Use of rab30-dt inhibitors in the preparation of medicaments for the prevention and treatment of tumors

Abstract

The application discloses use of a RAB30-DT inhibitor in preparation of a drug for preventing and / or treating tumors. It is found for the first time that RAB30-DT lncRNA can specifically bind to a p53-R175H mutant, stabilize the p53-R175H protein level, and thus play a role in promoting tumor growth. It is proved that after knocking down the expression level of RAB30-DT lncRNA, the tumor growth can be effectively inhibited, and overexpression of RAB30-DT lncRNA promotes tumor growth, which indicates that the RAB30-DT lncRNA inhibitor can be used as a potential drug target for treatment of p53-R175H mutation related tumors.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of RAB30-DT inhibitors in the preparation of drugs for the prevention and treatment of tumors. Background Technology

[0002] The TP53 gene (Gene ID: 7157) is an important tumor suppressor gene, widely hailed as the "guardian of the human genome" due to its central role in regulating key biological processes such as cell cycle and apoptosis. The p53 protein encoded by this gene acts as a transcription factor, recognizing specific binding sites in the promoters or enhancers of target genes and controlling gene expression by regulating transcriptional activity. Furthermore, it directly regulates key biological processes such as cell cycle, apoptosis, and DNA repair through transcription-independent mechanisms. Despite its crucial role in tumor suppression, TP53 exhibits a remarkably high mutation rate in human cancers. Statistics show that mutations in the TP53 gene can be detected in over 50% of human cancers. Analysis reveals that these mutations are primarily concentrated within the core DNA-binding domain of the p53 protein, including known hotspot mutations such as R175H, R248W, R273H, R245S, R249S, and R282W. Further research indicates that p53 proteins carrying these mutations typically exhibit three distinct functional changes: First, they may completely lose the normal function of wild-type p53 (loss of function, LOF). Second, some mutants can suppress the function of remaining wild-type p53 within the same cell through a dominant negative effect (DNE). Finally, in rare cases, these mutant proteins may also acquire novel oncogenic functions (gain of function, GOF), thereby significantly promoting tumor development and progression. Given its central role in cancer development and its unique molecular mechanisms, p53 is undoubtedly a highly attractive target for anti-tumor drug development.

[0003] As the most prevalent form of p53 mutation, p53-R175H ranks first in incidence among p53 missense mutations in tumors. Studies have shown that this mutant exhibits functional characteristics distinct from wild-type p53 through interactions with multiple proteins, including promoting malignant phenotypes such as cancer cell proliferation, migration, invasion, metabolic reprogramming, angiogenesis, and chemotherapy resistance. Specifically, p53-R175H can specifically bind to the transcription factor BACH1, relieving its transcriptional repression of SLC7A11 ferroptosis-related genes, thereby antagonizing ferroptosis and promoting tumor growth. In addition, p53-R175H can also inhibit the function of other tumor suppressor factors (such as p63 and p73) by binding to them, thereby promoting cancer metastasis and invasion. Interestingly, although wild-type p53 protein remains at low levels under non-stress conditions through MDM2-mediated ubiquitination and degradation, p53 mutant proteins, including p53-R175H, often accumulate at high levels in tumors, which is crucial for their GOF (Goal of Function) in tumorigenesis. Therefore, there is an urgent need to explore the potential mechanisms by which p53-R175H accumulates in cancer, so as to provide a theoretical basis for the development of specific p53-R175H inhibitors.

[0004] Currently, most drugs targeting p53 mutants are small molecule compounds that exhibit good tolerability in in vitro experiments, but have failed to demonstrate significant therapeutic responses in preclinical models and early clinical trials. The core reason is that wild-type p53 and its mutants lack stable drug-binding pockets and dynamically conformational target sites, leading to significant technical bottlenecks in structure-based small molecule drug development and making this target long considered an "undrugable" target.

[0005] In recent years, breakthroughs in high-throughput sequencing technology have revealed the widespread presence of transcriptionally active non-coding regions in the human genome, whose products, long non-coding RNAs (lncRNAs), significantly outnumber protein-coding genes by orders of magnitude. As a crucial component of the genome's "dark matter," lncRNAs precisely regulate gene expression networks through multi-dimensional mechanisms, including epigenetic regulation (such as chromatin remodeling and DNA methylation), transcriptional regulation (regulation of RNA polymerase II activity), and post-transcriptional regulation (mRNA stability and translational regulation). They play a pivotal role in key biological processes such as cell fate determination, developmental programming, and disease occurrence. Notably, the functional realization of lncRNAs is highly dependent on their subcellular localization: nuclear lncRNAs can cis-regulate the transcriptional activity of neighboring genes and remotely regulate chromosome structure and gene expression through three-dimensional genome topological remodeling; while cytoplasmic lncRNAs competitively bind to microRNAs via the ceRNA mechanism to regulate target mRNA stability or form functional complexes with post-translational modifying enzymes to influence protein activity. This "molecular scaffold" characteristic suggests that lncRNAs are not direct effector molecules, but rather achieve their biological effects by dynamically regulating the expression levels or functional states of key proteins. This characteristic provides them with a unique advantage as novel drug targets, and several targeted therapy strategies based on lncRNA regulatory networks have entered the preclinical research and development stage. Summary of the Invention

[0006] The purpose of this invention is to provide the use of RAB30-DT inhibitors in the preparation of drugs for the prevention and / or treatment of tumors. This invention is the first to discover that RAB30-DT lncRNA can specifically bind to the p53-R175H mutant, stabilizing p53-R175H protein levels, thereby promoting tumor growth; that is, RAB30-DT lncRNA can serve as a potential drug target for the treatment of p53-R175H mutation-related tumors. To achieve the above objective, the technical solution adopted by this invention is as follows: A first aspect of the invention provides the use of a RAB30-DT inhibitor in the preparation of a medicament for the prevention and / or treatment of tumors, wherein the tumor has a p53-R175H mutation.

[0007] In some embodiments of the present invention, the RAB30-DT inhibitor comprises at least one of the following: (a1) Substances that silence the RAB30-DT gene; (a2) Substances that knock out the RAB30-DT gene; (a3) Substances that inhibit the expression of the RAB30-DT gene.

[0008] In some embodiments of the present invention, the RAB30-DT inhibitor includes nucleic acid molecules, protein molecules or peptides, small molecule-oligonucleotide conjugates, and small molecule compounds.

[0009] In some embodiments of the present invention, the nucleic acid molecule is microRNA, siRNA, shRNA, dsRNA, sgRNA and / or antisense oligonucleotides (ASO).

[0010] In this invention, "antisense oligonucleotides" can specifically bind to lncRNA based on the base complementary pairing principle, recruit RNase H to degrade it, or directly block the interaction between lncRNA and other molecules such as proteins, thereby inhibiting its biological function. RNAi technology (siRNA or shRNA) utilizes the RNA interference mechanism to induce intracellular enzymes to degrade specific lncRNAs.

[0011] In some embodiments of the present invention, the sequence of the siRNA is shown in at least one of SEQ ID NO: 3 to SEQ ID NO: 4.

[0012] In some embodiments of the present invention, the shRNA has a forward sequence as described in SEQ ID NO: 16 and a reverse sequence as described in SEQ ID NO: 17, or has a forward sequence as described in SEQ ID NO: 18 and a reverse sequence as described in SEQ ID NO: 19.

[0013] In some embodiments of the present invention, the small molecule compound is a small molecule inhibitor; the "small molecule inhibitor" can target specific secondary or three-dimensional domains of RAB30-DT lncRNA, thereby inhibiting lncRNA function by stabilizing or disrupting its structure or blocking its interaction with proteins.

[0014] In some embodiments of the present invention, the protein molecule or polypeptide includes peptides that can mimic the structure of the protein, competitively bind to RAB30-DT lncRNA, or block the key interaction between RAB30-DT lncRNA and the protein.

[0015] In some embodiments of the present invention, the small molecule-oligonucleotide conjugate includes a ligand conjugate, for example, the above-mentioned nucleic acid molecules (such as ASO, siRNA) are conjugated to a specific target ligand via a linker to achieve tissue-specific delivery.

[0016] In some embodiments of the present invention, the delivery vector of the RAB30-DT inhibitor includes, but is not limited to, plasmid vectors, cationic polymer vectors, chitosan, liposomes, nanoparticles, and viral vectors.

[0017] In some embodiments of the present invention, the nanoparticles include, but are not limited to, lipid nanoparticles (LNPs).

[0018] "Lipid nanoparticles (LNPs)" form nanoparticles by encapsulating negatively charged RNA with positively charged lipids, thereby protecting the RNA and facilitating its entry into cells. In some embodiments of the present invention, the positively charged lipids are composed of various lipid components such as ionizable cationic lipids, cationic auxiliary lipids, cholesterol, and PEGylated lipids, serving as a highly efficient delivery system that enables the safe and efficient delivery of therapeutic molecules to target cells.

[0019] In some embodiments of the present invention, the nanoparticles include, but are not limited to, polymer nanoparticles, gold nanoparticles, and silica nanoparticles; the polymer nanoparticles, gold nanoparticles, and silica nanoparticles are loaded with nucleic acid drugs through electrostatic adsorption or encapsulation.

[0020] In some embodiments of the present invention, the viral vector includes, but is not limited to, adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, and herpesvirus vectors.

[0021] In some embodiments of the present invention, the tumor includes all tumor types expressing the p53-R175H mutation, and specific tumor types include, but are not limited to, breast cancer, tongue cancer, bile duct cancer, lung cancer, ovarian cancer, head and neck squamous cell carcinoma, colorectal cancer, pancreatic cancer, liver cancer, and glioblastoma.

[0022] In some embodiments of the present invention, the types of tumors include, but are not limited to, breast cancer, tongue cancer, bile duct cancer, non-small cell lung cancer, lung adenocarcinoma, serous ovarian cancer, head and neck squamous cell carcinoma, colorectal cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, and glioblastoma.

[0023] In some embodiments of the present invention, the drug comprises pharmaceutically acceptable excipients.

[0024] In some embodiments of the present invention, the excipients include at least one of the following: diluent, binder, wetting agent, lubricant, disintegrant, emulsifier, cosolvent, solubilizer, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant, antibacterial agent, or buffer.

[0025] In some embodiments of the present invention, the dosage form of the drug includes at least one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pellets, injections, oral preparations, suppositories, enemas, aerosols, patches, or drops.

[0026] In some embodiments of the present invention, the route of administration of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulized administration, or transdermal administration.

[0027] In some embodiments of the present invention, the dosage range of the drug is 0.01-50 mg / kg / day.

[0028] In a second aspect, the present invention provides an sgRNA, a DNA molecule encoding the sgRNA, or an expression cassette, recombinant vector, or recombinant cell containing the DNA molecule.

[0029] In some embodiments of the present invention, the sequence of the sgRNA is shown in SEQ ID NO: 1.

[0030] In this invention, sgRNA is used in conjunction with CRRSPR / Cas9 to achieve gene knockout, wherein the gene is p53-R175H gene; the p53-R175H gene refers to the mutation of arginine (R) to histidine (H) at position 175 of wild-type p53 (Gene ID: 7157).

[0031] In some embodiments of the present invention, the vector includes a viral vector or a non-viral vector.

[0032] In some embodiments of the present invention, the viral vector includes at least one of lentiviral vector, adenovirus vector, baculovirus vector, retrovirus vector, poxvirus vector, Sendai virus vector, and herpes simplex virus vector.

[0033] In some embodiments of the present invention, the non-viral vector includes at least one of the following: plasmid vector, cationic polymer vector, chitosan, liposome, and nanoparticle vector.

[0034] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new plant or animal varieties.

[0035] In some embodiments of the present invention, the prokaryotic cells include bacteria well known in the art, such as Escherichia coli, Streptomyces, and Bacillus subtilis, which are capable of expressing the target protein.

[0036] In some embodiments of the present invention, the eukaryotic cells include at least one of yeast cells, mammalian cells, plant cells, and insect cells.

[0037] A third aspect of the invention provides the application of RAB30-DT lncRNA in predicting the prognosis of tumor patients, wherein the tumor has a p53-R175H mutation.

[0038] In some embodiments of the present invention, the tumor includes all tumor types expressing the p53-R175H mutation, and specific tumor types include, but are not limited to, breast cancer, tongue cancer, bile duct cancer, lung cancer, ovarian cancer, head and neck squamous cell carcinoma, colorectal cancer, pancreatic cancer, liver cancer, and glioblastoma.

[0039] In some embodiments of the present invention, the types of tumors include, but are not limited to, breast cancer, tongue cancer, bile duct cancer, non-small cell lung cancer, lung adenocarcinoma, serous ovarian cancer, head and neck squamous cell carcinoma, colorectal cancer, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, and glioblastoma.

[0040] The beneficial effects of this invention are: This invention is the first to discover that RAB30-DT lncRNA can specifically bind to the p53-R175H mutant, stabilizing p53-R175H protein levels and thus promoting tumor growth. This invention demonstrates that knocking down RAB30-DT lncRNA expression effectively inhibits tumor growth, while overexpression of RAB30-DT lncRNA promotes tumor growth, indicating that RAB30-DT lncRNA inhibitors can serve as potential drug targets for the treatment of p53-R175H mutation-related tumors. Attached Figure Description

[0041] Figure 1A This indicates a potential lncRNA that specifically binds to p53-R175H (fold change > 3).

[0042] Figure 1B This study demonstrates the effect of knocking down lncRNAs that potentially bind specifically to p53-R175H on p53-R175H protein levels.

[0043] Figure 1C The study showed that RAB30-DT lncRNA in three tumor cells (SK-BR3, Cal-33, and HuCCT1) with a natural p53-R175H mutation binds endogenously to p53.

[0044] Figure 1D The study showed that RAB30-DT lncRNA in HCT116 tumor cells with wild-type p53 binds endogenously to p53.

[0045] Figure 1E The study showed that RAB30-DT lncRNA in four types of tumor cells (LS1034, H1770, BT-549, and H1975) with natural p53-R245S, p53-R248W, p53-R249S, and p53-R273H mutations, respectively, binds endogenously to p53.

[0046] Figure 2 This shows the structural features of RAB30-DT lncRNA and its location in the genome.

[0047] Figure 3 This study demonstrates the molecular mechanism by which RAB30-DT lncRNA regulates the stability of p53-R175H. AB shows the expression of p53-R175H in SK-BR3 and Cal-33 cells under RAB30-DT lncRNA overexpression; CD shows the expression of p53-R175H in SK-BR3 and Cal-33 cells under RAB30-DT lncRNA knockdown; and E shows the tumor growth in four groups of mice: LFPM overexpression group, control group, p53-R175H knockout group, and LFPM overexpression + p53-R175H knockout group.

[0048] Figure 4 The results of a clinical correlation study between RAB30-DT lncRNA and p53-R175H are shown. A shows the correlation between the gene expression level of RAB30-DT lncRNA and the protein expression level of p53-R175H in clinical samples. B shows the Kaplan-Meier survival analysis results of p53-R175H mutant lung cancer tissue samples. C shows the survival analysis results grouped by the p53-R175H / LFPM expression axis. D shows the tumor growth after knocking down RAB30-DT lncRNA expression in a PDX tumor model constructed from p53-R175H mutant non-small cell lung cancer tissue samples.

[0049] Figure 1C-1E and Figure 3 In the middle, "*" represents p <0.05, "**" means p <0.01, "***" represents p <0.001, where “ns” indicates no significant difference. Detailed Implementation

[0050] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0051] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0052] Example 1: lncRNA screening To screen for lncRNAs that specifically bind to p53-R175H, the p53-R175H sequence was first knocked into the Tp53 site using CRISPR-Cas9-mediated genome editing to establish the HCT116-p53 R175H cell line. The specific experimental steps are as follows: First, targeting the flanking region of the mutation site (R175), a highly cleaving sgRNA sequence 5'-AGCACATGACGGAGGTTGTG-3' (SEQ ID NO: 1) was screened and inserted into the PX459 (addgene: Plasmid #62988) vector to construct a CRISPR-sgRNA recombinant plasmid. Next, a DNA repair template containing homologous arms on both sides was designed to ensure the target mutation site was located in the center of the template. The DNA repair template sequence is as follows: 5'-gtgaggaatcagaggcctggggaccctgggcaaccagccctgtcgtctctccagccccagctgctcacCATCGCTATCTGAGCAGCGCTCATGGTGGGGGCAGTGTCTCACAACCTCCGTCATGTGCTGTGACTGCTTGTAGATGGCCATGGCGCGGACGCGGGTGCCGGGGGTGTGGAATCAACCCACAGCTGCACAGGGCAGGTCTTGGCCAGTT-3' (SEQ ID NO: 2). Then, the CRISPR-sgRNA recombinant plasmid and the DNA repair template were co-transfected into the HCT116 (ATTC, CCL-247) cell line using an electroporator. Single clones were selected using puromycin for selection, cultured, and sequenced to verify the mutation status. Clones with homozygous mutations were selected for further culture to obtain positive clones.

[0053] Then, in HCT116-p53 WT or HCT116-p53 R175H cells, lncRNAs specifically interacting with p53-R175H were screened using an RNA-binding protein immunoprecipitation assay combined with RNA sequencing. The steps for the RNA-binding protein immunoprecipitation assay are as follows: Collect 3 million positive clones of both wild-type and homozygous mutant cells, add 600 μL of cell IP lysis buffer (Thermo Scientific: 87788) to each, and lyse on ice for 40 minutes; centrifuge at 12000 rpm for 15 minutes, and collect the supernatant; then add p53 antibody (proteintech, 60283-2-Ig) to the supernatant sample and incubate at 4°C for 2 hours; take 20 μL of Dynabeads... TM Protein G (Invitrogen, 10003D) was added to the above samples and incubated overnight at 4°C. The next day, the samples were washed with washing buffer (Beyotime: P0013) at 4°C for 5 minutes each time, for a total of 4 washes. Trizol reagent (Invitrogen: 15596018CN) was added to the precipitate to extract RNA, and the enrichment of the target RNA was detected by reverse transcription and PCR.

[0054] Based on RNA enrichment, lncRNAs with fold change > 3 (A in Figure 1) were selected for validation. Validation involved knocking down lncRNAs with fold change > 3 using corresponding siRNAs. The experimental steps were as follows: HCT116-p53R175H cell lines were selected and seeded in 6-well plates. After cell adhesion, transfection was performed. 200 μL of culture medium was used to dilute 2 μL of liposome Lipofectamine 2000 (Thermo Fisher, 11668019) and 20 nM of the corresponding siRNA, respectively. After mixing, the mixture was incubated for 15 min, and then slowly dripped into the wells containing cells. After 48 hours, p53 expression levels were detected by Western blot. The primary antibodies used were anti-p53 antibody (ab131442, Abcam) and GAPDH antibody (Proteintech, 60004-1-Ig); the secondary antibody used was HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (ab150116, Abcam). The siRNA sequences used in the experiment are shown in Table 1 below.

[0055] Table 1: siRNA sequences

[0056] Experimental results showed that only knocking down RAB30-DT could significantly inhibit the protein level of p53-R175H (B in Figure 1). This indicates that RAB30-DT (hereinafter referred to as LFPM) is the most likely lncRNA involved in the regulation of the p53-R175H protein.

[0057] The inventors selected cell lines naturally expressing different p53 mutants and performed an immunoprecipitation assay of endogenous RNA-binding proteins by overexpressing the target gene LFPM or a negative control (H19) in the cell lines. The cell lines used in the experiment included SK-BR3 (ATTC, HTB-30), Cal-33 (ATTC, CL-0952), HuCCT1 (Pronosa, CL-0725), HCT116 (ATTC, CCL-247), LS1034 (ATTC, CRL-2158), H1770 (ATTC, CRL-5893), BT-549 (ATTC, HTB-122), and H1975 (Pronosa, CL-0298). Among them, SK-BR3, Cal-33, and HuCCT1 have the p53-R175H mutation, HCT116 has wild-type p53, LS1034 has the p53-R245S mutation, H1770 has the p53-R248W mutation, BT-549 has the p53-R249S mutation, and H1975 has the p53-R273H mutation. The inventors used the overexpression plasmid pLV-LFPM (vector backbone pLKO.1-TRC Cloning Vector, target gene LFPM (SEQ ID NO: 15) linker site EcoRI) to overexpress LFPM, and used H19 as a lncRNA negative control. Following standard procedures in the art, the LFPM gene or the H19 negative control gene was inserted into the pLKO.1-TRC Cloning Vector. The specific experimental steps for transfecting the overexpression plasmid are as follows: The target cell line was seeded in a 6-well plate and cultured in a 37°C, 5% CO2 incubator. Transfection was performed when the cells adhered and reached approximately 70% confluence. Before transfection, 5 μL of liposome transfection reagent Lipofectamine™ 2000 (Thermo Fisher, 11668019) and 2.5 μg of pLV-LFPM overexpression plasmid were diluted with 200 μL of Opti-MEM medium. After standing at room temperature for 5 minutes, the two were mixed and incubated at room temperature for 15 minutes to form a transfection complex. Subsequently, the complex was slowly added dropwise to the corresponding wells, gently shaken, and then returned to the incubator for further culture. Forty-eight hours after transfection, when the cells were in good growth condition and the confluence reached over 90%, an RNA-binding protein immunoprecipitation assay was performed using the same method as above. The results showed that the binding of the two proteins could only be detected in cells naturally expressing the p53-R175H mutant (CE in Figure 1).

[0058] Example 2: Cellular level study of LFPM The lncRNA LFPM gene contains three exons and is relatively conserved in vertebrates. 5' and 3' RACE analysis revealed that the LFPM transcript is 673 nt in length and located at chr11: 83072066-83073712 (…). Figure 2 Two cell lines that naturally express p53-R175H, SK-BR3 (Shanghai Cell Bank, Chinese Academy of Sciences, SCSP-5243) and Cal-33 (Pronosa, GCL-0952), were selected to construct a stable transfected cell line overexpressing LFPM. The gene sequence of LFPM is as follows: 5'--3' (SEQ ID NO: 15).

[0059] The steps for constructing a stable transfected cell line are as follows: 1) Packaging the virus in 293T cells (Shanghai Cell Bank, Chinese Academy of Sciences, SCSP-M502) using liposome transfection: Seed cells in logarithmic growth phase at an appropriate density in 75cm cells. 2 After cells adhered to the culture flask, transfection was performed the following day. 10 μL of Lipofectamine 2000 transfection reagent and 12 μg of packaging plasmid (pLP / VSVG lentiviral packaging vector (Invitrogen, K4970-00) and overexpression plasmid pLV-LFPM or negative control plasmid (pLV-LFPM backbone identical to pLV-LFPM but without the LFPM sequence) were diluted separately in 1 mL of Opti-MEM medium. After thorough mixing, the mixture was incubated at room temperature for 20 minutes to form the transfection complex. The complex was then added dropwise to the cell culture system, and complete medium was added to a final volume of 10 mL. Viral supernatant was collected 48 hours after transfection. Cell debris was removed by centrifugation at 3000×g for 10 minutes, and the cells were aliquoted and frozen at -80℃ for later use; 2) In the stable transfection cell line construction stage, target cells were seeded at an optimized density in 6-well plates. After adhesion, 500 μL of the virus stock solution obtained in step 1) was added and the culture medium was brought up to 2 mL for infection. After 48 hours, the selective medium containing 2 μg / mL puromycin was replaced to start the selection program. Fresh selection medium was added every 3 days and the cell status was dynamically monitored. After two rounds of strict selection, cell lines with stable overexpression of the LFPM gene were obtained. The cell lines with stable overexpression of the LFPM gene were cultured for 4 days, and the p53 expression level was detected by Western blot every day, and cell growth was detected by CCK8 assay. The results showed that LFPM overexpression significantly promoted the expression level of p53-R175H, thereby promoting cell growth ( Figure 3 (AB in the middle).

[0060] Next, two naturally expressed p53-R175H cell lines, SK-BR3 (Shanghai Cell Bank, Chinese Academy of Sciences, SCSP-5243) and Cal-33 (Pronosa, GCL-0952), were selected to construct stable screening cell lines for LFPM knockdown using viral-packaged shRNA. The steps for lentiviral packaging and stable transfection of cell lines were as described above. The expression plasmid backbone used for knockdown expression was pLKO.1-TRC Cloning Vector (constructed by Miaoling Biotechnology Co., Ltd.). shNC was used as a negative control. To prevent off-target effects, two shRNA knockdown sequences, shLFPM-1 and shLFPM-2, were designed for LFPM, respectively. The specific sequences are shown in Table 2 below.

[0061] Table 2: shRNA sequences

[0062] The LFPM-knockdown stabilized cell lines were cultured for 4 days, and p53 expression levels were detected daily by Western blot, while cell growth was assessed using a CCK8 assay. The results showed that both shLFPM-1 and shLFPM-2 effectively knocked down LFPM, and LFPM knockdown inhibited p53-R175H expression, thereby suppressing tumor cell growth. Figure 3 (CD in the middle).

[0063] Example 3: Animal-level study of LFPM To further investigate the effect of LFPM on the tumorigenicity of tumor cells, the constructed stable transfected cell line Cal-33 was digested into a single-cell suspension with a cell concentration of 1×10⁻⁶ cells / cells. 7 100 μL of cells were injected subcutaneously into the right side of each 5-week-old male BALB / c nude mouse. The mice were divided into four groups of five. The four groups were injected with LFPM-overexpressing Cal-33 cells (LFPM overexpression group), wild-type Cal-33 cells (control group), p53-R175H knockout Cal-33 cells (p53-R175H knockout group), and Cal-33 cells that simultaneously overexpressed LFPM and knocked out p53-R175H (LFPM overexpression and p53-R175H knockout group). The specific experimental steps for constructing the p53-R175H knockout group cells are as follows: First, targeting the flanking region of the mutation site (R175), a highly cleaving sgRNA sequence 5'-AGCACATGACGGAGGTTGTG-3' (SEQ ID NO:1) was screened and inserted into the PX459 (addgene: Plasmid #62988) vector to construct a CRISPR-sgRNA recombinant plasmid. Then, the CRISPR-sgRNA recombinant plasmid was transfected into Cal-33 cell lines naturally expressing p53-R175H using an electroporator. Single clones were selected using puromycin for screening, cultured, and sequenced to verify the knockout, obtaining positive clones. The specific experimental steps for LFPM overexpression and p53-R175H knockout group construction are as follows: pLV-LFPM was stably transfected into p53-R175H knockout group cells using the method described in Example 2, and cell lines stably overexpressing the LFPM gene were screened and cultured.

[0064] The size of the tumor mass was measured weekly using calipers. After 5 weeks of cultivation, the tumors were removed and their weight and volume were measured. The results showed that, compared to the control group, LFPM overexpression significantly promoted tumor growth. However, when p53-R175H was knocked out, LFPM overexpression failed to promote tumor growth, indicating that LFPM exerts its pro-tumor function by activating p53-R175H. Figure 3 (E in the text).

[0065] Example 4: Clinical Relevance Analysis To further explore the clinical correlation between LFPM and p53-R175H, tissue samples from 76 cases of non-small cell lung cancer with p53-R175H mutation were collected (samples from the Cancer Hospital of the Chinese Academy of Medical Sciences). The gene expression level of LFPM was detected by qPCR (primer sequences are shown in Table 3). The protein expression level of p53-R175H was detected by Western blot. The primary antibodies used were anti-p53 antibody (Abcam, ab131442) and GAPDH antibody (Proteintech, 60004-1-Ig); the secondary antibody used was HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Abcam, ab150116). Correlation analysis was then conducted to explore the correlation between the LFPM gene and the expression level of p53-R175H. The results showed a significant positive correlation between the expression levels of LFPM and p53-R175H. Figure 4 (A) Based on LFPM expression levels, the samples were grouped into a high LFPM expression group and a low LFPM expression group according to the best cutoff value. Kaplan-Meier survival analysis results showed that in lung cancer tissue samples with p53-R175H mutation, patients with high LFPM expression levels had a poorer prognosis. Figure 4 (B in the text); Further grouping of the p53-R175H / LFPM expression axis according to the best cutoff value showed that patients with high co-expression of p53-R175H / LFPM had the worst prognosis. Figure 4 (C in the middle).

[0066] Table 3: Primer sequences

[0067] A patient-derived xenograft (PDX) tumor model was constructed by collecting p53-R175H mutant non-small cell lung cancer tissue samples. The steps are as follows: a. Collect tumor samples from patients with p53-R175H-mutant lung cancer (samples from the Cancer Hospital of the Chinese Academy of Medical Sciences), wash with pre-cooled PBS, and cut the tissue into 1-2 mm pieces. 3 It was then transplanted onto the back of the right hind limb of an NSG mouse, thus creating a P0 generation PDX animal model. b. Wait until the subcutaneous tumors in P0 generation mice grow to 1000 mm 3 When the tumor is in its left or right position, remove the tumor mass and cut it into multiple 1-2mm pieces. 3 The tissue blocks were transplanted into new NSG mice using the method described above, which is the P1 generation. c. When the subcutaneous tumors in P1 generation mice reach 300 mm 3 Around 5 weeks, cholesterol-modified LFPM siRNA was injected intratumorally every three days for five weeks. The sequence information is as follows: siRNA-1: 5'-GAAGGTGTCTGATGTGTAA-3' (SEQ ID NO: 3); siRNA-2: 5'-GGAACAAGTCAAGATGAAT-3' (SEQ ID NO: 4); siNC (negative control): 5'-CGTGACACGTTCGGAGAA-3' (SEQ ID NO: 14); The injection dose is 6 nmol / kg body weight; d. Detect tumor growth according to the above-described nude mouse xenograft tumor method. Experimental results showed that knocking down LFPM significantly inhibited tumor growth. Figure 4 (D in the middle).

[0068] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of a RAB30-DT inhibitor in the preparation of a medicament for the prevention and / or treatment of tumors, wherein the tumor has a p53-R175H mutation; the RAB30-DT inhibitor is siRNA or shRNA; the sequence of the siRNA is shown in at least one of SEQ ID NO: 3 to SEQ ID NO: 4; the shRNA has a forward sequence as described in SEQ ID NO: 16 and a reverse sequence as described in SEQ ID NO: 17, or has a forward sequence as described in SEQ ID NO: 18 and a reverse sequence as described in SEQ ID NO: 19; the tumor is breast cancer, squamous cell carcinoma of the tongue, or non-small cell lung cancer.

2. The use according to claim 1, characterized in that, The dosage form of the drug includes at least one of the following: suspension, granules, capsules, powders, tablets, emulsions, solutions, and pellets.

3. The use according to claim 1, characterized in that, The dosage form of the drug includes at least one of the following: injection, oral, suppository, enema, and patch.

4. The use according to claim 1, characterized in that, The delivery vectors for the RAB30-DT inhibitor include plasmid vectors, cationic polymer vectors, and viral vectors.

5. The use according to claim 1, characterized in that, The delivery carrier for the RAB30-DT inhibitor comprises nanoparticles.

6. The use according to claim 4, characterized in that, The cationic polymer carrier includes chitosan.

7. The use according to claim 5, characterized in that, The nanoparticles include liposomes and lipid nanoparticles.

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