Application of P5A ATPase as target spot in preparation of neuroblastoma treatment medicine

Abstract

The invention relates to the field of tumor treatment drugs, and discloses application of P5A ATPase as a target spot in preparation of neuroblastoma treatment drugs. It is found for the first time that P5A ATPase affects the expression level of LRP8 protein and endoplasmic reticulum translocation of an LRP8 signal peptide guide peptide chain, and then affects growth and proliferation of neuroblastoma cells, and a new target is provided for development of neuroblastoma treatment drugs. Besides, the invention also finds that two P5A ATPase inhibitors, namely sodium metavanadate and DCC, can inhibit endoplasmic reticulum translocation of an LRP8 signal peptide guide peptide chain and reduce the expression level of LRP8 protein, so that growth and proliferation of neuroblastoma cells are inhibited, and a new therapeutic drug is provided for neuroblastoma.

Description

Technical Field

[0001] The present invention relates to the field of tumor therapeutic drugs, and in particular to the application of P5A ATPase as a target in the preparation of neuroblastoma therapeutic drugs. Background Art

[0002] P-type ATPases are a class of ATPase pumps widely distributed throughout biological membranes. Named for the phosphorylated intermediates they catalyze during transport, they mediate the transmembrane transport of various ions and small molecules. Based on the differences in their substrates and the degree of sequence conservation within their substrate binding sites, the P-type ATPase family is divided into five subfamilies, P1 to P5. Each subfamily is further divided into subgroups such as A and B. P5 is present in all eukaryotic genomes and is currently the least well-studied subfamily of P-type ATPases. The signature sequence within the fourth transmembrane domain of P5 ATPases differs from that of other families. Compared to the conserved Pxxx(P / L) signature sequence of other families, P5 ATPases possess an additional proline (PPxxP). Based on this PPxxP signature, P5 ATPases are divided into two subfamilies, P5A and P5B. The P5A subfamily has only one member in humans, ATP13A1. Different from other P-type ATPases, P1-P3 ATPase mainly mediates Ca 2+ Mg 2+ 、Na + , K + P5A ATPase mediates the transmembrane transport of cations such as cations, P4 ATPase mediates the transport of lipids, P5B ATPase mediates the transport of polyamines, and P5A ATPase's substrates are large proteins. P5A ATPase facilitates the correct positioning of tail-anchored proteins, the membrane insertion of multi-spanning proteins, and the translocation of proteins to the endoplasmic reticulum (ER). However, the substrates and mechanisms by which P5A ATPase regulates protein translocation to the ER remain unclear. Studying the substrates and mechanisms of P5A ATPase transport can provide a theoretical basis and potential drug targets for the treatment of related diseases.

[0003] Neuroblastoma (NB) originates from neural crest cells of the sympathetic nervous system and is the most common extracranial malignancy in children. It is highly heterogeneous, can recur and progress, is highly malignant, and is prone to early metastasis and bone marrow metastasis, posing a serious threat to the life and health of children with the disease. In addition to traditional surgery, chemotherapy, radiotherapy, autologous stem cell transplantation, and 13-cis retinoic acid treatment, disialoganglioside (GD2) monoclonal antibody treatment has also been included in the NB multimodality treatment consensus. Despite this, the 5-year survival rate of children with high-risk NB is still less than 50%. Some of the discovered neuroblastoma treatment targets have limitations such as difficulty in direct targeting, poor treatment effect, and the possibility of drug resistance with long-term use. Therefore, the development of more therapeutic targets will be of great significance. Potential neuroblastoma therapeutic targets that have been discovered include SAAL1 (patent CN119220682A), OLFM3 (patent CN119061142A), PPEF1 (patent CN117568347A), etc., but no studies have found a link between P5A ATPase and neuroblastoma. Summary of the Invention

[0004] To address the above technical issues, the present invention provides the use of P5A ATPase as a target in the preparation of a drug for treating neuroblastoma. This invention, for the first time, discovers that P5A ATPase affects the expression level of LRP8 protein and the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain, thereby affecting the growth and proliferation of neuroblastoma cells. This provides a new target for the development of neuroblastoma therapeutics.

[0005] The specific technical solutions of the present invention are: In a first aspect, the present invention provides the use of P5A ATPase as a target in the preparation of a drug for treating neuroblastoma.

[0006] Neuroblastoma is highly dependent on low-density lipoprotein receptor-related protein 8 (LRP8), and inhibiting LRP8 can induce ferroptosis in neuroblastoma cells. The present invention found that knocking out P5A ATPase significantly downregulated LRP8 protein expression in cells and reduced the endoplasmic reticulum translocation of the LRP8 signal peptide-directed peptide chain. Furthermore, when cells were treated with the P5A ATPase inhibitors sodium metavanadate and N,N-dicyclohexylcarbodiimide, the endoplasmic reticulum translocation of the LRP8 signal peptide-directed peptide chain and LRP8 protein expression were inhibited, and the growth and proliferation of neuroblastoma cells were also suppressed. Therefore, P5A ATPase can be used as a therapeutic target for neuroblastoma and in the preparation of neuroblastoma treatment drugs.

[0007] In a second aspect, the present invention provides use of a P5A ATPase inhibitor in the preparation of a drug for treating neuroblastoma.

[0008] Preferably, the P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide (DCC).

[0009] Preferably, the P5A ATPase inhibitor is an ATP13A1 inhibitor.

[0010] Preferably, the neuroblastoma therapeutic drug is a drug that inhibits the growth and proliferation of neuroblastoma cells, or a drug that reduces the expression of LRP8 protein (low-density lipoprotein receptor-related protein 8), or a drug that inhibits the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain.

[0011] Preferably, the neuroblastoma therapeutic drug includes a P5A ATPase inhibitor.

[0012] Preferably, the neuroblastoma therapeutic drug further comprises a pharmaceutically acceptable excipient; the excipient comprises one or more of a carrier, an excipient and a solvent.

[0013] Preferably, the drug for treating neuroblastoma is in the form of an oral solution, capsule, tablet, pill or injection, and is administered orally, intravenously, subcutaneously or intramuscularly. The drug for treating neuroblastoma is intended for human or animal use.

[0014] In a third aspect, the present invention provides a method for constructing an in vitro cell model with low expression of LRP8 and CTSA proteins, comprising the following steps: adding a P5A ATPase inhibitor to a cell culture medium, culturing the cells, and obtaining a cell model with low expression of LRP8 and CTSA proteins.

[0015] The present invention discovered that treating cells with a P5A ATPase inhibitor reduces the expression of LRP8 and CTSA proteins within the cells. This method allows the construction of a cell model with low LRP8 and CTSA protein expression, which can be used to study the physiological functions of these proteins and develop related therapeutic drugs.

[0016] Preferably, the P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide.

[0017] Furthermore, the amount of sodium metavanadate added to the cell culture medium is 10-15 μM; the amount of N,N-dicyclohexylcarbodiimide added to the cell culture medium is 0.4-0.8 mM.

[0018] In a fourth aspect, the present invention provides a method for constructing an in vitro cell model of abnormal endoplasmic reticulum translocation of peptide chains directed by LRP8 and CTSA signal peptides, comprising the following steps: adding a P5A ATPase inhibitor to a cell culture medium, culturing the cells, and obtaining a cell model of abnormal endoplasmic reticulum translocation of peptide chains directed by LRP8 and CTSA signal peptides.

[0019] The successful translocation of proteins to the endoplasmic reticulum (ER) is crucial for protein biosynthesis. ER translocation can be divided into three key steps: cytoplasmic targeting factors specifically recognize the protein's signal sequence; the cytoplasmic targeting factor interacts with its receptor on the ER membrane to target the nascent protein to the ER translocation complex; and the translocation complex on the ER membrane translocates the nascent protein into the endoplasmic lumen or inserts it into the ER membrane. As the first step in protein translocation, signal sequence recognition is crucial for the proper functioning of protein ER translocation.

[0020] The present invention discovered that treating cells with a P5A ATPase inhibitor inhibits the endoplasmic reticulum translocation of LRP8 and CTSA signal peptide-guided peptide chains. This method constructs a cell model with abnormal endoplasmic reticulum translocation of LRP8 and CTSA signal peptide-guided peptide chains, which can be used to study these endoplasmic reticulum translocation processes, their physiological functions, and the development of related therapeutic drugs.

[0021] Preferably, the P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide.

[0022] Furthermore, the amount of sodium metavanadate added to the cell culture medium is 10-15 μM; the amount of N,N-dicyclohexylcarbodiimide added to the cell culture medium is 0.4-0.8 mM.

[0023] Compared with the prior art, the present invention has the following advantages: (1) The present invention found that P5A ATPase affects the expression level of LRP8 protein and the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain, thereby affecting the growth and proliferation of neuroblastoma cells, providing a new target for the treatment of neuroblastoma.

[0024] (2) The present invention found that two P5A ATPase inhibitors (sodium metavanadate and N,N-dicyclohexylcarbodiimide) inhibit the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain and reduce the expression level of LRP8 protein, thereby inhibiting the growth and proliferation of neuroblastoma cells, providing a new therapeutic drug for neuroblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The results are the experimental results of the effects of sodium metavanadate and DCC on the growth and proliferation of neuroblastoma cells. Figure 1 A is a microscopic picture of neuroblastoma cells after treatment with DMSO, sodium metavanadate, and DCC; Figure 1 B is the BrdU experiment to detect the effects of sodium metavanadate and DCC on the proliferation of neuroblastoma cells.

[0026] Figure 2 The results are from experiments on the effects of P5A ATPase (ATP13A1) on protein translocation and protein expression levels. Figure 2 A is WB detection of the endoplasmic reticulum translocation of different signal peptides in wild-type cells and P5A ATPase / ATP13A1 knockout cells; Figure 2 B is WB detection of LRP8::GFP protein levels in wild-type cells and P5A ATPase / ATP13A1 knockout cells; Figure 2 C is WB detection of the protein level of CTSA::GFP protein in wild-type cells and P5A ATPase / ATP13A1 knockout cells.

[0027] Figure 3 The experimental results show the effects of sodium metavanadate and DCC on the endoplasmic reticulum translocation and protein expression levels of LRP8 protein. Figure 3 A is WB detection of the effects of different drug treatments on the endoplasmic reticulum translocation of the LRP8 signal peptide guide peptide chain (LRP8SS::FLAG::DHFR::Opsin); Figure 3 B is WB detection of the effects of different drug treatments on the protein level of LRP8::GFP protein.

[0028] Figure 4 The experimental results show the effects of sodium metavanadate and DCC on CTSA protein-related endoplasmic reticulum translocation and protein expression levels. Figure 4 A is WB detection of the effects of different drug treatments on the endoplasmic reticulum translocation of the CTSA signal peptide guide peptide chain (CTSASS::FLAG::DHFR::Opsin); Figure 4 B is WB detection of the effects of different drug treatments on the protein level of CTSA::GFP protein. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the embodiments.

[0030] Overall embodiment First, the present invention relates to the use of P5A ATPase as a target in the preparation of a drug for treating neuroblastoma.

[0031] Second, the present invention relates to the use of a P5A ATPase inhibitor in the preparation of a drug for treating neuroblastoma.

[0032] In some embodiments, the P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide (DCC).

[0033] In some embodiments, the P5A ATPase inhibitor is an ATP13A1 inhibitor.

[0034] In some specific embodiments, the neuroblastoma therapeutic drug is a drug that inhibits the growth and proliferation of neuroblastoma cells, or a drug that reduces the expression of LRP8 protein, or a drug that inhibits the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain.

[0035] In some embodiments, the neuroblastoma therapeutic drug comprises a P5A ATPase inhibitor.

[0036] In some specific embodiments, the neuroblastoma therapeutic drug further comprises a pharmaceutically acceptable excipient; the excipient comprises one or more of a carrier, an excipient, and a solvent.

[0037] In some specific embodiments, the neuroblastoma therapeutic drug is in the form of an oral solution, capsule, tablet, pill or injection, and is administered orally, intravenously, subcutaneously or intramuscularly; the neuroblastoma therapeutic drug is intended for human or animal use.

[0038] Third, the present invention relates to a method for constructing an in vitro cell model with low expression of LRP8 and CTSA proteins, comprising the following steps: adding a P5A ATPase inhibitor to a cell culture medium, culturing the cells, and obtaining a cell model with low expression of LRP8 and CTSA proteins.

[0039] In some embodiments, the P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide. In one embodiment, the P5A ATPase inhibitor is sodium metavanadate, and the amount added to the cell culture medium is 10-15 μM; in another embodiment, the P5A ATPase inhibitor is N,N-dicyclohexylcarbodiimide, and the amount added to the cell culture medium is 0.4-0.8 mM.

[0040] Fourth, the present invention relates to an in vitro method for constructing a cell model in which LRP8 and CTSA signal peptides guide abnormal endoplasmic reticulum translocation of peptide chains, comprising the following steps: adding a P5A ATPase inhibitor to a cell culture medium, culturing the cells, and obtaining a cell model in which LRP8 and CTSA signal peptides guide abnormal endoplasmic reticulum translocation of peptide chains.

[0041] In some embodiments, the P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide. In one embodiment, the P5A ATPase inhibitor is sodium metavanadate, and the amount added to the cell culture medium is 10-15 μM; in another embodiment, the P5A ATPase inhibitor is N,N-dicyclohexylcarbodiimide, and the amount added to the cell culture medium is 0.4-0.8 mM. Specific embodiments The present invention is described below by way of specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, any changes and advantages that can be imagined by those skilled in the art are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used herein are conventional in the art and can be obtained from conventional commercial sources. The methods used herein are conventional in the art, unless otherwise specified.

[0044] Example 1: Effects of P5A ATPase / ATP13A1 on protein translocation and protein expression levels 1.1 Experimental Materials and Methods 1.1.1 Cell Source Human embryonic kidney cells (HEK293FT) were obtained from the American Type Culture Collection (ATCC) and are adherent cells.

[0045] 1.1.2 Construction of knockout cell lines Using CRISPR-Cas9 gene editing technology, a P5A ATPase / ATP13A1 knockout HEK293FT cell line was constructed as follows: (1) Construction of vector: Based on the sequence information of the target gene, search for available sgRNA targeting sequences (located on the exons of the gene) on the sgRNA design website Sequence Scan for CRISPR, and then compare the specificity of the sgRNA in the NCBI or UCSC database. Select 2-3 sgRNA sequences with high scores and good specificity. After determining the sgRNA targeting sequence, design and synthesize primers, and use Overlap PCR to insert the sgRNA targeting sequence into the pGL3-U6-sgRNA-PGK-puromycin (Plasmid #51133) vector.

[0046] (2) Cell inoculation: HEK293FT cell line was revived and passaged twice before being inoculated into a 6 cm culture dish.

[0047] (3) Transfection: Add 8 μL Lipofectamine 2000 (Invitrogen, 11668019) to 500 μL Opti-MEM, mix well by vortexing, and let it stand for 5 minutes. Add 1 μg of sgRNA and 3 μg of Cas9 to 500 μL Opti-MEM and mix well. Add the Lipofectamine 2000 mixture to the plasmid mixture, mix well by vortexing, centrifuge for 10 seconds, and incubate at room temperature for 20 minutes. Remove the cells inoculated the day before and replace half of the culture medium with fresh antibiotic-free DMEM. Mark the cells. Evenly add the incubated plasmid / Lipofectamine 2000 transfection reagent complex to the cell culture dish and gently shake the cell culture dish to evenly distribute it. After 6 hours of cell transfection, replace with fresh double-antibody DMEM culture medium.

[0048] (4) Drug screening: 24 hours after transfection, add blasticidin (Blasticidin) at a final concentration of 10 μg / mL and puromycin (Puromycin) at a final concentration of 1 μg / mL. Because the constructed sgRNA vector has a Puromycin resistance gene, and the Cas9 protein vector has a Blasticidin resistance gene. Therefore, when Blasticidin and Puromycin are added at the same time, cells that have not been successfully transfected with Cas9 and sgRNA or have only been successfully transfected with one of the plasmids can be killed. Cells containing both Cas9 and sgRNA plasmids are retained.

[0049] (5) Monoclonal cells: After the cells have been screened with blasticidin and puromycin and have grown to fill a 6cm culture dish, use an aspirator to remove the old culture medium; gently add 2.5mL of PBS buffer to wash the cells; then add 500μL of trypsin to detach the cells; then add 1mL of DMEM culture medium to terminate the trypsin reaction, and use a pipette to gently blow away the cells. Collect the cells into a 15mL centrifuge tube and centrifuge at 1000g for 3min to obtain a cell pellet. Resuspend the cells in double-antibody DMEM culture medium and use a cell sieve to separate the cells into single cells. Combined with a blood cell counting plate and gradient dilution method, dilute the cells to a concentration of about 10 cells per milliliter of culture medium. Then distribute the cells into 96-well plates, 100μL per well (i.e., about 1 cell per well). Three 96-well plates can basically meet the needs. In addition, you can also choose to use a flow cytometer to sort out single cells.

[0050] (6) Cell expansion: monoclonal cells with good growth are selected from 96-well plates and then passaged to 12-well plates after they grow up.

[0051] (7) Western Blot analysis: Continue passage the cell lines in the 12-well plate and collect the excess cells. Treat the collected cells with 0.5% NP40 cell lysis buffer and use Western Blot to detect the protein level of the target gene. If the protein level is significantly lower than that of wild-type cells or even absent, it indicates that the gene knockout may be successful.

[0052] (8) PCR identification: Screen out cell lines with significantly reduced or no protein expression in Western Blot results. Combine PCR and second-generation sequencing to determine whether the knockout is successful and the type of cell knockout.

[0053] (9) Cryopreservation: Select two cell lines that are correctly identified by Western Blot and PCR, amplify them and freeze them.

[0054] 1.1.3 Western blotting to detect the effects of P5A ATPase knockout on protein translocation and protein expression levels After viable cell counting, HEK293FT wild-type cells and P5A ATPase / ATP13A1 knockout cells were seeded with 5×10 5 and 8×10 5 Cells were plated in 35 mm culture dishes and cultured in a 37°C, 5% CO2 incubator for one day. The cells were then transiently transfected with the corresponding proteins. Fresh culture medium was replaced 6 hours after transfection. The following day, cells were harvested and processed for Western blotting.

[0055] 1.2 Experimental Results The experimental results of the effect of P5A ATPase / ATP13A1 on protein translocation and protein expression levels are as follows Figure 2 shown. Figure 2 A is the experimental result of WB detection of the endoplasmic reticulum translocation of LRP8 and CTSA signal peptide-guided peptide chains, among which: the Opsin tag contains N-glycosylation modification sites that can undergo glycosylation modification in the endoplasmic reticulum, which is used to indicate whether the protein has successfully translocated into the endoplasmic reticulum; "●" indicates the glycosylated band (successful translocation), and "○" indicates the unglycosylated band (unsuccessful translocation). Figure 2 B is the experimental result of WB detection of LRP8::GFP protein expression, where Actin is the internal control. Figure 2 C is the experimental result of WB detection of CTSA::GFP protein expression, where Actin is the internal control.

[0056] Studies have found that high-risk MYCN-amplified neuroblastomas are highly dependent on LRP8, and LRP8 has been identified as a key factor in selenium / selenocysteine ​​metabolism. Inhibition of LRP8 can induce ferroptosis in neuroblastoma cells.

[0057] The experimental results in this example show that knocking out P5A ATPase / ATP13A1 in human HEK293FT cells does not affect the normal growth of HEK293FT cells, but knocking out P5A ATPase causes the endoplasmic reticulum translocation of the LRP8 signal peptide guide peptide chain (LRP8SS::FLAG::DHFR::Opsin) in HEK293FT cells to be severely affected (e.g. Figure 2 To further confirm whether P5A ATPase can be used as a therapeutic target for high-risk MYCN-amplified neuroblastoma, the effect of P5A ATPase knockout on LRP8 protein was detected. The results showed that P5A ATPase knockout led to a significant downregulation of LRP8 protein (as shown in Figure 4A). Figure 2 These results indicate that P5A ATPase may serve as a new therapeutic target for neuroblastoma.

[0058] In addition, in this example, the P5A ATPase / ATP13A1 knockout cell line was used to detect the effect of P5A ATPase knockout on CTSA. The results showed that P5A ATPase knockout seriously affected the endoplasmic reticulum translocation of the CTSA signal peptide guide peptide chain (CTSASS::FLAG::DHFR::Opsin) (e.g. Figure 2 At the same time, this example also detected the effect of P5A ATPase knockout on the amount of CTSA protein. The results showed that P5A ATPase knockout resulted in a significant decrease in the amount of CTSA protein (as shown in Figure 2A). Figure 2 C).

[0059] Example 2: Effects of sodium metavanadate and DCC on LRP8 protein-related endoplasmic reticulum translocation and protein expression levels 2.1 Experimental materials and methods 2.1.1 Cell Source Human embryonic kidney cells (HEK293FT) were obtained from the American Type Culture Collection (ATCC) and are adherent cells.

[0060] 2.1.2 Western blotting to detect the effects of sodium metavanadate and DCC on LRP8 protein-related endoplasmic reticulum translocation and protein expression levels Viable cell count, 1.5×10 5 Viable cells were seeded into 35 mm culture dishes at 400 μg / mL and cultured in a 37°C, 5% CO2 incubator for one day before transient transfection with the corresponding proteins. Six hours after transfection, the culture medium was replaced with fresh media and treated with dimethyl sulfoxide (DMSO), N,N-dicyclohexylcarbodiimide (DCC) (0.5 mM in the culture medium), oligomycin A (1 μM in the culture medium), and sodium metavanadate (10 μM in the culture medium). Two days later, cells were harvested and processed for Western blotting.

[0061] 2.2 Experimental Results The experimental results of the effects of sodium metavanadate and DCC on LRP8 protein-related endoplasmic reticulum translocation and protein expression levels are as follows Figure 3 As shown, Figure 3 A is the experimental result of WB detection of the endoplasmic reticulum translocation of the LRP8 signal peptide-guided peptide chain under different treatments, among which: the Opsin tag contains an N-glycosylation modification site that can undergo glycosylation modification in the endoplasmic reticulum, which is used to indicate whether the protein has successfully translocated into the endoplasmic reticulum; "●" indicates the glycosylated band (successful translocation), and "○" indicates the unglycosylated band (unsuccessful translocation). Figure 3 B is the experimental result of WB detection of LRP8::GFP protein expression under different treatments, where Actin is the internal control.

[0062] To further demonstrate the application of P5A ATPase as a target in tumor therapy, this example treated HEK293FT cells with control solutions DMSO, oligomycin A, sodium metavanadate, and DCC for 24 hours, and then detected the endoplasmic reticulum translocation of the peptide chain guided by the LRP8 signal peptide. The results showed that both sodium metavanadate and DCC treatments caused severe defects in the endoplasmic reticulum translocation of the peptide chain guided by the LRP8 signal peptide (e.g., Figure 3In addition, the expression level of LRP8 protein was also detected in this example, and the results showed that both sodium metavanadate and DCC treatments resulted in a significant decrease in the protein level of LRP8 protein (as shown in FIG. Figure 3 The above results indicate that sodium metavanadate and DCC affect the function of P5A ATPase, thereby reducing the expression of LRP8 and inhibiting the endoplasmic reticulum translocation of the peptide chain guided by the LRP8 signal peptide.

[0063] Example 3: Effects of sodium metavanadate and DCC on CTSA protein-related endoplasmic reticulum translocation and protein expression levels 3.1 Experimental materials and methods 3.1.1 Cell Source Human embryonic kidney cells (HEK293FT) were obtained from the American Type Culture Collection (ATCC) and are adherent cells.

[0064] 3.1.2 Western blotting to detect the effects of sodium metavanadate and DCC on CTSA protein-related endoplasmic reticulum translocation and protein expression levels Viable cell count, 1.5×10 5 Viable cells were seeded at 1 μg / mL in a 35 mm culture dish and cultured in a 37°C, 5% CO2 incubator for one day before transient transfection with the corresponding proteins. Six hours after transfection, the culture medium was replaced with fresh media and treated with DMSO, DCC (0.5 mM in the culture medium), oligomycin A (1 μM in the culture medium), and sodium metavanadate (10 μM in the culture medium). Two days later, cells were harvested and processed for Western blotting.

[0065] 3.2 Experimental Results The experimental results of the effects of sodium metavanadate and DCC on CTSA protein-related endoplasmic reticulum translocation and protein expression levels are as follows Figure 4 As shown, Figure 4 A shows the experimental results of WB detection of the endoplasmic reticulum translocation of the CTSA signal peptide-guided peptide chain under different treatments, where: the Opsin tag contains an N-glycosylation modification site that can undergo glycosylation modification in the endoplasmic reticulum, which is used to indicate whether the protein has successfully translocated into the endoplasmic reticulum; "●" indicates a glycosylated band (successful translocation), and "○" indicates an unglycosylated band (unsuccessful translocation). Figure 4 B is the experimental result of WB detection of CTSA::GFP protein expression under different treatments, where Actin is the internal reference.

[0066] In this example, HEK293FT cells were treated with control solutions DMSO, oligomycin A, sodium metavanadate, and DCC for 24 hours, and then the endoplasmic reticulum translocation of the peptide chain guided by the CTSA signal peptide was detected. The results showed that both sodium metavanadate and DCC treatments resulted in severe defects in the endoplasmic reticulum translocation of the peptide chain guided by the CTSA signal peptide (e.g., Figure 4 In addition, the expression level of CTSA protein was also detected in this example, and the results showed that both sodium metavanadate and DCC treatments resulted in a significant decrease in the protein level of CTSA protein (as shown in FIG. Figure 4 The above results indicate that sodium metavanadate and DCC affect the function of P5A ATPase, thereby reducing the expression of CTSA and inhibiting the endoplasmic reticulum translocation of the peptide chain guided by the CTSA signal peptide.

[0067] Example 4: Effects of sodium metavanadate and DCC on the growth and proliferation of neuroblastoma cells 4.1 Experimental Materials and Methods 4.1.1 Cell Source Neuroblastoma cells (SH-SY5Y) were obtained from the American Type Culture Collection (ATCC) and are adherent cells.

[0068] 4.1.2 Photographic examination of the effects of sodium metavanadate and DCC on neuroblastoma cell growth. Live cell counts (1.5 × 10 5 / mL of viable cells were seeded into a 35 mm diameter culture dish and cultured in a 37°C, 5% CO2 cell culture incubator for 1 day. DMSO, DCC (0.5 mM in the culture medium), and sodium metavanadate (10 μM in the culture medium) were then added for 2 days, and the cell growth status was analyzed by photographing.

[0069] 4.1.3 BrdU assay to detect the effects of sodium metavanadate and DCC on neuroblastoma cell proliferation 1.5×10 5Cells were seeded at 100 μg / mL in a 35 mm diameter culture dish (with a coverslip inside) and cultured for one day. Synchronized cells were then synchronized for three days using 0.4% FBS-containing medium, allowing the majority of cells to enter the G0 phase. Synchronized cells were then treated with DMSO, DCC (0.5 mM in the culture medium), and sodium metavanadate (10 μM in the culture medium) for 24 hours. BrdU (stock solution: 1.0 mg / mL, final concentration: 0.03 μg / mL) was added and incubated at 37°C for 40 minutes. The culture medium was discarded, and the slides were washed three times with PBS. The slides were fixed with methanol / acetic acid for 10 minutes. The fixed slides were air-dried and inactivated with 0.3% H2O2-methanol for 30 minutes. The slides were blocked with 5% normal rabbit serum. Nucleic acids were denatured with formamide at 100°C for 5 minutes. After cooling in an ice bath, the slides were washed with PBS and the primary antibody, an anti-mouse BrdU monoclonal antibody, was added at a working concentration of 1:50. Negative controls were treated with PBS or serum. The cells were detected by ABC method and stained with hematoxylin or eosin. The total number of cells and the number of BrdU-positive cells in 10 high-power fields were randomly counted under a microscope to calculate the labeling index (LI).

[0070] 4.2 Experimental Results The experimental results of the effects of sodium metavanadate and DCC on the growth and proliferation of neuroblastoma cells are as follows Figure 1 shown. Figure 1 A is a microscope photo of neuroblastoma cells under different treatments. Figure 1 B shows the experimental results of BrdU assay to detect the proliferation of neuroblastoma cells under different treatments, where: the quantification of cell proliferation is from 4 biological replicates and is expressed as mean ± SEMs; "***" indicates p < 0.001, and "****" indicates p < 0.0001 (t-test).

[0071] In order to further explore the role of sodium metavanadate and DCC in targeting P5A ATPase in treating tumors, this example tested whether DCC and sodium metavanadate had an effect on the growth of neuroblastoma cells SH-SY5Y. The results showed that both DCC and sodium metavanadate seriously affected the growth of neuroblastoma cells SH-SY5Y (e.g., Figure 1 In addition, the proliferation of neuroblastoma cells SH-SY5Y was detected by BrdU assay in this example. The results showed that both DCC and sodium metavanadate had a significant inhibitory effect on the proliferation of neuroblastoma cells SH-SY5Y (as shown in Figure 2A). Figure 1 (B) This indicates that P5A ATPase can be used as a drug target for the treatment of neuroblastoma, and DCC and sodium metavanadate are potential drugs.

[0072] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0073] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. Application of P5A ATPase as a target in the preparation of therapeutic drugs for neuroblastoma.

2. Application of P5A ATPase inhibitors in the preparation of drugs for the treatment of neuroblastoma.

3. The use according to claim 2, characterized in that The P5A ATPase inhibitor is sodium metavanadate and / or N,N-dicyclohexylcarbodiimide.

4. The use according to claim 2 or 3, characterized in that The P5A ATPase inhibitor is an ATP13A1 inhibitor.

5. The use according to claim 2 or 3, characterized in that The neuroblastoma therapeutic drug is a drug that inhibits the growth and proliferation of neuroblastoma cells, or a drug that reduces the expression of LRP8 protein, or a drug that inhibits the endoplasmic reticulum translocation of the peptide chain guided by the LRP8 signal peptide.

6. The use according to claim 2 or 3, characterized in that The neuroblastoma therapeutic drug includes a P5A ATPase inhibitor.

7. The use according to claim 6, characterized in that The neuroblastoma therapeutic drug further comprises pharmaceutically acceptable excipients; the excipients include one or more of a carrier, an excipient and a solvent.

8. The use according to claim 2 or 3, characterized in that The dosage form of the neuroblastoma therapeutic drug is oral solution, capsule, tablet, pill or injection, and the administration method is oral, intravenous, subcutaneous or intramuscular injection; the neuroblastoma therapeutic drug is applicable to humans or animals.

9. A method for constructing an in vitro cell model with low expression of LRP8 and CTSA proteins, characterized in that: The following steps are involved: A P5A ATPase inhibitor was added to the cell culture medium, and the cells were cultured to obtain a cell model with low expression of LRP8 and CTSA proteins.

10. A method for constructing an in vitro cell model of abnormal endoplasmic reticulum translocation of peptide chains guided by LRP8 and CTSA signal peptides, characterized in that: The following steps are involved: A P5A ATPase inhibitor was added to the cell culture medium and the cells were cultured to obtain a cell model in which LRP8 and CTSA signal peptides directed abnormal endoplasmic reticulum translocation of peptide chains.

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