b) the amount of a second complex formed by SLC25A10 isoform 3 and CEBPB, B1;

[0108] and in the control group, under the same conditions except that the culture system does not contain the test compound, the hepatoma cells are cultured, and the data selected from the group consisting of:

[0109] (c) the amount of a first complex formed by SLC25A10 isoform 3 and IPO7, A0; and / or

[0110] (d) the amount of a second complex formed by SLC25A10 isoform 3 and CEBPB, B0; and

[0111] (s2) comparing the amount of the first complex and / or the second complex in the test group and the control group,

[0112] wherein if the amount of the first complex in the test group, A1, is significantly lower than the amount of the first complex in the control group, A0, and / or the amount of the second complex in the test group, B1, is significantly lower than the amount of the second complex in the control group, B0, it indicates that the test compound is a candidate compound for treating hepatoma.

[0113] The main advantages of the present application include:

[0114] 1. The present application first discovers that SLC25A10 promotes hepatoma drug resistance, indicating that it can be used as a target for developing drugs for inhibiting hepatoma drug resistance.

[0115] 2. The present application provides the use of SLC25A10 inhibitors in inhibiting hepatoma drug resistance.

[0116] 3. The present application also provides a method for screening drugs for treating hepatoma drug resistance, which can achieve rapid, low-cost, high-throughput screening of drugs for inhibiting hepatoma drug resistance by using SLC25A10 as a drug target.

[0117] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out under conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight.

[0118] 1. Materials and Methods

[0119] 1.1 Cell lines and reagents

[0120] All cells were cultured at 37 °C in a humidified incubator with 5% CO2. In particular, hypoxic cells were cultured at 37 °C in a humidified incubator with 1% O2 and 5% CO2. Human hepatocarcinoma cell lines HepG2 and Huh7 and HEK-293T cells were cultured in Dulbecco’s Modified Eagle Medium (Cat# L110KJ, BasalMedia, Shanghai, China) supplemented with 10% fetal bovine serum (Cat# 10270-106, Gibco, New York, USA) and 50 IU of penicillin / streptomycin (Cat# S110JV, BasalMedia, Shanghai, China).

[0121] Commercial antibodies used include: Bcl2a1 (Cat# 14093, CST, Massachusetts, USA), CEBPB (Cat# PAS-27244, Invitrogen, California, USA), IPO7 (Cat# 28289-1-AP, Proteintech, Hubei, China), SAFB2 (Cat# A4330, Abclonal, Hubei, China), SLC25A10 (Cat# WG03144D, Abclonal, Hubei, China), Pol II (Cat# sc-899, Santa Cruz, Texas, USA), Lamin A / C (Cat# 2032T, CST, Massachusetts, USA), Histone H3 (Cat# AM8433, Abeepta, California, USA), β-actin (Cat# sc-47778, Santa Cruz, Texas, USA), a-Tubulin (Cat# 11224-1-AP, Proteintech, Hubei, China).

[0122] 1.2 Plasmids and transfection

[0123] To express Flag-SLC25A10 isoform 3 protein, the ORF complementary DNA (NM_001270953.2) was cloned into the lentivirus recombinant pCDH-CMV-MCS-EF1-Puro vector. SLC25A10-V3[P232-252A] and SLC25A10-V3A232-252 were generated using AccuPrime Pfx DNA Polymerase (Invitrogen, 12344024) following the manufacturer’s instructions.

[0124] The shRNAs used in this study include: SLC25A10 isoform 3 shRNA (TGCTAGCTCTGCACTTCGTGT, SEQ ID NO: 1), Bcl2a1 shRNA (GCCAGAACACTATTCAACCAA, SEQ ID NO: 2), IPO7 shRNA1 (GCTAACAAGAAGATGTCTGAT, SEQ ID NO: 3), IPO7 shRNA2 (GCACTGACTCACGGTCTTAAT, SEQ ID NO: 4), CEBPB shRNA1 (CCCGTGGTGTTATTTAAAGAA, SEQ ID NO: 5), CEBPB shRNA2 (CCTGCCTTTAATCCATGGAA, SEQ ID NO: 6), SAFB2 shRNA1 (GCCACCATGTTGTAGCTCAAT, SEQ ID NO: 7), SAFB2 shRNA2 (CGGACATTGAAGAATCCCTTT, SEQ ID NO: 8), and control sequence (GAATTACTCCTAGAACCGC, SEQ ID NO: 9), which were synthesized as 58 bp stem-loop structures and cloned into pLKO.1-puro vector, respectively. Lentivirus was generated by co-transfecting the above recombinant plasmids with packaging plasmids (psPAX2 and pMD2G) into HEK-293T cells. After 48 hours of lentivirus infection, puromycin (1 pg / mL) or blasticidin (10 pg / mL) was added to select stable cells. The efficiency of overexpression or knockdown was analyzed by real-time PCR and Western blot.

[0125] 1.3 Western blot

[0126] Cells cultured to 80-90% confluency were collected in PBS. The cytoplasmic and nuclear fractions were extracted using a nuclear and cytoplasmic protein extraction kit (Cat# PK10014, Proteintech, Hubei, China) according to the manufacturer’s protocol. Total proteins were extracted using RIPA lysis buffer containing protease inhibitors (1 mM PMSF, 1 mg / L aprotinin, 1 mg / L leupeptin, and 1 mg / L pepstatin) and phosphatase inhibitors (1 mM Na3V04 and 10 mM NaF). After centrifugation to remove cell debris, the protein concentration was measured using a BCA assay kit (Cat# MA0082-1, Meilunbio, Liaoning, China). Subsequently, 40 pg of protein was separated on a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane, which was incubated with the indicated antibodies, respectively. The LAS 4000 instrument (GE Healthcare) was used for development.

[0127] 1.4 Real-time PCR

[0128] Total RNA was extracted from cultured cells using TRIzol reagent and cDNA was synthesized from 1000 ng of total RNA using Evo M-MLV RT reaction mix Ver.2 (Cat# AG11728, AG, Hunan, China). Quantitative real-time PCR was performed using 2X SYBR Green master mix (Cat# AG11719, AG, Hunan, China). Primers used included: SLC25A10 isoform 3 forward primer: 5'-GGGGAGTATCAGGGCGTTTT-3' (SEQ ID NO: 10), reverse primer: 5'- CAAAAGTGAGCACGGTGTGG-3' (SEQ ID NO: 11); Bcl2a1 forward primer: 5'- GCGGGAAATCGTGCGTGACATT-3' (SEQ ID NO: 12), reverse primer: 5'- GATGGAGTTGAAGGTAGTTTCG-3' (SEQ ID NO: 13); GAPDH forward primer: 5'- ACCCAGAAGACTGTGGATGG-3' (SEQ ID NO: 14), reverse primer: 5'- CAGTGAGCTTCCCGTTCAG-3' (SEQ ID NO: 15). Data were collected and analyzed using an ABI7500 sequence detector (Applied Biosystems, UK). Ct values for SLC25A10 and Bcl2a1 were normalized to GAPDH as an internal control.

[0129] 1.5 Cell proliferation analysis

[0130] Cells were seeded at a density of 2 x 10 5 cells / dish in 60 mm dishes and incubated at 37 °C in 5% CO2. Cell numbers were counted at different time points using Countless cell counter (Invitrogen, Grand Island, NY, USA). For colony formation experiments, cells were seeded at a density of 1 x 10 3 cells / well in 6-well plates. Cells were incubated at 37 °C for 10 days. Subsequently, cells were fixed with paraformaldehyde for 30 min and stained with 2.5% crystal violet for 15 min. Clusters containing > 50 cells were counted as one colony.

[0131] 1.6 Co-immunoprecipitation

[0132] Samples were homogenized in single-detergent lysis buffer (1% NP40, 0.25% sodium deoxycholate, 50 mM Tris-HCl pH 7.4, 150 mM NaCl) and proteinase and phosphatase inhibitor cocktails were added. Extracts were pre-cleared with Protein A / G beads (Cat# sc-2003, Santa Cruz, Texas, USA) and then incubated with the appropriate antibody (1-2 pg) overnight at 4°C, followed by the addition of 20 pL of Protein A / G beads for 2 hours at 4°C. For Flag immunoprecipitation, cell lysates were incubated with anti-Flag beads (Cat# B23101, Selleck, Texas, USA) for 4 hours at 4°C. Beads were washed 3 times with lysis buffer and resuspended in 1x SDS loading buffer. Samples were further analyzed by Western blot.

[0133] 1.7 Immunohistochemistry staining

[0134] HCC tissue sections were incubated with SLC25A10 antibody (1:250) overnight at 4°C. Subsequently, biotin-labeled IgG secondary antibody was incubated for 30 minutes at 37°C. Finally, color development was performed using diaminobenzidine (DAB).

[0135] 1.8 Immunofluorescence staining

[0136] Cells were seeded at a density of 1 x 10 5 cells / well on glass coverslips in 6-well plates. Cells were incubated at 37°C for 48 hours. Subsequently, cells were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes, then washed with PBS, permeabilized with 0.4% Triton X-100 / PBS for 15 minutes, and blocked with 5% BSA in 0.1% Triton / PBS for 30 minutes. Cells were stained with SLC25A10 antibody (1:50, Cat# HPA023048, Altas, Bromma, Sweden) and DAPI. Immunofluorescence imaging was performed on a Leica TCS SP8 X confocal microscope using a 40x oil objective to take images.

[0137] 1.9 Flow cytometry

[0138] All cells were seeded in 6-well plates, then washed with PBS and stained with Annexin V-APC / PI apoptosis kit (Cat# 70-AP107-100, Multisciences, Zhejiang, China). About 10 4 cells were analyzed by CytoFlex S (Beckman, California, USA). Cells were gated according to forward and side scatter properties.

[0139] 1.10 RNA Sequencing Analysis

[0140] RNA sequencing analysis was performed by BGI Genomics. After total RNA extraction, mRNA was separated using Oligo magnetic beads and cleaved into small fragments for cDNA synthesis. Libraries were generated in the Illumina system using the NEB Next Ultra™ RNA Library Preparation Kit (New England Biolabs, Ipswich, Massachusetts, USA) according to the manufacturer's instructions. Sequencing was performed using the Illumina Hiseq XTEN platform.

[0141] 1.11 ChIP Sequencing Analysis

[0142] HepG2 cells were fixed with 1% formaldehyde and then... Chromatin preparation was performed according to the instructions of the Enzyme Chromatin IP Kit (Cat#9003, CST, Massachusetts, USA). The purified DNA was then subjected to ChIP sequencing.

[0143] 1.12 Tissue Samples

[0144] Clinical liver cancer samples were obtained from Ruijin Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. The clinical ethics were approved by the Ruijin Hospital Medical Ethics Committee. All patients in this study underwent pathological breast cancer diagnosis and signed informed consent forms before surgery.

[0145] 1.13 Xenograft Mouse Model

[0146] Five to six-week-old congenital athymic nude mice (athymic Ncr-nu / nu) (SLAC, Shanghai, China). The protocol for xenograft tumor models was approved by the Animal Care and Use Committee of Shanghai Jiao Tong University. 5 × 10 6 One Huh7 cell was subcutaneously injected into the axilla of a nude mouse.

[0147] 2. Results

[0148] 2.1 SLC25A10 is closely related to HCC

[0149] Figure 1 Image A shows the expression profile of hepatocellular carcinoma (HCC) cells under hypoxic conditions. The heatmap illustrates the expression differences among three HCC cell lines (GepG2, Huh7, and Huh7). The volcano plot indicates that the mitochondrial vector SLC25A10 is one of the upregulated genes. Figure 1 (B). In addition, Western blot ( Figure 1 (C) and immunohistochemical staining (IHC) ( Figure 1MID) verified that SLC25A10 expression was significantly higher in clinical HCC than in paracarcinoma tissues. The inventors also examined the expression of SLC25A10 in several HCC cell lines in the laboratory Figure 1 MID E).

[0150] Furthermore, according to TCGA data, SLC25A10 gene amplification or mutation and SLC25A10 mRNA transcription were enhanced in most tumor types, including HCC. Survival analysis showed that HCC patients with higher SLC25A10 expression levels had a poorer prognosis Figure 1 MID G), indicating that SLC25A10 plays a key role in HCC progression.

[0151] 2.2 Hypoxia-induced SLC25A10 isoform 3 splicing into the nucleus through binding with IPO7

[0152] To investigate the effect of hypoxia on SLC25A10 expression, ChIP sequencing analysis was first performed using a HIF1a antibody. As shown in Figure 2 MID A, hypoxia enhanced HIF1a binding to the SLC25A10 promoter. In addition, through transcriptome analysis of HCC cells, it was found that hypoxia triggered a splicing variation of SLC25A10 from isoform 1 to isoform 3 Figure 2 MID B), and further verified by Q-PCR Figure 2 MID C). In addition, protein sequence analysis showed that SLC25A10 isoform 3 can enter the nucleus through its nuclear localization sequence PPWPWPP (SEQ ID NO: 16). Western blot of nuclear fraction showed that SLC25A10 was present in the nucleus Figure 2 MID D). Confocal immunofluorescence images also showed the nuclear localization of SLC25A10 Figure 2 MID E). Immunohistochemical images showed that SLC25A10 was increased in HCC cells compared to paracarcinoma tissues, especially in the nucleus Figure 2 MID F).

[0153] To more accurately determine whether the PPWPWPP sequence in SLC25A10 isoform 3 promotes its nuclear localization, the cellular distribution of truncated SLC25A10 isoform 3 with PAWAWAP (SEQ ID NO: 17) deleted was examined. As shown in Figure 2 MID G, overexpression of SLC25A10 isoform 3 enhanced its nuclear localization, while truncated isoform 3 was almost unable to enter the nucleus. Immunofluorescence images also confirmed that truncated isoform 3 prevented its nuclear entry. However, the SLC25A10 transport inhibitor butanedioic acid (BMA) (Hlouschek et al., 2018) did not prevent this nuclear transport Figure 2(H).

[0154] To determine which nuclear transporter facilitates the entry of SLC25A10 isoform 3 into the nucleus, mass spectrometry was performed to identify the nuclear transporter that binds to SLC25A10. Although nuclear transporters IPO7 and IPO13 are two potential candidates for binding to SLC25A10 (…),… Figure 2 (I), but Western blot results confirmed that IPO7 did indeed bind to SLC25A10 ( Figure 2 (J). The absence of IPO7 leads to a reduction in SLC25A10 in the cell nucleus, which was confirmed by Western blot and immunofluorescence staining analysis. Figure 3 The presence of K and L indicates that IPO7 facilitates the core localization of the SLC25A10 isomer 3.

[0155] 2.3 SLC25A10 isoform 3 reduces etoposide-induced apoptosis in HCC cells.

[0156] To investigate the effect of SLC25A10 isoform 3 on HCC growth, GO (gene ontology) analysis was performed on differentially expressed genes in HCC cells under hypoxic conditions. Figure 3 The results showed that apoptotic signaling was activated in HCC cells under hypoxic conditions. Furthermore, knockdown of the SLC25A10 isoform 3 reduced the number of HepG2 cells. Figure 7 China B and Figure 3 (A), while increased SLC25A10 expression increased the number of Huh7 cells (A). Figure 7 C and Figure 3 (B) Colony formation assays showed that knockdown of SLC25A10 isoform 3 led to a decrease in the number of HepG2 cell colonies. Figure 3 In the middle D), overexpression of SLC25A10 isoform 3 increased the number of Huh7 cell colonies (D). Figure 3 (E).

[0157] To determine the mechanism by which SLC25A10 isoform 3 regulates HCC growth, flow cytometry was used to detect the apoptosis rate of HCC cells under hypoxic or normoxic conditions. The results showed that knockdown of SLC25A10 isoform 3 increased the apoptosis rate of HepG2 cells from 8.91% to 11.31%. Figure 3 In contrast, overexpression of the SLC25A10 isoform 3 reduced the apoptosis rate of Huh7 cells from 13.74% to 10.14%. Figure 3Figure 6. SLC25A10 isoform 3 upregulates BCL2A1 expression. Figure 6A. SLC25A10 isoform 3 upregulates BCL2A1, one of the most upregulated genes. Figure 6B and 6C. qPCR and Western blot results also validate this upregulation. Figure 4 Figure 6. SLC25A10 isoform 3 upregulates BCL2A1 expression. Figure 6A. SLC25A10 isoform 3 upregulates BCL2A1, one of the most upregulated genes. Figure 6B and 6C. qPCR and Western blot results also validate this upregulation.

[0158] 2.4 SLC25A10 isoform 3 upregulates BCL2A1 expression

[0159] To investigate how SLC25A10 isoform 3 regulates etoposide-induced apoptosis, the transcriptional profile of apoptosis-related genes was analyzed in Huh7 cells overexpressing SLC25A10 isoform 3 (Figure 5A). Figure 4 Figure 6. SLC25A10 isoform 3 upregulates BCL2A1 expression. Figure 6A. SLC25A10 isoform 3 upregulates BCL2A1, one of the most upregulated genes. Figure 6B and 6C. qPCR and Western blot results also validate this upregulation. Figure 4 Figure 6. SLC25A10 isoform 3 upregulates BCL2A1 expression. Figure 6A. SLC25A10 isoform 3 upregulates BCL2A1, one of the most upregulated genes. Figure 6B and 6C. qPCR and Western blot results also validate this upregulation.

[0160] Figure 6. SLC25A10 isoform 3 upregulates BCL2A1 expression. Figure 6A. SLC25A10 isoform 3 upregulates BCL2A1, one of the most upregulated genes. Figure 6B and 6C. qPCR and Western blot results also validate this upregulation. Figure 4 Figure 6. SLC25A10 isoform 3 upregulates BCL2A1 expression. Figure 6A. SLC25A10 isoform 3 upregulates BCL2A1, one of the most upregulated genes. Figure 6B and 6C. qPCR and Western blot results also validate this upregulation. Figure 4 Figure 6. SLC25A10 isoform 3 upregulates BCL2A1 expression. Figure 6A. SLC25A10 isoform 3 upregulates BCL2A1, one of the most upregulated genes. Figure 6B and 6C. qPCR and Western blot results also validate this upregulation.

[0161] To determine whether SLC25A10 attenuates apoptosis in a function-dependent manner in mitochondria, the effect of BMA, a SLC25A10 transport inhibitor, on etoposide-induced apoptosis was tested. As shown in Figure 7A, BMA treatment increased the levels of cleaved PARP, Caspase 3 and Caspase 9 in Huh7 cells overexpressing SLC25A10 isoform 3, compared to the parental Huh7 cells. Figure 8As shown in F and G, although BMA did attenuate mitochondrial function in HCCHuh7 cells, it did not eliminate SLC25A10-induced BCL2A1 expression in HCC cells, regardless of whether the overexpression was of the wild-type SLC25A10 isoform 3 or the truncated isoform 3 lacking PAWAP. The mutation did not inhibit the growth of untreated Huh7 cells. Figure 4 Flow cytometry analysis showed that BMA did not affect the attenuating effect of SLC25A10 on etoposide-induced apoptosis in HCC cells. Figure 5 (H).

[0162] The above results indicate that the regulation of apoptosis by SLC25A10 does not depend on its transport function in mitochondria, while isoform 3 plays a key role.

[0163] 2.5SLC25A10 isoform 3 binds to transcription factor CEBPB to regulate BCL2A1 expression.

[0164] To investigate which transcription factor mediates the upregulation of BCL2A1 by SLC25A10 isoform 3, mass spectrometry analysis was performed on cell lysates from the nuclear region.

[0165] like Figure 5 As shown in Figure A, SLC25A10 isoform 3 may bind to seven transcription-related proteins, including two transcription factors, CEBPB and SAFB2. However, Western blot results showed that only CEBPB bound to SLC25A10 isoform 3. Figure 5 (B), and hypoxia increases this binding (B). Figure 5 (C). Furthermore, ChIP sequencing analysis showed that CEBPB was enriched in the promoter region of the BCL2A1 gene in HCC cells overexpressing SLC25A10 isoform 3. Figure 5 (D).

[0166] To investigate whether the apoptosis reduction of SLC25A10 isoform 3 is dependent on CEBPB, the expression of BCL2A1 in CEBPB-knockdown Huh7 cells was first examined. Western blot results showed that CEBPB knockdown reduced the enhanced BCL2A1 expression in SLC25A10 isoform 3. Figure 5 Furthermore, knockdown of CEBPB eliminated the alleviating effect of SLC25A10 isoform 3 on etoposide-induced apoptosis, as shown in colony formation assays, flow cytometry analysis, and TUNEL assays. Figure 6 (FH). The results showed that SLC25A10 isoform 3 regulates BCL2A1 expression by binding to CEBPB.

[0167] 2.6 Disruption of SLC25A10 binding to IPO7 enhances sensitivity to etoposide in mouse models

[0168] To assess the impact of SLC25A10 isoform 3 on the effectiveness of HCC chemotherapy, the impact of SLC25A10 isoform 3 on the sensitivity of HCC to etoposide was investigated using a xenograft mouse model.

[0169] As shown in Figure 10 A and Figure 6 B, HCC Huh7 cells were injected into the axillary and inguinal grooves of mice, and it was observed that Huh7-derived tumors responded to etoposide treatment regardless of whether SLC25A10 isoform 3 was overexpressed. However, when SLC25A10 isoform 3 was overexpressed, the efficacy of etoposide was attenuated. Consistently, the protein levels of BCL2A1 were elevated in tumors overexpressing SLC25A10 isoform 3 Figure 6 B).

[0170] Knocking down BCL2A1 did indeed enhance the sensitivity of HCC xenografts to etoposide compared to controls, even in cells overexpressing SLC25A10 isoform 3 Figure 10 C and Figure 6 B). Consistently, multicolor IHC analysis of these xenografts showed that SLC25A10 isoform 3 overexpression upregulated BCL2A1 expression and reduced cleaved caspase 3; however, SLC25A10 isoform 3 did not reduce the cleaved caspase 3 enhanced by BCL2A1 knockdown Figure 6 C).

[0171] To further explore the role of CEBPB in isoform 3 regulation of apoptosis, Huh7 cells with CEBPB knocked down were injected. Consistent with the above results, overexpression of isoform 3 reduced the sensitivity of HCC xenografts to etoposide. However, knocking down CEBPB increased the sensitivity of xenografts to etoposide compared to controls and abolished the inhibitory effect of isoform 3 on apoptosis Figure 10 D and Figure 6 C). Multicolor IHC analysis of these xenografts also validated this phenotype, indicating that SLC25A10 isoform 3 could not counteract the increased cleaved caspase 3 caused by CEBPB knockdown.

[0172] Finally, it was tested whether disrupting the binding of isoform 3 to IPO7 would enhance the sensitivity of HCC to etoposide. As shown in Figure 10 E and ​As shown in FIG. 12D, deletion of the PPWPWPP (Δ232-252) motif in SLC25A10 isoform 3 that binds to IPO7 increased the sensitivity of HCC xenografts to etoposide compared to the wild-type isoform 3 overexpression group. Multicolor IHC analysis of these xenografts showed that overexpression of mutant SLC25A10 isoform 3 did not significantly reduce the levels of cleaved caspase 3. These findings suggest that hypoxia-induced splicing of SLC25A10 isoform 3 promotes its nuclear regulation of BCL2A1 expression, and disruption of the interaction of isoform 3 with IPO7 can potentially enhance the chemotherapeutic effect of HCC.

[0173] 3. Discussion

[0174] SLC25A10 is spliced into isoform 3 in HCC, and hypoxia enhances this splicing. This isoform 3 enters the nucleus by binding to IPO7 and increases the expression of BCL2A1 by binding to the transcription factor CEBPB. Ultimately, this isoform 3 enhances the resistance of HCC to etoposide through BCL2A1. In addition, disruption of the binding of SLC25A10 to IPO7 can enhance the sensitivity of HCC to etoposide, laying the foundation for improving the chemotherapeutic effect of HCC.

[0175] In summary, the present application discloses a new function of mitochondrial dicarboxylate carrier SLC25A10 in transcriptional regulation, which is different from its function in mitochondria.

[0176] Amino acid sequence of SLC25A10 protein isoform 3: MAAEARVSRWYFGGLASCGAACCTHPLDLLKVHLQTQQEVKLRMTGMALRVVRTDGILALYSGLSASLCRQMTYSLTRFAIYETVRDRVAKGSQGPLPFHEKVLLGSVSGLAGGFVGTPADLVNVRMQNDVKLPQGQRRNYAHALDGLYRVAREEGLRRLFSGATMASSRGALVTVGQLYCRWMCHVPVPAPGCAEDSPDELQGGVSGRFPLRRGDSEARASGLLQGPRPSWHPPHPPHRAHFCVSGTATQKLWHQSAILTSRGNGWAARPDTLGSSKESQAQHLLLGPRPPWPWPPVLRSRPLLSPHLLAELLLASSPLSCSCTTPALATRLSRLGTAWPCPSPAGSSSGEQGLPEADFSPLLGQGRGIIPASCPRCPKQHLPALSIEDLGGRVWVQPGCCSPKC (SEQ ID NO: 18)

[0177] All documents referred to in the present application are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to readers of the application from the teaching- presented herein.

Claims

1. A reagent combination for screening candidate compounds for the treatment of liver cancer, characterized in that, comprises: (1) a first detection reagent set for detecting a first complex, the first complex being a complex of SLC25A10 isoform 3 and IPO7; and (2) a second detection reagent set for detecting a second complex, the second complex being a complex of SLC25A10 isoform 3 and CEBPB; and, the amino acid sequence of the SLC25A10 isoform 3 is shown as SEQ ID NO: 18; the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a second antibody against IPO7; the second detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a third antibody against CEBPB.

2. The agent combination of claim 1, wherein The reagent combination is also used for screening candidate compounds for reversing drug resistance and / or sensitizing drug-resistant liver cancer cells, the drug-resistant liver cancer cells being resistant to etoposide.

3. The agent combination of claim 1, wherein The first antibody, the second antibody, and the third antibody each independently carries a detectable label, and / or is coupled to a magnetic bead.

4. A method of screening candidate compounds for the treatment of liver cancer, characterized in that, comprises steps of: (s1) in a test group, culturing liver cancer cells in a culture system containing a test compound, and measuring data of the test group selected from the group consisting of: (a) the number of a first complex of SLC25A10 isoform 3 and IPO7 A1; and / or (b) the number of a second complex of SLC25A10 isoform 3 and CEBPB B1; and in a control group, culturing liver cancer cells in a culture system under the same conditions except that the test compound is not contained, and measuring data of the control group selected from the group consisting of: (c) the number of a first complex of SLC25A10 isoform 3 and IPO7 A0; and / or (d) the number of a second complex of SLC25A10 isoform 3 and CEBPB B0; and (s2) comparing the number of the first complex and / or the second complex in the test group and the control group, wherein if the number of the first complex A1 in the test group is significantly lower than the number of the first complex A0 in the control group, and / or the number of the second complex B1 in the test group is significantly lower than the number of the second complex B0 in the control group, it indicates that the test compound is a candidate compound for treating liver cancer; and, the amino acid sequence of the SLC25A10 isoform 3 is shown as SEQ ID NO: 18; The liver cancer cells in the test group and the control group are drug-resistant liver cancer cells, the drug-resistant liver cancer cells being resistant to etoposide.

5. The method of claim 4, wherein, The "significantly lower" means A1 / A0≤1 / 2 and / or B1 / B0≤1 / 2.

6. The method of claim 4, wherein, The first complex is detected by a first detection reagent set, wherein the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a second antibody against IPO7; The second complex is detected by a second detection reagent set, wherein the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a third antibody against CEBPB.

7. The method of claim 4, wherein, The test compound is selected from the group consisting of an antibody or a binding fragment thereof, a small molecule compound, a nucleic acid, a PROTEC compound.

8. A kit characterized in that, The kit comprises: (f1) a first container and a first detection reagent group for detecting a first complex in the first container, the first complex being a SLC25A10 isoform 3 and IPO7 complex; and (f2) a second container and a second detection reagent group for detecting a second complex in the second container, the second complex being a SLC25A10 isoform 3 and CEBPB complex; and the amino acid sequence of the SLC25A10 isoform 3 is shown as SEQ ID NO: 18; the first detection reagent group comprises a first antibody against SLC25A10 isoform 3 and a second antibody against IPO7; the second detection reagent group comprises a first antibody against SLC25A10 isoform 3 and a third antibody against CEBPB.

9. The kit of claim 8, wherein The kit is used for screening candidate compounds for reversing drug resistance of drug-resistant liver cancer cells and / or sensitizing drug-resistant liver cancer cells; the drug-resistant liver cancer cells are resistant to etoposide.

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