SLC25A10 for promoting drug resistance to liver cancer drugs

Abstract

The invention relates to the fact that SLC25A10 causes liver cancer cells to generate drug resistance, in particular, the liver cancer cells are located in an oxygen-deficient environment, and oxygen deficiency enhances expression of the SLC25A10 and triggers splicing variation of the SLC25A10 from an isomer 1 to an isomer 3. An isomer 3 of the SLC25A10 enters a cell nucleus by being combined with a nuclear transporter IPO7 and is combined with a transcription factor CEBPB to up-regulate the expression of BCL2A1, so that the drug resistance of HCC cells to etoposide is enhanced. According to the mechanism, a screening method and a kit of the drug for treating the drug resistance of the liver cancer are developed.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically relates to SLC25A10 promoting drug resistance to liver cancer drugs. Background Art

[0002] Liver cancer, especially hepatocellular carcinoma (HCC), is one of the malignant tumors with high mortality and increasing incidence worldwide. Chemotherapy, targeted therapy, and immunotherapy are the main current treatment methods for liver cancer. Although radiotherapy and chemotherapy can kill cancer cells, they can also cause damage to normal cells and trigger a series of side effects. Moreover, liver cancer cells are prone to develop drug resistance to a variety of chemotherapy drugs and targeted therapy drugs, which greatly limits the treatment effect.

[0003] In recent years, with the progress of molecular biology and drug R & D technology, targeted therapy and immunotherapy have become new hotspots 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 phenomenon of multidrug resistance (MDR) in liver cancer remains one of the main difficulties faced in clinical treatment. Immunotherapy kills cancer cells by activating the patient's own immune system. Although immunotherapy has brought new hope to patients with advanced liver cancer, its efficacy is still limited and may trigger immune-related adverse reactions.

[0004] Therefore, there is an urgent need in this field for a screening method for drugs for treating liver cancer drug resistance. Summary of the Invention

[0005] The purpose of the present invention is to provide a screening method for drugs for treating liver cancer drug resistance.

[0006] In the first aspect of the present invention, a method for screening candidate compounds for treating liver cancer is provided, including the steps of:

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

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

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

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

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

[0012] (d) The number B0 of the second complex 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 A1 of the first complex in the test group is significantly lower than the number A0 of the first complex in the control group, and / or the number B1 of the second complex in the test group is significantly lower than the number B0 of the second complex in the control group, this indicates that the test compound is a candidate compound for treating liver cancer.

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

[0016] In another preferred example, each of the first antibody and the second antibody independently bears a detectable label and / or is conjugated to magnetic beads.

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

[0018] In another preferred example, each of the first antibody and the third antibody independently bears a detectable label and / or is conjugated to magnetic beads.

[0019] In another preferred example, each of the first antibody, the second antibody, and the third antibody independently bears a detectable label and / or is conjugated to magnetic beads.

[0020] In another preferred example, the detection of the first complex and / or the detection of the second complex adopt a method selected from the following group: ELISA, immunoprecipitation, or a combination thereof.

[0021] In another preferred example, the test compound is selected from the following group: an antibody or its binding fragment, a small molecule compound, a nucleic acid, a PROTEC compound.

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

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

[0024] In another preferred example, the method further includes step (s3a):

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

[0026] In the drug resistance control group, the test compound is not added to the culture system of liver cancer cells, and the number and / or growth of liver cancer cells are observed;

[0027] Among them, if the number or growth rate of 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 drug-resistant liver cancer cells and / or sensitize drug-resistant liver cancer cells.

[0028] In another preferred example, the method further includes step (s3b): further administering the candidate compound to a non-human mammalian model, and determining its therapeutic effect on liver cancer in the non-human mammal.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0043] In another preferred example, each of the first antibody, the second antibody, and the third antibody independently bears a detectable label and / or is conjugated to magnetic beads.

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

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

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

[0047] In a third aspect of the present invention, a kit is provided, and the kit includes:

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

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

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

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

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

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

[0054] In another preferred example, the kit is further used for screening candidate compounds that can reverse the drug resistance of drug-resistant liver cancer cells and / or sensitize drug-resistant liver cancer cells.

[0055] In another preferred example, the drug-resistant liver cancer cells are resistant to drugs 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 invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Brief Description of the Drawings

[0057] Figure 1 Shows that SLC25A10 is closely related to HCC. A: Heatmap of expression profiles of three HCC cell lines under hypoxic or normoxic conditions. RNA sequencing was performed in three cell lines, Huh7, HepG2, and MHCC-97H. 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 multiple tumor types and their corresponding normal tissues based on the TCGA database. G: Analysis of SLC25A10 gene mutation frequency based on the TCGA database. H: Kaplan-Meier (KM) curve analysis of patient survival (total of 476 cases).

[0058] Figure 2It shows that hypoxia-induced SLC25A10 isoform 3 splicing enters the nucleus by binding to IPO7. A: Analysis of the binding of HIF1α to the SLC25A10 promoter in Huh7 cells under hypoxic conditions. B: Splicing analysis of SLC25A10 expression under hypoxic conditions. C: Detection of the expression of SLC25A10 isoform 3 under hypoxic conditions by Q-PCR. D: Detection of SLC25A10 in cell fractionation. E: Immunofluorescence confocal imaging of SLC25A10 (magnification: 400). F: Immunohistochemical analysis of SLC25A10 in clinical samples (magnification: 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 after immunoprecipitation of Huh7 cell lysates using SLC25A10. J: Confirmation of candidate proteins after immunoprecipitation of Huh7 cell lysates using SLC25A10. The candidate proteins were selected by mass spectrometry. K: Immunofluorescence confocal imaging of SLC25A10 in Huh7 cells with IPO7 knockdown. L: Detection of SLC25A10 in cell fractionation of Huh7 cells with IPO7 knockdown.

[0059] Figure 3 It shows that 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 SLC25A10 isoform 3 knockdown. 10 4 cells were seeded at the start of the experiment. C: Growth analysis of Huh7 cells overexpressing SLC25A10 isoform 3. 10 4 cells were seeded at the start of the experiment. D: Colony formation assay of HepG2 cells with SLC25A10 isoform 3 knockdown. 10 3 cells were seeded at the start of the experiment. E: Colony formation assay of Huh7 cells overexpressing SLC25A10 isoform 3. 10 3 cells were seeded at the start of the experiment. F: Detection of apoptotic cells in HepG2 cells with SLC25A10 isoform 3 knockdown by flow cytometry. G: Detection of apoptotic cells in Huh7 cells overexpressing SLC25A10 isoform 3 by flow cytometry. H: Detection of apoptotic signals in HepG2 cells with SLC25A10 isoform 3 knockdown. I: Detection of apoptotic signals in Huh7 cells overexpressing SLC25A10 isoform 3.

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

[0061] Figure 5 Shown is that SLC25A10 isoform 3 binds to the 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: Immunoblot analysis of potential transcription factors binding to SLC25A10, performed after immunoprecipitation. C: Immunoblot analysis of potential transcription factors binding to SLC25A10 in Huh7 cells under hypoxic conditions. D: ChIP-seq analysis of CEBPB at the BCL2A1 gene locus. E: Immunoblot analysis of BCL2A1 in Huh7 cells with CEBPB knocked down. F: Detection of the effect of CEBPB knockdown on apoptosis regulated by isoform 3 by colony formation assay. G: Detection of the effect of CEBPB knockdown on apoptosis regulated by isoform 3 by flow cytometry. H: Analysis of the effect of CEBPB knockdown on apoptosis regulated by isoform 3 by TUNEL assay.

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

[0063] Figure 7 In A, the knockout efficiency of SLC25A10 detected in HepG2 cells was shown, which was upregulated compared with other HCC cells; in B, the overexpression efficiency of SLC25A10 iso3 detected in Huh7 cells was shown.

[0064] Figure 8 The effects of SLC25A10 isoform 3 or ΔISO3 on the growth of Huh7 cells (representative images of colony formation) were shown.

[0065] Figure 9 In A, the effects of SAFB2 knockout on Huh7 cells were evaluated by flow cytometry analysis; in B, the effects of SAFB2 knockout on Huh7 cells were detected by colony formation.

[0066] Figure 10 Representative tumor photos of each group in the examples were shown. Detailed implementation manners

[0067] After extensive and in-depth research, through a large number of experiments and screenings, the present inventors provided the use of SLC25A10 and SLC25A10 isoform 3 as targets in screening drugs for treating liver cancer. The present invention unexpectedly found for the first time that in vitro and in mouse models, SLC25A10 promoted the resistance of HCC to etoposide. Specifically, hypoxia enhanced the expression of SLC25A10 and induced the splicing variation of SLC25A10 from isoform 1 to isoform 3. Isoform 3 of SLC25A10 entered the nucleus by binding to the nuclear transporter IPO7 and bound to the transcription factor CEBPB, upregulating the expression of BCL2A1, thereby enhancing the resistance of HCC cells to etoposide. Based on this, the present invention was completed.

[0068] Terms

[0069] To facilitate a better understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0070] As used herein, the term "comprising" or variations thereof such as "including" or "having" etc. are understood to include the stated element or component, without excluding other elements or other components.

[0071] The term "about" may refer to a value or a component within an acceptable error range of a specific value or component determined by those of ordinary skill in the art, which will depend in part on how the value or component is measured or determined.

[0072] As used herein, unless otherwise specified, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the stated range and, where appropriate, fractional values thereof (e.g., one-tenth and one-hundredth of an integer).

[0073] As used herein, the term "and / or" relates 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 for nutrient uptake and transport, is a large class of transport proteins within cells and plays an important role in tumorigenesis and development.

[0076] SLC25A10 is a transport protein located on the inner mitochondrial membrane, also known as mitochondrial dicarboxylate carrier (DIC). It mainly transports dicarboxylates such as malate and succinate from inside the mitochondria to outside the mitochondria to exchange for phosphate, sulfate and thiosulfate, thereby providing substrates for processes such as gluconeogenesis and urea synthesis, and further maintaining the distribution and homeostasis of the intermediates in the TCA cycle inside and outside the mitochondria.

[0077] IPO7

[0078] The 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 the human chromosome and has multiple splice variants and orthologues. The IPO7 protein is one of the importin-beta-like transport receptors and has specific structural and functional domains that are essential for its involvement in the nuclear transport process.

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

[0080] CEBPB

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

[0082] CEBPB is abnormally expressed in various tumors and may 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 upregulates 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 drug resistance

[0084] The inventor of the present invention unexpectedly found that SLC25A10 promotes drug resistance in liver cancer. Specifically, when liver cancer cells are in a hypoxic environment, hypoxia enhances the expression of SLC25A10 and triggers splicing variation of SLC25A10 from isoform 1 to isoform 3. Isoform 3 of SLC25A10 enters the nucleus by binding to the nuclear transporter IPO7, binds to the transcription factor CEBPB, upregulates the expression of BCL2A1, thereby enhancing the drug resistance of HCC cells to etoposide.

[0085] The screening method of the candidate compound for treating liver cancer of the present invention includes the steps:

[0086] (S1) In the test group, liver cancer cells are cultured in a culture system containing a test compound, and the data selected from the following groups are measured for the test group:

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

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

[0089] And in the control group, liver cancer cells are cultured in a culture system with the same other conditions and without the test compound, and the data selected from the following groups are measured for the control group:

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

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

[0092] (S2) Compare the numbers of the first complex and / or the second complex in the test group and the control group,

[0093] wherein, if the number A1 of the first complex in the test group is significantly lower than the number A0 of the first complex in the control group, and / or the number B1 of the second complex in the test group is significantly lower than the number B0 of the second complex in the control group, this 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 drugs selected from the following groups: 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 includes 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 includes 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 includes 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 includes 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 each independently carry a detectable label and / or are conjugated to magnetic beads.

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

[0099] The kit of the present invention is used for screening candidate compounds for treating liver cancer, and includes:

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

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

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

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

[0104] Generally, the kit further includes an instruction manual, etc. The instruction manual records the following content:

[0105] (s1) In the test group, liver cancer cells are cultured in a culture system containing a test compound, and data selected from the following groups are measured for the test group:

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

[0107] (b) The quantity B1 of the second complex formed by SLC25A10 isoform 3 and CEBPB;

[0108] And in the control group, hepatoma cells were cultured in a culture system with the same other conditions and without the test compound, and the data selected from the following group in the control group were measured:

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

[0110] (d) The quantity B0 of the second complex formed by SLC25A10 isoform 3 and CEBPB; and

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

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

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

[0114] 1. The present invention discovers for the first time that SLC25A10 promotes hepatoma drug resistance, indicating that it can be used as a target for developing drugs to inhibit hepatoma drug resistance.

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

[0116] 3. The present invention also provides a method for screening drugs for treating hepatoma drug resistance. By using SLC25A10 as a drug target, high-throughput screening of drugs for inhibiting hepatoma drug resistance can be achieved quickly and at low cost.

[0117] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0118] 1. Materials and Methods

[0119] 1.1 Cell lines and reagents

[0120] All cells were cultured in a humidified incubator at 37 °C with 5% CO2. In particular, hypoxic cells were cultured in a humidified incubator at 37 °C with 1% O2 and 5% CO2. Human hepatocellular carcinoma cell lines HepG2 and Huh7, as well as 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] The commercial antibodies used included: 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), α-Tubulin (Cat#11224-1-AP, Proteintech, Hubei, China).

[0122] 1.2 Plasmids and transfection

[0123] To express the Flag-SLC25A10 isoform 3 protein, the ORF complementary DNA (NM_001270953.2) was cloned into the lentiviral recombinant pCDH-CMV-MCS-EF1-Puro vector. SLC25A10-V3[P232-252A] and SLC25A10-V3△232-252 were generated using AccuPrime PfxDNA polymerase (Invitrogen, 12344024) according to the manufacturer's instructions.

[0124] The shRNAs used in this study included: 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 a control sequence (GAATTACTCCTAGAACCGC, SEQ ID NO: 9). These sequences were synthesized into 58-bp stem-loop structures and cloned into the pLKO.1-puro vector respectively. Lentiviruses were 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 μg / mL) or bleomycin (10 μg / mL) was added to screen for stable cells. The overexpression or knockdown efficiency was analyzed by real-time PCR and Western blot.

[0125] 1.3 Western blot

[0126] Cells cultured to 80 - 90% confluence 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 protein was 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 Na3VO4 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 μg of protein was separated on a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane, which was incubated with the designated antibodies respectively. Development was performed using a LAS 4000 instrument (GE Healthcare).

[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 the Evo M-MLV RT reaction mixture Ver.2 (Cat#AG11728, AG, Hunan, China). Quantitative real-time PCR was performed using the 2X SYBR Green master mix (Cat#AG11719, AG, Hunan, China). The 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). The Ct values of SLC25A10 and Bcl2a1 were normalized to GAPDH as an internal reference.

[0129] 1.5 Cell proliferation assay

[0130] Cells were seeded in 60-mm culture dishes at a density of 2×10 5 cells / dish and cultured at 37 °C in 5% CO2. The cell number was counted at different time points using a Countless cell counter (Invitrogen, Grand Island, NY, USA). For the colony formation assay, cells were seeded in 6-well plates at a density of 1×10 3 cells / well. The cells were cultured at 37 °C for 10 days. Subsequently, the cells were fixed with paraformaldehyde for 30 minutes and stained with 2.5% crystal violet for 15 minutes. Clusters containing ≥50 cells were counted as a colony.

[0131] 1.6 Co-immunoprecipitation

[0132] Samples were homogenized in a single detergent lysis buffer (1% NP40, 0.25% sodium deoxycholate, 50 mM Tris-HCl pH 7.4, 150 mM NaCl), and a protease inhibitor and phosphatase inhibitor mixture was added. The extracts were pre-cleared with Protein A / G beads (Cat#sc-2003, Santa Cruz, Texas, USA), then incubated overnight at 4°C with the appropriate antibody (1 - 2 μg), and subsequently 20 μL of Protein A / G beads were added and incubated 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. The beads were washed 3 times with lysis buffer and resuspended in 1× SDS loading buffer. Samples were further analyzed by Western blot.

[0133] 1.7 Immunohistochemical staining

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

[0135] 1.8 Immunofluorescent staining

[0136] Cells were seeded at a density of 1×10 5 cells / well on glass coverslips in 6-well plates. Cells were cultured 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% TritonX-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, Atlas, Bromma, Sweden) and DAPI. Immunofluorescent imaging was performed on a Leica TCS SP8 X confocal microscope, and images were taken using a 40× oil immersion lens.

[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). Approximately 10 4 cells were analyzed by CytoFlex S (Beckman, California, USA). Cells were gated based on forward and side scatter characteristics.

[0139] 1.10 RNA sequencing analysis

[0140] RNA sequencing analysis was performed by BGI. After total RNA was extracted, mRNA was isolated using Oligo magnetic beads and cut into small fragments for cDNA synthesis. Libraries were generated in the Illumina system using the NEB Next UltraTM 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] 1.11ChIP sequencing analysis

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

[0143] 1.12 Tissue samples

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

[0145] 1.13 Xenograft Mouse Model

[0146] Congenital athymic nude mice (athymic Ncr-nu / nu) aged 5 to 6 weeks (SLAC, Shanghai, China) were used. The animal care and use committee of Shanghai Jiao Tong University approved the protocol of xenograft tumor model. 5 × 10 6 Huh7 cells were injected subcutaneously into the axilla of nude mice.

[0147] 2. Results

[0148] 2.1 SLC25A10 is closely related to HCC

[0149] Figure 1 Figure A shows the expression profile of hepatocellular carcinoma (HCC) cells under hypoxic conditions. The heat map shows the expression differences between three HCC cell lines (GepG2, Huh7, and Huh7). The volcano plot indicates that the mitochondrial carrier SLC25A10 is one of the upregulated genes ( Figure 1 In addition, Western blot ( Figure 1 C) and immunohistochemistry (IHC) ( Figure 1D) It was verified that the expression of SLC25A10 in clinical HCC was significantly higher than that in adjacent tissues. The inventors also detected the expression of SLC25A10 in several HCC cell lines in the laboratory ( Figure 1 E).

[0150] In addition, according to the data of TCGA, gene amplification or mutation of SLC25A10 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 G), indicating that SLC25A10 plays a key role in HCC progression.

[0151] 2.2 Hypoxia-induced splicing of SLC25A10 isoform 3 enters the nucleus by binding to IPO7

[0152] To study the effect of hypoxia on the expression of SLC25A10, ChIP-seq analysis was first performed using an anti-HIF1α antibody. As Figure 2 shown in A, hypoxia enhanced the binding of HIF1α to the SLC25A10 promoter. In addition, through transcriptome analysis of HCC cells, it was found that hypoxia induced a splicing variation of SLC25A10 from isoform 1 to isoform 3 ( Figure 2 B), and it was further verified by Q-PCR ( Figure 2 C). In addition, protein sequence analysis showed that SLC25A10 isoform 3 might enter the nucleus through its nuclear localization sequence PPWPWPP (SEQ ID NO:16). Western blot of nuclear isolation showed that SLC25A10 was present in the nucleus ( Figure 2 D). Confocal immunofluorescence images also showed the nuclear localization of SLC25A10 ( Figure 2 E). Immunohistochemical images showed that SLC25A10 increased in HCC cells compared with adjacent tissues, especially in the nucleus ( Figure 2 F).

[0153] To more precisely determine whether the PPWPWPP sequence in SLC25A10 isoform 3 promoted its nuclear localization, the cellular distribution of truncated SLC25A10 isoform 3 lacking PAWAWAP (SEQ ID NO:17) was detected. As Figure 2 shown in G, overexpression of SLC25A10 isoform 3 enhanced its nuclear localization, while the truncated isoform 3 could hardly enter the nucleus. Immunofluorescence images also confirmed that the truncated isoform 3 blocked its nuclear entry. However, the SLC25A10 transport inhibitor butanedioic acid (BMA) (Hlouschek et al., 2018) did not prevent this nuclear transport ( Figure 2H).

[0154] To determine which nuclear transport protein promotes the entry of SLC25A10 isoform 3 into the nucleus, mass spectrometry was performed to identify the nuclear transport proteins that bind to SLC25A10. Although the nuclear transport proteins IPO7 and IPO13 are two potential candidates that bind to SLC25A10 ( Figure 2 in I), Western blot results confirmed that IPO7 does bind to SLC25A10 ( Figure 2 in J). The depletion of IPO7 led to a decrease in SLC25A10 in the nucleus, as confirmed by Western blot and immunofluorescence staining analyses ( Figure 2 in K and L), indicating that IPO7 contributes to the nuclear localization of SLC25A10 isoform 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 the differentially expressed genes in HCC cells under hypoxic conditions. Figure 3 Shown in A, the apoptotic signal was activated in HCC cells under hypoxic conditions. In addition, the knockdown of SLC25A10 isoform 3 reduced the number of HepG2 cells ( Figure 3 in B and Figure 7 in A), while increasing SLC25A10 expression increased the number of Huh7 cells ( Figure 3 in C and Figure 7 in B). The colony formation assay showed that the knockdown of SLC25A10 isoform 3 led to a decrease in the number of colonies of HepG2 cells ( Figure 3 in D), while overexpressing SLC25A10 isoform 3 increased the number of colonies of Huh7 cells ( Figure 3 in 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 the knockdown of SLC25A10 isoform 3 increased the apoptosis rate of HepG2 cells from 8.91% to 11.31% ( Figure 3 in F), while overexpressing SLC25A10 isoform 3 decreased the apoptosis rate of Huh7 cells from 13.74% to 10.14% ( Figure 3G). In addition, apoptotic signals in HCC cells were also investigated. Compared with HepG2 cells, parental Huh7 cells showed higher levels of cleaved PARP, Caspase 3, and Caspase 9 after etoposide treatment, while the expression of SLC25A10 was higher in HepG2 cells (as shown in the left and right blue boxes in Figure 3 H and 3I). Consistently, knockdown of SLC25A10 in HepG2 cells led to increased levels of cytochrome C, cleaved PARP, Caspase 3, and Caspase 9 compared with parental HepG2 cells (as shown in the left blue box and the left red box). Conversely, overexpression of SLC25A10 in Huh7 cells led to decreased levels of cytochrome C, cleaved PARP, Caspase 3, and Caspase 9 compared with parental Huh7 cells (as shown in the right blue box and the right red box). These results indicate that SLC25A10 isoform 3 promotes resistance of HCC cells to etoposide-induced apoptosis.

[0158] 2.4 SLC25A10 isoform 3 upregulates BCL2A1 expression

[0159] To explore how SLC25A10 isoform 3 regulates etoposide-induced apoptosis, the transcriptional profiles of apoptosis-related genes in Huh7 cells overexpressing SLC25A10 were analyzed ( Figure 4 in A). SLC25A10 isoform 3 upregulated BCL2A1, which was one of the most upregulated genes. The results of qPCR and Western blot also verified this upregulation ( Figure 4 in B and C).

[0160] In addition, colony formation assays showed that knockdown of BCL2A1 increased the sensitivity of Huh7 cells to etoposide, even in Huh7 cells overexpressing SLC25A10 isoform 3 ( Figure 4 in D). Flow cytometry experiments showed that knockdown of BCL2A1 increased etoposide-induced apoptosis in HCC cells, and overexpression of SLC25A10 isoform 3 did not significantly reduce this apoptosis rate ( Figure 4 in E).

[0161] To determine whether SLC25A10 alleviates apoptosis dependent on its function in mitochondria, the effect of the SLC25A10 transport inhibitor BMA on etoposide-induced apoptosis was examined. As Figure 4As shown in F and G, although BMA did weaken mitochondrial function in HCCHuh7 cells, it did not eliminate the SLC25A10-induced expression of BCL2A1 in HCC cells. Whether the overexpressed was the wild-type SLC25A10 isoform 3 or the truncated isoform 3 lacking PAWAWAP, its mutation did not inhibit the growth of untreated Huh7 cells( Figure 8 ). Flow cytometry analysis showed that BMA did not affect the attenuation of etoposide-induced HCC cell apoptosis by SLC25A10( Figure 4 in H).

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

[0163] 2.5 SLC25A10 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 the nuclear fraction of cell lysates.

[0165] As Figure 5 shown in 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 in B), and hypoxia increased this binding( Figure 5 in C). In addition, 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 in D).

[0166] To investigate whether the apoptosis attenuation by SLC25A10 isoform 3 depends on CEBPB, the expression of BCL2A1 in Huh7 cells with knocked-down CEBPB was first detected. Western blot results showed that knocking down CEBPB decreased the BCL2A1 expression enhanced by SLC25A10 isoform 3( Figure 5 in E). In addition, knocking down CEBPB eliminated the alleviating effect of SLC25A10 isoform 3 on etoposide-induced apoptosis, as shown by colony formation assay, flow cytometry analysis, and TUNEL assay( Figure 5 in F-H). The results indicate that SLC25A10 isoform 3 regulates the expression of BCL2A1 by binding to CEBPB.

[0167] 2.6 Disrupting the binding of SLC25A10 to IPO7 enhances sensitivity to etoposide in a mouse model

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

[0169] As Figure 6 shown in A and Figure 10 A, HCC Huh7 cells were injected into the axilla and groin of mice, and Huh7-derived tumors were observed to respond to etoposide treatment regardless of whether SLC25A10 isoform 3 was overexpressed. However, when SLC25A10 isoform 3 was overexpressed, the efficacy of etoposide was attenuated. Consistent with this, the protein level of BCL2A1 was increased in tumors overexpressing SLC25A10 isoform 3 ( Figure 6 shown in B).

[0170] Knockdown of BCL2A1 did enhance the sensitivity of HCC xenografts to etoposide compared with the control group, even in cells overexpressing SLC25A10 isoform 3 ( Figure 6 shown in C and Figure 10 shown in B). Consistent with this, multicolor IHC analysis of these xenografts showed that overexpression of SLC25A10 isoform 3 upregulated the expression of BCL2A1 and reduced cleaved caspase 3; however, SLC25A10 isoform 3 did not reduce the enhanced cleaved caspase 3 caused by BCL2A1 knockdown( Figure 6 shown in C).

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

[0172] Finally, it was examined whether disrupting the binding of isoform 3 to IPO7 would enhance the sensitivity of HCC to etoposide. As Figure 6 shown in E and Figure 10As shown in D, deletion of the PPWPWPP (Δ232-252) motif that binds to IPO7 in SLC25A10 isoform 3 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 the mutant SLC25A10 isoform 3 did not significantly reduce the level 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 between isoform 3 and IPO7 may 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. Finally, this isoform 3 enhances the resistance of HCC to etoposide through BCL2A1. In addition, disruption of the binding between SLC25A10 and IPO7 can enhance the sensitivity of HCC to etoposide, laying a foundation for improving the chemotherapeutic effect of HCC.

[0175] In summary, the present invention reveals a new function of the 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 mentioned in this invention are cited herein as references, as if each document was cited individually as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A reagent combination for screening candidate compounds for treating liver cancer, characterized in that, Comprising: (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 (2) A second detection reagent set for detecting a second complex, wherein the second complex is a complex of SLC25A10 isoform 3 and CEBPB.

2. The reagent combination according to claim 1, wherein 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.

3. The reagent combination according to claim 2, wherein The first antibody, the second antibody, and the third antibody each independently carry a detectable label and / or are conjugated to magnetic beads.

4. A method for screening candidate compounds for treating liver cancer, characterized in that, Including the steps of: (s1) In the test group, culturing hepatoma cells in a culture system containing a test compound, and measuring data selected from the following groups in the test group: (a) The number A1 of the first complex formed by SLC25A10 isoform 3 and IPO7; and / or (b) The number B1 of the second complex formed by SLC25A10 isoform 3 and CEBPB; And in the control group, culturing hepatoma cells in a culture system with the same other conditions and without the test compound, and measuring data selected from the following groups in the control group: (c) The number A0 of the first complex formed by SLC25A10 isoform 3 and IPO7; and / or (d) The number B0 of the second complex formed by SLC25A10 isoform 3 and CEBPB; and (s2) Comparing the numbers of the first complex and / or the second complex in the test group and the control group, wherein, if the number A1 of the first complex in the test group is significantly lower than the number A0 of the first complex in the control group, and / or the number B1 of the second complex in the test group is significantly lower than the number B0 of the second complex in the control group, this indicates that the test compound is a candidate compound for treating hepatoma.

5. The method according to claim 4, wherein The hepatoma cells in the test group and the control group are drug-resistant hepatoma cells.

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

7. The method according to claim 4, characterized in that, The first complex is detected by the 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 the second detection reagent set, wherein the first detection reagent set comprises a first antibody against SLC25A10 isoform 3 and a third antibody against CEBPB.

8. The method according to claim 4, characterized in that, The test compound is selected from the following groups: antibodies or their binding fragments, small molecule compounds, nucleic acids, PROTEC compounds.

9. A kit, characterized in that, The kit comprises: (f1) A first container and a first detection reagent set for detecting a first complex located in the first container, wherein the first complex is a SLC25A10 isoform 3 and IPO7 complex; and (f2) A second container and a second detection reagent set for detecting a second complex located in the second container, wherein the second complex is a SLC25A10 isoform 3 and CEBPB complex.

10. The kit according to claim 9, characterized in that, The first detection reagent group includes a first antibody against SLC25A10 isoform 3 and a second antibody against IPO7; The second detection reagent group includes a first antibody against SLC25A10 isoform 3 and a third antibody against CEBPB.

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