Use of rbm25 inhibitors in the preparation of drugs for tumor radiotherapy sensitization

Abstract

This invention relates to the field of biomedical technology, providing a novel use for RBM25, specifically the application of RBM25 inhibitors in the preparation of tumor radiosensitizing drugs. The invention further provides a recombinant vector for an RBM25 inhibitor, and a tumor radiosensitizing drug with an RBM25 inhibitor or its recombinant expression vector as the active component. Through clonogenic assays, apoptosis assays, comet electrophoresis assays, and immunofluorescence assays, it was demonstrated that shRNA targeting the RBM25 gene can significantly inhibit the survival rate of colorectal cancer cells after irradiation, increase the degree of DNA damage after irradiation, and make cells more sensitive to radiation. The shRNA targeting the RBM25 gene provided by this invention can effectively improve the radiosensitivity of tumor cells during radiotherapy, especially effectively improving the radiosensitivity of colorectal cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of RBM25 as a radiosensitizing target for tumor radiotherapy. Specifically, it relates to the application of RBM25 inhibitors in the preparation of radiosensitizing drugs for tumor radiotherapy, especially in the preparation of radiosensitizing drugs for colorectal cancer. Background Technology

[0002] Colorectal cancer is the third most common malignant tumor and the second leading cause of cancer-related death. Neoadjuvant radiotherapy combined with surgery has become a routine treatment strategy for stage II / III rectal cancer patients. Preoperative radiotherapy can effectively reduce tumor volume and increase the rate of anal preservation, thus significantly improving patient prognosis. However, in clinical practice, some rectal cancer patients remain insensitive to or even resistant to radiotherapy; radioresistance of tumor cells is a major obstacle to effective treatment. Elucidating the molecular mechanisms of tumor cell radiosensitivity has become a major challenge in the field of radiation oncology, and in-depth research in this area is of significant guiding importance for improving the efficacy of radiotherapy.

[0003] The inventors previously used a CRISPR Cas9 whole-genome library to screen for radiosensitizing targets. Combining bioinformatics analysis and high-content technology, they identified RNA-binding motif protein 25 (RBM25), a molecular marker that significantly affects the radiosensitivity of colorectal cancer cells. RBM25 is a member of the RNA-binding protein family and plays a crucial role in the assembly of mRNA splice bodies and the regulation of alternative splicing. Alternative splicing (AS) is the process of processing a pre-mRNA into multiple mRNAs of the same type by including or removing different introns or exons. AS greatly enriches the structural and functional diversity of the proteome by splicing the same gene into different gene subtypes.

[0004] Numerous studies have reported on the role of apoptosis (AS) in malignant tumors, highlighting its close relationship with tumor cell proliferation, differentiation, apoptosis, migration, and drug resistance. RBM25, a highly conserved splicing factor in eukaryotic cell lines, is increasingly revealing its role in mediating important AS genes and regulating crucial biological functions. Studies have shown that RBM25 and LUC7L3 mediate SCN5AmRNA splicing, leading to a decrease in Na channel currents and consequently, heart failure in humans. Other research has found that RBM25 binds to the U1snRNP-related factor hLuc7A, thereby regulating the Bcl-x pre-mRNA 5' splicing site and promoting apoptosis. Mechanistic studies have revealed that the PWI domain of RBM25 plays a crucial role in the regulation of Bcl-x pre-mRNA alternative splicing; this article also reports on the PWI domain and its flanking basic regions. The above work explored the role of RBM25 in selective splicing events in heart failure and apoptosis, as well as the PWI domains involved in selective splicing, and preliminarily revealed the role of RBM25 in the occurrence and development of various diseases. However, there are no reports on the role of RBM25 in radiation damage and the regulation of radiosensitivity. Summary of the Invention

[0005] This invention builds upon the aforementioned research and aims to provide new uses for RBM25, exploring its applications in radiation damage and radiosensitivity regulation. It also provides the application of RBM25 inhibitors in the preparation of radiosensitizing drugs for tumor radiotherapy.

[0006] This invention first designed three shRNAs (shRNA-1, shRNA-2, and shRNA-3) targeting RBM25. After transfecting HCT116 (human colon cancer cells) cells, the optimal shRNA—shRNA-1—was selected by RT-qPCR and Western blot electrophoresis. Then, HCT116 cells transfected with shRNA-1 were irradiated with different doses. 60 Coγ-rays were used to detect cell clonogenic ability, apoptosis, cell proliferation, and DNA damage repair efficiency. The results showed that after irradiation, the clonogenic and proliferative abilities of RBM25 knockdown cells were significantly inhibited, while the apoptosis rate and DNA damage were significantly higher than in the non-knockdown group. This finding provides experimental evidence for the development of RBM25 as a potential radiosensitization target for tumor radiotherapy and related drugs.

[0007] Specifically, based on the research of this invention, the following technical solution is provided:

[0008] In a first aspect, the invention provides the application of RBM25 as a radiosensitizing target for tumor radiotherapy.

[0009] In a second aspect, the invention provides the use of RBM25 inhibitors in the preparation of tumor radiosensitizing drugs.

[0010] Preferably, RBM25 is selected from any of the following substances: RBM25 gene, RBM25 mRNA or cDNA, with RBM25 gene being preferred.

[0011] Preferably, the RBM25 inhibitor is selected from reagents that inhibit or downregulate the expression of the RBM25 gene, including any one of RBM25 sgRNA and CRISPR-CAS9 mRNA, small interfering RNA molecules, short hairpin RNA, antisense nucleotides, or nanoparticles, viral vectors, PEG-modified proteins, protein microspheres, liposomes, or extracellular vesicles carrying any of the above substances.

[0012] Furthermore, the RBM25 inhibitor is selected from reagents that silence the RBM25 gene via RNA.

[0013] In a preferred embodiment of the present invention, the RBM25 gene is silenced using shRNA targeting the RBM25 gene, comprising a sense strand and an antisense strand. The nucleotide sequence of the sense strand is shown in SEQ ID NO. 1; the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 2.

[0014] Chain of Justice: 5'- GCGCCTTAAGAATTGGGAAATTT-3' (SEQ ID NO. 1);

[0015] Antisense chain: 5'-AUUUCCCAATTCTTAAGGCGCTT-3' (SEQ ID NO. 2).

[0016] Preferably, the RBM25 shRNA can promote tumor cell apoptosis, inhibit tumor cell growth, promote irradiation-induced DNA damage to tumor cells, and improve the sensitivity of tumor cells to radiotherapy.

[0017] Furthermore, the aforementioned tumor radiosensitizing drugs refer to radiosensitizing drugs used for colorectal cancer.

[0018] It should be understood that the RBM25 molecule described herein is preferably derived from humans. Other RBM25 molecules derived from other animals that are highly homologous to human RBM25 (e.g., having more than 70%, 75%, 80%, more preferably more than 85%, such as 85%, 90%, 95%, 98%, or even 99% or more sequence identity) are also within the scope of the preferred consideration in this invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0019] In a second aspect, the present invention provides a recombinant vector for an RBM25 inhibitor, comprising an expression vector and a coding sequence for inhibiting RBM25 gene expression inserted into the expression vector. The coding sequence is preferably an RBM25 shRNA, the nucleotide sequences of which are shown in SEQ ID NO. 1 and SEQ ID NO. 2 above, respectively.

[0020] In a third aspect, the present invention provides the application of the above-mentioned RBM25 inhibitor recombinant vector in the preparation of tumor radiosensitizing drugs.

[0021] In a fourth aspect, the present invention provides a tumor radiosensitizing pharmaceutical composition, comprising an active component and a medically acceptable excipient, carrier, or diluent. The active component is the aforementioned RBM25 inhibitor or a recombinant RBM25 inhibitor carrier.

[0022] That is, the present invention provides a pulmonary hypertension treatment product, comprising:

[0023] (A) Inhibitors of RBM25;

[0024] (B) Pharmaceutically or immunologically acceptable carriers or excipients;

[0025] (C) Optionally, one or more other active ingredients that enhance the radiosensitivity of tumors.

[0026] The term "pharmaceutically / immunologically acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. As used herein, the term "effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0027] The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0028] Pharmaceutically acceptable carriers in the composition may contain liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffers, etc. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, with a pH usually around 5-8, preferably around 6-8.

[0029] The compositions of the present invention can be in solid form (such as granules, tablets, lyophilized powders, suppositories, capsules) or liquid form (such as oral liquids, injectable formulations) or other suitable forms. The administration routes can be: (1) direct naked DNA / RNA injection; (2) linking RBM25 molecule-related sgRNA and CRISPR-Cas9 mRNA with transferrin / poly-L-lysine complex to enhance their biological effects; (3) RBM25 sgRNA and CRISPR-Cas9 mRNA forming complexes with positively charged lipids to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) encapsulating RBM25 sgRNA and CRISPR-Cas9 mRNA with liposomes to mediate their entry into the cell, which is conducive to the smooth entry of macromolecules and avoids the hydrolysis of various extracellular enzymes; (5) binding RBM25 sgRNA and CRISPR-Cas9 mRNA with cholesterol increases their cytoplasmic retention time by 10 times; (6) transporting RBM25 sgRNA and CRISPR-Cas9 mRNA with immunoliposomes can specifically transport them to target tissues and target cells; (7) combining RBM25 sgRNA and CRISPR-Cas9 mRNA with liposomes can enhance their biological effects. mRNA transfection into regenerating cells (such as fibroblasts) can also effectively deliver related drugs into target cells; (8) Electroporation, which uses electric current to introduce RBM25 sgRNA and CRISPR-Cas9 mRNA into target cells.

[0030] As used in this invention, the term "unit dosage form" refers to a dosage form in which the composition of this invention is prepared for a single dose for ease of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release formulations.

[0031] It should be understood that the effective dose of the active substance used can vary depending on the severity of the condition of the patient being treated. The specific dosage is determined based on the individual patient's circumstances (e.g., weight, age, physical condition, and desired outcome), within the judgment of a skilled physician.

[0032] In some embodiments, the present invention also provides a treatment method for enhancing the radiosensitization of tumors, the method comprising administering a therapeutically effective amount of an RBM25 inhibitor to a subject in need.

[0033] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0034] This invention provides a reagent for inhibiting or downregulating RBM25 gene expression, namely, shRNA targeting the RBM25 gene. Through clonogenic assays, apoptosis assays, cell proliferation assays, comet electrophoresis assays, and immunofluorescence assays, it has been demonstrated that shRNA targeting the RBM25 gene can significantly inhibit the survival rate of HCT116 colorectal cancer cells after irradiation, increase the degree of DNA damage after irradiation, and make the cells more sensitive to radiation. The shRNA targeting the RBM25 gene provided by this invention can effectively improve the radiosensitivity of tumor cells during radiotherapy, especially for colorectal cancer. Attached Figure Description

[0035] Figure 1 This is a schematic diagram showing the expression of RBM25 protein and mRNA in HCT116 cells transfected with RBM25 knockdown and NC sequences in Example 1. A and B show the Western blot and RT-qPCR statistical results of RBM25 inhibition group and control group cells, respectively.

[0036] Figure 2 This is a schematic diagram of the colony formation rate of HCT116 cells transfected with RBM25 knockdown and NC sequences after irradiation in Example 2. A and B show the colony formation ability and statistical results of RBM25 inhibition group and control group cells under different irradiation doses, respectively.

[0037] Figure 3 This is a schematic diagram of the apoptosis changes in HCT116 cells transfected with RBM25 knockdown and NC sequences after irradiation in Example 3. A and B show the detection and statistical results of cell apoptosis in the RBM25 inhibition group and the control group under different irradiation doses, respectively.

[0038] Figure 4 The statistical results show the proliferation changes of HCT116 cells transfected with RBM25 knockdown and NC sequences in Example 4.

[0039] Figure 5 This is a schematic diagram and statistical results of DNA damage changes in HCT116 cells transfected with RBM25 knockdown and NC sequences in Example 5 after imaging. Detailed Implementation

[0040] The present invention will now be described in detail with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0041] All reagents and raw materials used in this invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer.

[0042] The following description uses HCT116 cells (human colon cancer cells) as an example to illustrate the radiosensitizing effect of RBM25 inhibitors on colorectal cancer. However, this invention is not limited to radiosensitizing colorectal cancer; it is also suitable for radiosensitizing other tumors.

[0043] The materials used in each embodiment are as follows:

[0044] Cell line and cell culture: HCT116 (human colon cancer cells) were cultured in DMEM containing 10% fetal bovine serum at 37°C and 5% CO2.

[0045] Drugs and main reagents: DMEM medium, fetal bovine serum, and trypsin were purchased from Gibco; Annexin V-FITC / PI were purchased from Invitrogen. Crystal violet, standard protein molecular weight markers, SDS-PAGE loading buffer, RIPA protein lysis buffer, TE electrophoresis buffer, transfer buffer, 30% acrylamide / methylenebisacrylamide solution, Tris-HCl, ammonium persulfate (AP), sodium dodecyl sulfate (SDS), tetramethylethylenediamine (TEMED), and propidium iodide (PI) were purchased from Jiangsu Beyotime Biotechnology Research Institute; dimethyl sulfoxide (DMSO), agarose, and Tween-20 were from Shanghai High-Tech Biotechnology Co., Ltd.; anti-RBM25 and GAPDH antibodies were purchased from ProteinTech (25297-1-AP) and Cell Signaling Technology (2118).

[0046] Example 1: Construction and Application of RBM25 shRNA

[0047] In this invention, the shRNA of RBM25 (the shRNA of the negative control (NC) is named shNC) was transfected into HCT116 colon cancer cells using riboFECT™ CP transfection reagent. The cells were then returned to the incubator and cultured for 24-48 hours before the medium was changed. Subsequently, RT-qPCR and Western blot were used to detect the RNA and protein of RBM25 in HCT116 colon cancer cells.

[0048] Cell culture: HCT116 (human colon cancer cells) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics; the cells were cultured in a 37°C, 5% CO2 incubator and passaged every 2-3 days. Cells in the logarithmic growth phase were used for experiments.

[0049] Cell transfection: The day before transfection, trypsinize and count the cells, then seed them at an appropriate density into 6-well plates. Incubate the plates overnight in a CO2 incubator to allow cell attachment, achieving 30%–50% confluence by the day of transfection. Avoid using antibiotics during seeding and transfection. (Add 120 μl of 1x riboFECT) TM Add 5 μl of 20 μM shNC storage solution, shRNA-1 storage solution, shRNA-2 storage solution, and shRNA-3 storage solution (v3) to CP Buffer (v2) and mix gently; then add 12 μl of riboFECT. TM Mix CP Reagent (v4) gently by pipetting and incubate at room temperature for 0–15 minutes to prepare the transfection complex. Add the transfection complex dropwise to cells containing an appropriate amount of antibiotic-free complete medium (v1) and mix gently. Incubate the culture plate in a CO2 incubator at 37°C for 24–48 hours.

[0050] shRNA Stock Solution: Before use, briefly centrifuge the shRNA tube to allow the powder to settle to the bottom. Prepare RNase-free water (such as DEPC water) or sterile ddH2O, calculating the dissolution volume based on the amount of shRNA and the desired concentration. For example, to prepare a 20 μM stock solution, for a double-stranded shRNA of approximately 21 bases, 1 OD (approximately 2.5 nmol) is typically added to 125 μl of water to obtain a 20 μM stock solution. Dissolve the shRNA by repeatedly pipetting or vortexing at low speed, ensuring complete mixing. In this invention, 125 μl of DEPC water is added to the shRNA tube to obtain a 20 μM shRNA stock solution.

[0051] In preparing the shRNA storage solution, this invention uses three shRNAs targeting RBM25, named shRNA-1, shRNA-2, and shRNA-3, respectively.

[0052] The shRNA (i.e., shRNA-1 above) comprises a sense strand and an antisense strand, the nucleotide sequence of which is shown in SEQ ID NO. 1; the nucleotide sequence of which is shown in SEQ ID NO. 2;

[0053] Chain of Justice: 5'- GCGCCTTAAGAATTGGGAAATTT-3' (SEQ ID NO. 1);

[0054] Antisense chain: 5'-AUUUCCCAATTCTTAAGGCGCTT-3' (SEQ ID NO. 2).

[0055] shRNA-2 comprises a sense strand and an antisense strand, the nucleotide sequence of which is shown in SEQ ID NO. 3; the nucleotide sequence of which is shown in SEQ ID NO. 4.

[0056] Chain of Justice: 5'-TTATACTCACCAGGTACAAATTT-3' (SEQ ID NO. 3).

[0057] Antonym: 5'-AUUUGUACCUGGUGAGUAATATT-3' (SEQ ID NO. 4).

[0058] shRNA-3 comprises a sense strand and an antisense strand, the nucleotide sequence of which is shown in SEQ ID NO. 5; the nucleotide sequence of which is shown in SEQ ID NO. 6.

[0059] Chain of Justice: 5'-GCTGGATGAATGGAAAGCAAATT-3' (SEQ ID NO. 5);

[0060] Antonym: 5'-UUUGCUUUCCAUUCAUCCAGCTT-3' (SEQ ID NO. 6).

[0061] shNC comprises a sense strand and an antisense strand, the nucleotide sequence of which is shown in SEQ ID NO. 7; the nucleotide sequence of which is shown in SEQ ID NO. 8.

[0062] Chain of Justice: 5'-TTCTCCGAACGTGTCACGTTT-3' (SEQ ID NO. 7);

[0063] Antisense chain: 5'-ACUGUGACACGUUCGGAGAATT-3' (SEQ ID NO. 8).

[0064] Results validation: RNA was extracted 24 hours after transfection and subjected to RT-qPCR. Protein was extracted 48 hours after transfection and subjected to Western blot electrophoresis.

[0065] The results are as follows Figure 1 As shown, Figure 1This diagram illustrates the expression of RBM25 protein in HCT116 cells transfected with RBM25 knockdown and NC sequences in Example 1. Figure A shows the Western blot electrophoresis results, and Figure B shows the RT-qPCR results. The results show that the protein expression level and mRNA level of RBM25 decreased most significantly after transfection with shRNA-1, indicating that RBM25 was knocked down. In conclusion, shRNA-1 targeting RBM25 can effectively inhibit the expression of RBM25 protein in HCT116 cells.

[0066] The following examples all use transfected shRNA-1, abbreviated as shRBM25.

[0067] Example 2: Detection of cell colony-forming ability

[0068] Cell culture and cell transfection were performed in the same manner as in Example 1.

[0069] Cell colony formation method: HCT116 cells transfected with shRNA-1 (shRBM25) for 48 hours were irradiated with 0, 2, 4, and 6 Gy. 60 Co-γ rays were used to seed 500, 1000, 2000, and 4000 cells into six-well plates. Cells were simultaneously irradiated with γ rays at a dose rate of 1 Gy / min for 0, 2, 4, and 6 min, respectively, and irradiation continued until clearly visible cell clumps appeared in the culture dish (approximately 2 weeks). Culture was then terminated. The culture medium was discarded, the cells were washed twice with PBS, fixed with paraformaldehyde for 30 min, stained with crystal violet for 30 min, gently rinsed with running water, air-dried, photographed, and counted to calculate cell viability.

[0070] Groups: shRBM25 group and shNC group (shRNA of negative control group is named shNC).

[0071] The shRBM25 group was prepared as follows: One day before transfection, cells were trypsinized, counted, and seeded into 6-well plates at an appropriate density. The plates were then placed in a CO2 incubator overnight to allow cell attachment, achieving 30%–50% confluence on the day of transfection. Antibiotics were avoided during seeding and transfection. 120 μl of 1xriboFECT was added. TM Add 5 μl of 20 μM shRNA-1 stock solution (v3) to CPBuffer (v2) and mix gently; then add 12 μl of riboFECT. TMMix CP Reagent (v4) gently by pipetting and incubate at room temperature for 0–15 minutes to prepare the transfection complex. Add the transfection complex dropwise to cells containing an appropriate amount of antibiotic-free complete medium (v1) and mix gently. Incubate the culture plate in a CO2 incubator at 37°C for 24–48 hours.

[0072] The shNC group was prepared as follows: One day before transfection, cells were trypsinized, counted, and seeded into 6-well plates at an appropriate density. The plates were then placed in a CO2 incubator overnight to allow cell attachment, achieving 30%–50% confluence on the day of transfection. Antibiotics were avoided during seeding and transfection. 120 μl of 1xriboFECT was added. TM Add 5 μl of 20 μM shNC stock solution (v3) to CP Buffer (v2) and mix gently; then add 12 μl of riboFECT. TM Mix CPReagent (v4) gently by pipetting and incubate at room temperature for 0–15 minutes to prepare the transfection complex. Add the transfection complex dropwise to cells containing an appropriate amount of antibiotic-free complete medium (v1) and mix gently. Incubate the culture plate in a CO2 incubator at 37°C for 24–48 hours.

[0073] The results are as follows Figure 2 As shown, Figure 2 This is a schematic diagram illustrating the colony formation rate of HCT116 cells transfected with RBM25 knockdown and NC sequences after irradiation, as shown in Example 2. Figure A shows cell clumps of HCT116 cells transfected with the RBM25 shRNA sequence after irradiation for 2 weeks, and Figure B is a quantitative representation of these cell clumps. The figures show that, under different irradiation doses, the number of cell clumps in the shRBM25 group was less than that in the shNC group, and the cell death rate in the RBM25 knockdown group was significantly higher than that in the shNC group. This indicates that RBM25 knockdown significantly reduces the colony formation ability of cells after irradiation.

[0074] Example 3 Apoptosis Detection

[0075] Cell culture and cell transfection were performed in the same manner as in Example 1.

[0076] Flow cytometry detection of apoptosis: 2 × 10⁻⁶ HCT116 cells in logarithmic growth phase transfected with shRNA-1 (shRBM25) were used. 5 Cells were seeded into six-well plates at a concentration of 8 Gy / mL and administered to the cells. 60Irradiate with Co γ rays and then continue culturing for 24 hours. Since apoptotic cells are also present in the supernatant, the cell supernatant should be collected. Digest the cells with trypsin without EDTA and terminate the digestion with the collected supernatant. After centrifugation at 1000 rpm for 5 minutes, wash twice with PBS. After double staining with Annexin V-FITC and PI, cell apoptosis is detected by flow cytometry.

[0077] Groups: shRBM25 group and shNC group (shRNA of negative control group is named shNC).

[0078] The shRBM25 group was prepared as follows: One day before transfection, cells were trypsinized, counted, and seeded into 6-well plates at an appropriate density. The plates were then placed in a CO2 incubator overnight to allow cell attachment, achieving 30%–50% confluence on the day of transfection. Antibiotics were avoided during seeding and transfection. 5 μl of 20 μM shRNA-1 stock solution (v3) was added to 120 μl of 1xriboFECT™ CP Buffer (v2) and gently mixed. Then, 12 μl of riboFECT™ CP Reagent (v4) was added and gently mixed by pipetting. The mixture was incubated at room temperature for 0–15 minutes to prepare the transfection complex. The transfection complex was added dropwise to cells containing an appropriate amount of antibiotic-free complete medium (v1) and gently mixed. The culture plates were incubated in a CO2 incubator at 37°C for 24–48 hours.

[0079] The shNC group was prepared as follows: One day before transfection, cells were trypsinized, counted, and seeded into 6-well plates at an appropriate density. The plates were then placed in a CO2 incubator overnight to allow cell attachment, achieving 30%–50% confluence on the day of transfection. Antibiotics were avoided during seeding and transfection. 5 μl of 20 μM shNC stock solution (v3) was added to 120 μl of 1xriboFECT™ CP Buffer (v2) and gently mixed. Then, 12 μl of riboFECT™ CPReagent (v4) was added and gently mixed by pipetting. The mixture was incubated at room temperature for 0–15 minutes to prepare the transfection complex. The transfection complex was added dropwise to cells containing an appropriate amount of antibiotic-free complete medium (v1) and gently mixed. The culture plates were incubated in a CO2 incubator at 37°C for 24–48 hours.

[0080] The results are as follows Figure 3 As shown, Figure 3This is a schematic diagram illustrating the apoptosis changes in HCT116 cells transfected with RBM25 knockdown and NC sequences after irradiation, as shown in Example 3. Figure A shows the apoptosis changes detected by flow cytometry after cell irradiation, and Figure B is a quantitative diagram of the apoptosis changes. As can be seen from the figures, under no irradiation, there was no significant difference in the apoptosis rate between the shNC group and the shRBM25 group. Cells irradiated with 8 Gy... 60 After Co γ-ray irradiation, the apoptosis rate of cells in the shRBM25 group was significantly higher than that in the shNC group. The results showed that the apoptosis rate of HCT116 cells with RBM25 knockdown was significantly higher than that of cells without knockdown after irradiation.

[0081] Example 4 Cell proliferation detection

[0082] Cell culture and cell transfection were performed in the same manner as in Example 1.

[0083] Cell proliferation method: HCT116 cells transfected with shRNA-1 (shRBM25) in the logarithmic growth phase (8 × 10⁻⁶ cells) were harvested. 4 Inoculate each well into a 96-well plate, with 8 replicates per group. Perform 8 Gy injections the day after inoculation. 60 Treatment was performed with Co γ irradiation, followed by continuous monitoring for 3 days. Before detection, 100 μL of culture medium containing 10 μL of CCK-8 reagent was added to each well, and the mixture was incubated at 37°C in the dark for 2 hours. The absorbance (OD) value at 450 nm was measured using a BIO-RAD microplate reader.

[0084] Groups: shRBM25 group; shNC group (shRNA of the negative control group was named shNC); shNC+IR group indicates that cells transfected with the negative control group were irradiated; shRBM25+IR group indicates that cells transfected with shRNA-1 were irradiated.

[0085] The results are as follows Figure 4 As shown, Figure 4 This is a schematic diagram illustrating the proliferation changes of HCT116 cells transfected with RBM25 knockdown and NC sequences after irradiation in Example 4. As can be seen from the figure, the absorbance (OD) values ​​72 hours after irradiation show that the proliferation rate of the shRBM25 group cells was significantly lower than that of the shNC group cells, indicating that the proliferation rate of the RBM25 knockdown group cells was significantly lower than that of the non-knockdown group after irradiation.

[0086] Example 5: DNA Damage Repair Efficiency Detection

[0087] Cell culture and cell transfection were performed in the same manner as in Example 1.

[0088] Immunofluorescence assay for DNA damage repair efficiency: A sterile coverslip was placed in a 12-well plate, and the cell suspension was incubated at 2 × 10⁻⁶ cells / well. 5Inoculate the culture dish at a density of 100 cells / ml. Perform 8 Gy injection the following day. 60 Cells were irradiated with Coγ and treated at specific time points. Cells were fixed with paraformaldehyde and washed with PBS. Wells were punched on ice using Triton-X 100, blocked with goat serum at room temperature for 1 hour, and incubated overnight at 4°C with γH2AX primary antibody. γH2AX was selected to indicate the degree of double-strand breaks in cells, thus analyzing the efficiency of DNA damage repair. After washing with PBS, cells were incubated with secondary antibody at room temperature for 1 hour, followed by another PBS wash. DAPI was incubated at room temperature in the dark, followed by PBS wash. Finally, the slides were removed and air-dried on absorbent paper. Glycerol was placed in the center of a slide, sealed with nail polish, and stored at -20°C in the dark. Images were taken using a confocal microscope within two weeks, and the data were statistically analyzed.

[0089] Comet electrophoresis to detect DNA damage changes: Cells are electrophoresed at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1 / ml in 12-well plates and cultured in 1 ml of DMEM medium. Clean glass slides were immersed in molten 1% high-melting-point agarose and immediately wiped to prepare comet electrophoresis pre-smears. The next day, both cell lines were administered 8 Gy of [a specific treatment / treatment]. 60 Irradiated with Co gamma rays, digested with trypsin at 0h and 4h respectively, centrifuged at 1000rpm for 5min; washed once with PBS, supernatant discarded; then digested with Ca-free PBS. 2+ Mg 2+ The concentration of the single-cell suspension was adjusted to 2 × 10⁻⁶ using PBS. 4 Cells / ml were collected, and 0.4 ml of the cell solution was then immersed in 1.2 ml of 0.65% low-melting-point agarose and incubated at 40°C. 1.6 ml of the cell suspension was mixed and quickly pipetted onto the surface of a pre-smear, spreading it evenly. After drying, the slide was gently immersed in freshly prepared and pre-chilled cell lysis buffer and lysed at 4°C in the dark for 2 hours. The slide was removed from the lysis buffer, rinsed with electrophoresis buffer for 5 seconds, and then transferred to a horizontal electrophoresis tank, which was filled with electrophoresis buffer. Electrophoresis was performed at 25V for 30 minutes. After electrophoresis, the slide was removed and gently washed with double-distilled water. The gel was stained with SYBR Green for 20 minutes and then gently rinsed with double-distilled water. All gels were observed under a fluorescence microscope (Olympus BX60) at 10x magnification.

[0090] Statistical analysis: All experiments in the above embodiments were repeated at least three times, and the results are expressed as mean ± standard deviation (S). SAS statistical software was used to perform t-tests on the relevant data, with P < 0.05 considered statistically significant.

[0091] Groups: shRBM25 group and shNC group (shRNA of negative control group is named shNC).

[0092] The results are as follows Figure 5 As shown, Figure 5This is a schematic diagram of DNA damage changes in HCT116 cells transfected with RBM25 knockdown and NC sequences after irradiation, as shown in Example 5. In the diagram, A is an immunofluorescence illustration of γH2AX in cells; the RBM25 knockdown group showed significantly more γH2AX focal points than the non-knockdown group. B is a quantitative map of γH2AX focal points; the RBM25 knockdown group showed significantly more γH2AX in cells than the non-knockdown group. C is a schematic diagram of DNA damage changes in HCT116 cells transfected with shNC or shRBM25 nucleotide sequences after irradiation—detected by a comet electrophoresis experiment; the DNA tailing in the RBM25 knockdown group was significantly more severe than in the non-knockdown group after irradiation. D is a quantitative map of the tail moments detected by comet electrophoresis in HCT116 cells transfected with shNC or shRBM25 nucleotide sequences after irradiation; the DNA tail moments in the RBM25 knockdown group were significantly longer than in the non-knockdown group after irradiation.

[0093] like Figure 5 As shown in Figures A and B, after irradiation, the number of γH2AX focal points in the RBM25 knockdown group was significantly higher than that in the non-knockdown group, indicating that the DNA damage repair efficiency of cells in the RBM25 knockdown group decreased.

[0094] like Figure 5 As shown in C and D, both tail length and tail phase increased after irradiation, with the tailing effect being more pronounced in the RBM25 knockdown group than in the control group. This indicates that DNA damage in RBM25 knockdown cells was aggravated after irradiation.

[0095] The results showed that DNA damage was more severe and DNA damage repair efficiency was reduced in HCT116 cells with RBM25 knockdown.

[0096] Therefore, the reagents of this invention that inhibit or downregulate the RBM25 gene can be used as radiosensitizing agents for tumor radiotherapy. By inhibiting or downregulating the RBM25 gene, the sensitivity to radiotherapy can be improved, thereby enhancing the efficacy of radiotherapy.

[0097] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. Application of RBM25 as a radiosensitizing target for tumor radiotherapy.

2. Application of RBM25 inhibitors in the preparation of tumor radiosensitizing drugs.

3. The application according to claim 1 or 2, characterized in that: in, The tumor was selected from colorectal cancer.

4. The application according to claim 2, characterized in that: in, The RBM25 is selected from any one of the following: the RBM25 gene, RBM25 mRNA or cDNA. The RBM25 inhibitor is selected from substances that inhibit or silence RBM25 expression levels.

5. The application according to claim 4, characterized in that: in, The RBM25 inhibitors include any one of RBM25 sgRNA and CRISPR-CAS9 mRNA, small interfering RNA molecules, shRNA, antisense nucleotides, or nanoparticles, viral vectors, PEG-modified proteins, protein microspheres, liposomes, or extracellular vesicles carrying any of the above substances, and also include small molecule drugs.

6. The application according to claim 5, characterized in that, in, The shRNA of RBM25 includes a sense strand and an antisense strand, the nucleotide sequence of which is shown in SEQ ID NO. 1; the nucleotide sequence of which is shown in SEQ ID NO.

2.

7. A recombinant vector for an RBM25 inhibitor, characterized in that, The invention includes an expression vector and RBM25 sgRNA, CRISPR-CAS9 mRNA, RBM25 siRNA, RBM25 shRNA, or RBM25 antisense nucleotides inserted into the expression vector. The RBM25 shRNA comprises a sense strand and an antisense strand, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the nucleotide sequence of which is shown in SEQ ID NO.

2.

8. The use of the RBM25 inhibitor recombinant vector according to claim 7 in the preparation of tumor radiosensitizing drugs.

9. A tumor radiosensitizing drug composition, characterized in that, This includes the active ingredient and pharmaceutically or immunologically acceptable excipients, carriers, or diluents. The active component is the RBM25 inhibitor according to any one of claims 3 to 5 or the RBM25 inhibitor recombinant vector according to claim 7.

10. The tumor radiosensitizing drug according to claim 9, characterized in that, The tumor is colorectal cancer.

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