Application of sgRNA with metallothionein gene knocked out in preparation of disulfiram medicine for treating tumors

Abstract

The invention discloses an application of a metallothionein gene knockout sgRNA in preparation of a drug for treating tumor disulfiram, a molecular mechanism influencing DSF drug resistance is found through RNA sequencing and data analysis, and a sequencing result shows that multiple enriched genes of up-regulated genes of a DSF treatment group belong to a metallothionein gene MTs family; real-time PCR detection finds that mRNA expression of tumor cells MTs treated by DSF is increased, and the result is consistent with a sequencing result; in order to determine whether up-regulation of MTs expression is related to tumor cell DSF drug resistance, expression of MT1X and MT2A in an MTs gene family is down-regulated by using a gene knockout technology, and sensitivity of a tumor cell line in which MT1X and MT2A are knocked out to DSF is increased, so that MTs can be used as a target spot of DSF anti-tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a sgRNA for knocking out a metallothionein gene in the preparation of a disulfiram drug for treating tumors. Background Art

[0002] A tumor is a disease characterized by excessive proliferation of abnormal cells, and if the abnormal proliferation is not controlled, it will lead to tumor metastasis. Before the tumor metastasizes, the initial treatment includes surgical resection of the primary site and adjacent lymph nodes, drug chemotherapy and radiotherapy to prevent the tumor from continuing to grow and metastasize. However, in many cases, tumor cells that have undergone initial treatment still exist in the body and evolve into more malignant tumors, making them more difficult to treat and more likely to metastasize. The limitations of chemotherapy and radiotherapy lie in their toxicity. Although high-dose chemotherapy can induce tumor cell death, it has greater toxic side effects on the human body. Therefore, it is crucial to develop specific anti-tumor drugs that can selectively induce tumor cell death while being non-lethal to normal cells.

[0003] The development of new anti-tumor drugs must undergo preclinical and clinical trial evaluations, which is a time-consuming and expensive process. Currently, combination therapy is an important way to treat tumors. This method can achieve anti-tumor effects through a variety of different mechanisms and can also reduce the adverse reactions of drugs. In addition, "new uses of old drugs" has opened up a new perspective for tumor treatment. Drug reuse can shorten the development time by 5 to 7 years and reduce research and development funds. Disulfiram (DSF) is a drug widely used to treat alcohol addiction. It has been used for more than 70 years. The safety of human administration has been fully confirmed. It is cheap and has mild toxic and side effects. Studies in recent years have shown that DSF plays an important role in tumor treatment. As more and more studies have confirmed the anti-cancer activity of DSF, a large number of clinical trials have been carried out to evaluate the feasibility of DSF in treating tumors. As a drug for quitting alcohol, DSF currently plays a role in anti-tumor research and has attracted much attention. Its advantages are safety, low cost and few side effects, which has great advantages in anti-tumor treatment research.

[0004] Although DSF has great application potential in tumor treatment, chemotherapy drugs alone are prone to drug resistance, and only a very small number of patients have significant therapeutic effects. Natural resistance and acquired resistance are the key factors that hinder the widespread clinical application of DSF. Studies have shown that hepatocellular carcinoma is not effective in DSF combined with copper treatment, and its mechanism is related to the increase in PDL1 stability caused by the drug, which leads to immunosuppression. At present, the mechanism of DSF resistance is not completely clear. Previous studies used DSF to treat a variety of blood system tumor cells including lymphoma and myeloma cells, and found that most blood tumor cells were not sensitive to DSF alone, suggesting that these cells may have natural resistance to DSF alone; some cells are sensitive to low concentrations and resistant to high concentrations after DSF drug treatment, suggesting that such tumor cells may develop acquired resistance if DSF is used for long-term anti-tumor treatment. Only a very small number of cells are completely sensitive to DSF. Studying the mechanism of DSF tumor resistance and improving the sensitivity of tumor cells to DSF, and enhancing the toxicity of DSF to tumor cells are the key points and difficulties that need to be solved by the present invention for the clinical application of DSF in treating tumors. Summary of the invention

[0005] The present invention provides an application of sgRNA for knocking out metallothionein gene in preparing a disulfiram drug for treating tumors, a method for treating tumors and a disulfiram drug composition for treating tumors, which solve the problems raised by the background technology.

[0006] Technical solution: The use of the sgRNA for knocking out the metallothionein gene of the present invention in the preparation of a disulfiram drug for treating tumors. The disulfiram drug composition for treating tumors at least includes the sgRNA for knocking out the metallothionein gene and a disulfiram component.

[0007] A method for treating tumors, comprising administering a therapeutically effective amount of a disulfiram drug for treating tumors to a subject, wherein the disulfiram drug can induce enhanced expression of metallothionein genes MTs in tumor cells, and selecting appropriate MTs family genes for knockout to increase the sensitivity of tumor cells to the disulfiram drug and improve the ability to kill tumor cells.

[0008] Compared with the prior art, the present invention has at least the following outstanding advantages: 1. The present invention uses RNA sequencing and data analysis to find the molecular mechanism that affects DSF resistance. The sequencing results show that there are multiple enriched genes in the upregulated genes of the DSF treatment group that belong to the metallothionein gene MTs family. Real-time PCR detection found that the mRNA expression of MTs in tumor cells treated with DSF increased, and the result was consistent with the sequencing result. In order to clarify whether the upregulation of MTs expression is related to the DSF resistance of tumor cells, the expression of MT1X and MT2A in the MTs gene family was downregulated by gene knockout technology. The tumor cell lines with MT1X and MT2A knocked out increased sensitivity to DSF, suggesting that MTs can be used as a target for DSF anti-tumor treatment. 2. Studies have shown that different tumor cells have different sensitivities to DSF, and some tumor cells are even resistant to high concentrations of disulfiram, suggesting that there is a certain mechanism in tumor cells that induces DSF resistance. The present invention studies the mechanism of DSF tumor resistance and improves the sensitivity of tumor cells to DSF. Enhancing the toxicity of DSF to tumor cells is very important for the clinical application of DSF to treat tumors; 3. The present invention uses RNA sequencing and data analysis to find the molecular mechanism that affects DSF resistance. RNA sequencing found that DSF can induce increased expression of metallothionein gene MTs in lymphoma cells, suggesting that MTs upregulation may be related to DSF resistance. Real-time PCR analysis results further verify that MTs gene mRNA expression in various tumor cells treated with DSF is upregulated. 4. The present invention uses gene knockout technology to downregulate the expression of MT1X and MT2A. Knockout of MT1X and MT2A increases the sensitivity of tumor cell lines to DSF, indicating that MTs can be used as a target for DSF anti-tumor therapy; 5. DSF has great application potential in tumor treatment, but the development of drug resistance limits its therapeutic effect. The research results of this invention show that MTs can be used as a target for DSF anti-tumor treatment. The development of drugs targeting MTs combined with disulfiram to treat tumors has a good market prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a line graph of the toxic effect of disulfiram DSF on different cells in the embodiment of the present invention (A is the viability of suspended cells measured using CCK-8 after DSF treatment; B is the viability of adherent cells measured using CCK-8 after DSF treatment).

[0010] Figure 2The differentially expressed gene results and GO enrichment analysis diagram of RNA sequencing of BJAB cells treated with DSF in the embodiment of the present invention (A is a volcano plot of RNA sequencing results of BJAB cells treated with 0.5 μM DSF and DMSO; B is a volcano plot of the top 10 upregulated genes shown by the sequencing results of A; C is a volcano plot of RNA sequencing results of BJAB cells treated with 5 μM DSF and DMSO; D is a volcano plot of the top 10 upregulated genes shown by the sequencing results of C; E is an enrichment analysis of upregulated or downregulated genes in DSF-treated cells using an online gene set enrichment analysis program (GSEA)).

[0011] Figure 3 This is a bar graph of MT1G, MT1X and MT2A gene expression in tumor cells treated with DSF detected by real-time PCR in an embodiment of the present invention.

[0012] Figure 4 This is a line graph showing the effect of knocking out MT1X and MT2A in HeLa cells on cell viability using CCK-8 to detect DSF.

[0013] Figure 5 This is a picture of PI staining to detect apoptosis in DSF-treated stable cell lines observed by fluorescence microscopy in the embodiment of the present invention (SgV, SgMT1X-1, SgMT1X-2, and SgMT2A stable cell lines in HeLa cells were treated with (0, 10 μM) DSF, and cells in each treatment group were stained for 10 minutes under light-proof conditions and then photographed under fluorescence microscopy). DETAILED DESCRIPTION

[0014] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the following will be combined with the attached Figures 1 to 5 The technical solutions of the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0015] Example 1: Use of the sgRNA for knocking out the metallothionein gene of the present invention in the preparation of a disulfiram drug for treating tumors. The disulfiram drug composition for treating tumors at least includes the sgRNA for knocking out the metallothionein gene and a disulfiram component.

[0016] Example 2: A method for treating tumors, comprising administering to a subject a therapeutically effective amount of a disulfiram drug for treating tumors, wherein the disulfiram drug can induce enhanced expression of metallothionein genes MTs in tumor cells, and selecting appropriate MTs family genes for knockout to increase the sensitivity of tumor cells to the disulfiram drug and enhance the ability to kill tumor cells.

[0017] The present invention searches for the molecular mechanism affecting DSF resistance through RNA sequencing and data analysis, and the sequencing results show that there are multiple enriched genes belonging to the metallothionein gene MTs family among the genes upregulated in the DSF treatment group, and Real-time PCR detection finds that the mRNA expression of MTs in tumor cells treated with DSF increases, and the result is consistent with the sequencing result; in order to clarify whether the upregulation of MTs expression is related to the DSF resistance of tumor cells, the expression of MT1X and MT2A in the MTs gene family is downregulated by gene knockout technology, and the sensitivity of tumor cell lines knocked out of MT1X and MT2A to DSF is increased, indicating that MTs can be used as a target for DSF anti-tumor treatment, and the development of drugs targeting MTs combined with disulfiram to treat tumors has a good market prospect.

[0018] (I) The specific test process of the embodiment of the present invention is as follows: 1. Use Cell Counting Kit (CCK-8) and microscope to observe the effect of different doses of DSF on the vitality of various tumor cells (lymphoma, cervical cancer, prostate cancer, breast cancer, hepatocellular carcinoma, pancreatic cancer, mouse melanoma, etc.). Specific steps for treating tumor cells with DSF: 1) Suspension cells: The present invention uses six blood system tumor cells, including U266, HBL-1, BJAB, OCI-LY10, SU-DHL-2, and Raji. The cell lines are centrifuged, washed once with PBS, resuspended in PRMI 1640 complete medium, and inoculated into 96-well plates according to experimental requirements. The volume of each well is 100 μL and the cell density is 2×10 5 / mL, DSF prepared with PRMI 1640 culture medium, different concentration groups were set up, and 3 replicates were set up in each concentration group. DSF was added to the tumor cells to make the final concentrations of 0μM, 0.2μM, 0.5μM, 1μM, 2μM, 5μM, and 10μM, respectively. At the same time, a blank control group without cells was set up, and the DSF drug and tumor cells were evenly mixed and cultured in an incubator.

[0019] 2) Adherent cells: The present invention uses Huh-7, HepG2, SUM159, MDA-MB-231, PC-3, PANC-1, HeLa, B16 and tool cell 293T. The above cell lines are inoculated in 96-well plates one day before DSF treatment, with a cell density of 1×10 ５ / mL, the volume of each well is 100μL, and the cells are cultured overnight. The next day, DSF is prepared with DMEM medium, and DSF drugs are added to the cells to make the final concentrations of 0μM, 0.1μM, 0.5μM, 1μM, 5μM, and 10μM. After the cells treated with the above drugs are cultured for 48 hours, 10% CCK-8 solution is added to each well of the suspended cells and mixed; the DMEM medium in the wells of the adherent cells is aspirated, and a 10% concentration of CCK-8 detection solution is prepared with PRMI 1640 medium, added to the 96-well plate, and incubated at 37°C for 2 hours. The 96-well cell culture plate is taken out, and the optical density (OD) value of each well at a wavelength of 450nm is detected by an ELISA instrument, and the results are recorded, and the cell viability is calculated at the same time.

[0020] 2. RNA sequencing to detect differentially expressed genes in tumor cells treated with DSF and those not treated with DSF: In the present invention, the tumor cells BJAB were treated with low concentration 0.5 μM and high concentration 5 μM DSF drugs, total RNA was extracted from the tumor cells, and RNA sequencing was performed. The differentially expressed genes between different samples in the sequencing results were analyzed using an online GSEA program.

[0021] 3. Real-time PCR technology was used to verify the effect of DSF treatment on MTs gene expression in various tumor cells.

[0022] 4. Construction and transfection of MT1X and MT2A specific sgRNA plasmids: 1) The specific experimental scheme for the construction and transfection of specific sgRNA vectors is as follows: The specific sgRNA sequence for MT1X-1 was synthesized by GeneWeiZ, with the following specific sequences: MT1X-1 sense strand: ccgGCATTTGCAGGAGCCGGCAC, MT1X-1 antisense strand: aacGTGCCGGCTCCTGCAAATGC. The specific sgRNA sequence for MT1X-2: MT1X-2 sense strand: ccgGCAGGAGCCGGCACAGGCAC, MT1X-2 antisense strand: aacGTGCCTGTGCCGGCTCCTGC. MT2A sense strand: ccgGCAGGAGCCGGCGCAGGTGC, MT2A antisense strand: aacGCACCTGCGCCGGCTCCTGC.

[0023] 2) Clone the sequence into the epiCRISPR vector: First, the BSPQI enzyme was used for digestion at 50°C for 4 hours, the forward primer and the reverse primer were annealed together at 94°C for 20 minutes, and then cooled to room temperature for 1 hour; the annealed primers were cloned into the vector using T4 DNA ligase at 16°C overnight; the above plasmids were verified by DNA sequencing, and the MT1X and MT2A target genes were knocked out using Lipo2000 transfection reagent. After transfection for 48 hours, 2 μg / ml puromycin was added to the culture medium and cultured for 7 days to screen stable transfection cell lines.

[0024] 5. CCK-8 was used to detect the sensitivity of cell lines transfected with specific sgRNA plasmids to DSF.

[0025] 6. PI staining was used to detect apoptosis of sgRNA plasmid-transfected cell lines treated with DSF: The cell line was transfected with sgRNA plasmid treated with DSF drug. After culturing for 48 hours, PI dye was added at a final concentration of 50 μg / mL. The whole operation was protected from light and placed in an incubator for staining for 10 minutes. The culture plate was taken out and observed and photographed using a fluorescence microscope.

[0026] (II) Analysis of test results: 1. Figure 1 This is a line graph showing the toxic effects of disulfiram on different tumor cells. Figure 1 (A) After DSF treatment, the viability of suspended cells was measured using CCK-8. Figure 1 (B) After DSF treatment, the viability of adherent cells was measured using CCK-8.

[0027] Depend on Figure 1 It can be seen that different tumor cells have different sensitivities to DSF treatment, which can be divided into three types: ① U266, Huh-7, HepG2, SUM159, MDA-MB-231, PC-3, PANC-1, HeLa and B16 cells did not show a significant decrease in cell viability after 48 hours of DSF treatment. ② After DSF treatment, the cell viability of SU-DHL-2 cells gradually decreased with the increase of drug concentration. ③ After DSF treatment, the cell viability of HBL-1, BJAB, Raji, OCI-Ly10 and tool cell 293T cells decreased significantly at low concentrations (0.2µM, 0.5µM), but did not decrease significantly at high concentrations (5µM, 10µM). The above results indicate that most cells are insensitive to DSF treatment, a very small number of tumor cells are completely sensitive to DSF, and some cells are sensitive to low concentrations of DSF but resistant to high concentrations.

[0028] 2. Figure 2 The results of differentially expressed genes and GO enrichment analysis of RNA sequencing of BJAB cells treated with DSF drugs are shown in Figure 2. Figure 2 (A) Volcano plot showing RNA sequencing results of BJAB cells treated with 0.5 μM DSF and DMSO, with the most upregulated genes marked. Figure 2 (B) Figure 2 The results in (A) show the top 10 upregulated genes. Figure 2 (C) Volcano plot showing RNA results of BJAB cells treated with 5 μM DSF and DMSO. Figure 2 (D) Figure 2 (C) The results show the top 10 upregulated genes. Figure 2 (E) Enrichment analysis of up-regulated or down-regulated genes in cells treated with DSF drug was performed using the online gene set enrichment analysis program (GSEA).

[0029] like Figure 2 As shown in Figure 2, RNA sequencing results showed that DSF drugs induced upregulation of mRNA expression of MT gene family members in tumor cells. Tumor cells BJAB were treated with low concentration (0.5μM) and high concentration (5μM) DSF drugs, and RNA sequencing was performed. After data analysis of differential genes in different samples, the results showed that compared with the control group, there were multiple enriched genes in the DSF drug treatment group that belonged to the metallothionein gene MTs family, including MT1X, MT1G, MT2A and MT1H (such as Figure 2 (A) Figure 2 (B) Figure 2 (C) Figure 2 (D)). In addition, the results of gene GO enrichment analysis showed that genes such as MT1X, MT1G, MT2A and MT1H were related to stress responses involving metal ions ( Figure 2 (E)).

[0030] 3. Figure 3In order to detect the MT1G, MT1X and MT2A gene expression bar graphs in DSF-treated tumor cells by real-time PCR, Real-time PCR technology was used to verify whether DSF drugs can upregulate MTs gene expression in tumor cells. HBL, U266, HeLa and SUM159 cells were treated with different concentrations of DSF drugs. The results of Real-time PCR analysis showed that compared with the DSF drug-untreated group, the gene expression of MTs family members MT1G, MT1X, and MT2A was significantly upregulated after DSF drug treatment. Samples were collected at different time points after DSF drug treatment of HeLa and B16 cells. The results showed that the expression of MT1X and MT1G in HeLa cells and MT1 and MT2 mRNA (MT family genes in mice) in B16 cells increased with the treatment time, and the highest expression was at 12h of treatment. The above results show that DSF treatment can induce upregulation of MTs expression.

[0031] 4. Figure 4 The figure is a line chart showing the effect of knocking out MT1X and MT2A in HeLa cells and detecting DSF on cell viability using CCK-8. MT1X and MT2A were targetedly knocked out in HeLa cells using CRISPR-Cas9 gene editing and molecular cloning technology, and the sensitivity of HeLa cell lines to DSF drugs after knocking out MT1X and MT2A was detected. The stable transfected cell lines of SgV, SgMT1X-1, SgMT1X-2, and SgMT2A were treated with DSF drugs, and the sensitivity of the stable transfected cell lines to DSF drugs was detected using CCK-8. The results showed that the cell viability of SgV did not change significantly after treatment with DSF drugs, while the cell lines of SgMT1X-1, SgMT1X-2, and SgMT2A knocked out had significantly lower viability than the control group SgV at high concentrations of DSF drugs (5μM, 10μM), indicating that knocking out MT1X and MT2A can enhance the sensitivity of drug-resistant HeLa cells to DSF drugs.

[0032] 5. Figure 5In order to detect the apoptosis of DSF-treated stable cell lines by PI staining and observe the cell apoptosis by fluorescence microscope, HeLa cells SgV, SgMT1X-1, SgMT1X-2, and SgMT2A stable cell lines were treated with DSF drugs (0, 10 μM), and the cells of each treatment group were stained for 10 minutes under light-proof conditions and then photographed by fluorescence microscope. The HeLa stable cell lines SgV, SgMT1X-1, SgMT1X-2, and SgMT2A treated with the above DSF drugs were stained with PI. The results showed that HeLa After treatment with 10 μM DSF, the red fluorescence of PI staining of SgV stably transfected cell lines did not increase significantly, and the cells were in good condition in the bright field. However, after treatment with DSF, the red fluorescence of PI staining of SgMT1X-1, SgMT1X-2 and SgMT2A stably transfected cell lines increased significantly at high DSF concentrations (10 μM) compared with the untreated group, and some cells shrank after DSF treatment could be seen in the bright field. The results showed that compared with the control group cells (transfected with SgV), DSF could induce apoptosis of more HeLa cells after MT1X and MT2A were knocked out in HeLa cells.

[0033] In order to study the molecular mechanism of DSF resistance, the present invention found through RNA sequencing that DSF can induce increased expression of metallothionein gene MTs family gene members in lymphoma cells, including MT1X, MT1G, MT2A and MT1H, and these genes are related to stress responses involving metal ions. MTs is a highly conservative, low molecular weight (6-7KD) cysteine-rich cytoplasmic protein. In the human body, the MTs gene is located on chromosome 16q13, and its family members include MT1, MT2 (i.e., MT2A), MT3, and MT4; among them, MT1 includes MT1A, MT1B, MT1E, MT1F, MT1G, MT1H and MT1M, while MT1C, MT1D, MT1I, ​​MT1J and MT1L are pseudogenes that cannot encode MT proteins; among them, MT1 and MT2 are widely expressed in multiple tissues, while MT3 and MT4 account for a small proportion and have tissue differences in expression. MTs play an important role in maintaining metal ion homeostasis and anti-toxicity. This type of protein regulates metal homeostasis by binding heavy metal ions through the sulfhydryl groups in their cysteine ​​residues. MTs have a high affinity for heavy metals and can bind exogenous heavy metals to protect against the cytotoxicity caused by metal stress. When MTs bind to heavy metals that normally exist in the human body, such as zinc and copper, they participate in cell growth, proliferation, and anti-oxidative stress response. MTs are involved in tumor growth, metastasis, and resistance to chemotherapy drugs. MTs participate in chemotherapy resistance in different tumors through different mechanisms. Studies have shown that overexpression of MTs causes tumor cells to acquire resistance to chemotherapy drugs. MTs overexpression can promote cell growth, angiogenesis, tumor metastasis, and chemotherapy resistance in a variety of tumors. Therefore, knocking out the MT gene to treat these tumors has broad application prospects.

[0034] In summary, MTs can be used as a target for anti-tumor therapy. Combined with the sequencing results, the expression of MTs in tumor cells treated with DSF drugs was upregulated, indicating that MTs played an important role in the anti-tumor treatment of DSF drugs. To further verify the relationship between MTs and DSF resistance of tumor cells, Real-time PCR was used to detect the expression changes of MTs in tumor cells treated with DSF drugs. The results were consistent with the sequencing results. The expression of MT1G, MT1X, and MT2A genes in U266, HeLa, and SUM159 cells was significantly upregulated after DSF drug treatment. MTs in HeLa and B16 were upregulated in a time-dependent manner after DSF drug treatment, indicating that DSF drug treatment can induce upregulation of MTs expression. Then, is the upregulation of MTs expression related to the DSF resistance of tumor cells? In the present invention, two genes, MT1X and MT2A, in the MTs family were selected for knockout based on the sequencing results, and stable cell lines were constructed in HeLa and 293T cells. The stable cell lines were treated with DSF drugs, and the sensitivity of cells to DSF drugs was detected by CCK-8. The results showed that the sensitivity of cell lines with MT1X and MT2A knockout to DSF drugs was significantly improved, and DSF drugs induced more tumor cell death with MT1X and MT2A knockout, indicating that MTs are involved in the occurrence of DSF resistance of tumor cells and play an important role. The targeted knockout of MT1X and MT2A using CRISPR-Cas9 gene editing technology can increase the sensitivity of tumor cells to DSF, providing new ideas for clinical tumor treatment.

[0035] The above are preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of a sgRNA for knocking out the metallothionein gene in the preparation of disulfiram drugs for treating tumors.

2. The use of the sgRNA for knocking out the metallothionein gene according to claim 1 in the preparation of a disulfiram drug for treating tumors, characterized in that: The disulfiram drug for treating tumors at least includes sgRNA for knocking out the metallothionein gene and a disulfiram component.

3. Use of the sgRNA for knocking out the metallothionein gene according to claim 2 in the preparation of a disulfiram drug for treating tumors, characterized in that: Disulfiram can induce enhanced expression of metallothionein genes MTs in tumor cells. Appropriate MTs family genes can be knocked out to increase the sensitivity of tumor cells to disulfiram and enhance the ability to kill tumor cells.

4. A method for treating tumors, characterized in that: The method comprises administering to the subject a therapeutically effective amount of a tumor-treating disulfiram drug.

5. A disulfiram pharmaceutical composition for treating tumors, characterized in that: At least it includes sgRNA and disulfiram components for knocking out the metallothionein gene.

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