Methods, systems, and devices for computer-aided screening of drugs that enhance radiosensitivity in non-small cell lung cancer based on miR-320a and RAD51.

Abstract

This invention discloses a method, system, and device for computer-aided screening of drugs that enhance radiosensitivity in non-small cell lung cancer (NSCLC) based on miR-320a and RAD51. This invention provides a computer-readable storage medium and a method for screening drugs that enhance radiosensitivity in NSCLC based on miR-320a and RAD51. This invention offers an efficient, accurate, and rapid screening method for the research and development of related drugs that enhance radiosensitivity in NSCLC, with broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of intelligent medical technology, specifically relating to a method, system, and device for computer-aided screening of drugs that promote radiosensitivity in non-small cell lung cancer based on miR-320a and RAD51. Background Technology

[0002] Lung cancer is one of the most common malignant tumors worldwide, with high morbidity and mortality rates. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all cases, and treatment outcomes and prognoses vary significantly among individuals. However, due to the lack of obvious early symptoms and signs, most patients are already in advanced stages at the time of initial treatment. Radiotherapy is applicable to all stages of NSCLC, and approximately 50% of NSCLC patients receive this treatment. However, due to radiotherapy tolerance, patients frequently experience metastasis or progression, and those skilled in the art currently know very little about the key factors associated with radioresistance. Therefore, there is an urgent need to screen for new and more effective therapeutic targets to increase the radiosensitivity of NSCLC and improve patient survival.

[0003] Non-coding RNAs (ncRNAs) are a class of ribonucleic acids that lack translational function but regulate diverse biological behaviors at the RNA level. Currently, some ncRNAs, such as microRNAs (miRNAs), play crucial roles in both normal cellular function and cancer progression. miRNAs are small, stable, and readily diffuse into various bodily fluids, such as serum, plasma, urine, and saliva. The expression levels of circulating miRNAs are associated with the occurrence and development of malignant tumors. Although studies have shown that miRNAs can predict patient prognosis and guide clinical treatment plans, it remains unclear which miRNAs can be effectively screened to promote radiosensitivity in non-small cell lung cancer research.

[0004] Non-small cell lung cancer (NSCLC), one of the most common and deadliest malignant tumors globally, is primarily treated with surgery, radiotherapy, and chemotherapy. However, tumor cell resistance to radiotherapy severely limits treatment efficacy, leading to low patient survival rates. With ongoing clinical research, radiotherapy resistance has become a key bottleneck hindering long-term survival for NSCLC patients, urgently requiring innovative drugs to overcome this challenge. Screening or developing drugs that enhance radiosensitivity in NSCLC can not only improve the efficacy of radiotherapy and reduce the risk of tumor recurrence, thus increasing patients' chances of survival, but also fill gaps in current treatment strategies and effectively alleviate clinical treatment difficulties. Computer-aided drug design (CADD), based on computer computation and simulation technology, learns from the prior knowledge contained in massive amounts of drug data to uncover the interaction relationships between target molecules and candidate drugs. This allows for the rapid selection of drug-like active molecules from millions of molecules. This significantly reduces the blind spots in screening candidate drug molecules and improves research and development efficiency. Summary of the Invention

[0005] In view of the above-mentioned technical problems existing in the field of screening or development of drugs that promote radiosensitivity in non-small cell lung cancer, the purpose of this invention is to provide a method, system and device for computer-aided screening of drugs that promote radiosensitivity in non-small cell lung cancer based on miR-320a and RAD51.

[0006] The method, system, and equipment provided by this invention utilize computer-aided drug screening technology combined with miR-320a and RAD51 to screen for drugs that can effectively promote radiosensitivity in non-small cell lung cancer. The invention has irreplaceable practical significance for improving the clinical treatment level of non-small cell lung cancer and improving the current public health situation.

[0007] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0008] A first aspect of the present invention provides a computer-aided screening method based on miR-320a and RAD51 for screening drugs that promote radiosensitivity in non-small cell lung cancer, the method comprising:

[0009] Obtain secondary structure data of miR-320a and structure data of RAD51 protein;

[0010] The spatial structure of the miR-320a secondary structure and the RAD51 protein complex was selected, and the binding site of the miR-320a secondary structure and the RAD51 protein complex was determined as the binding site of the targeted drug.

[0011] A computer-aided drug screening method was used to obtain candidate drugs that target the binding site, wherein the candidate drugs are drugs that promote radiosensitivity in non-small cell lung cancer.

[0012] Furthermore, the method for determining the binding site of the miR-320a secondary structure and the RAD51 protein complex includes:

[0013] Construct n truncated expression mutants of RAD51, where n is a natural number greater than or equal to 1;

[0014] Based on the n truncated expression mutants, dual-luciferase reporter analysis was performed to detect the luciferase activity of the n truncated expression mutants after co-transfection with miR-320a, and the key region of the miR-320a secondary structure and RAD51 protein complex binding was located.

[0015] Single-point mutations were performed on each base in the key region, and the changes in luciferase activity after each mutation were detected. The resulting key complementary base region is the binding site of the miR-320a secondary structure and the RAD51 protein complex.

[0016] Furthermore, the computer-aided drug screening method includes:

[0017] Obtain the secondary structure of miR-320a and the binding site of the RAD51 protein complex;

[0018] Based on the spatial structure of the miR-320a secondary structure and the binding site of the RAD51 protein complex, small molecule compounds with similar structures were screened from molecular databases.

[0019] The small molecule compounds selected through screening are molecularly docked with the RAD51 protein to calculate the affinity or binding energy of the small molecule compounds to the RAD51 protein receptor, and a score is obtained for each small molecule compound. Candidate drugs are obtained by sorting the small molecule compounds according to the scores.

[0020] Furthermore, the small molecule compound is selected from siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic small molecule compound.

[0021] Furthermore, the method also includes:

[0022] Obtain a system expressing RAD51;

[0023] The effectiveness of the small molecule compounds was verified by treating the RAD51-expressing system with the small molecule compounds obtained from the screening.

[0024] Optionally, the system expressing RAD51 includes a cell system expressing RAD51, a subcellular system expressing RAD51, a tissue system expressing RAD51, a solution system expressing RAD51, an organ system expressing RAD51, or an animal system expressing RAD51.

[0025] Optionally, small molecule compounds that inhibit RAD51 expression can be screened as candidate drugs.

[0026] A second aspect of the present invention provides a computer-aided screening system based on miR-320a and RAD51 for screening drugs that enhance radiosensitivity in non-small cell lung cancer, the system comprising:

[0027] Data acquisition unit: Acquires secondary structure data of miR-320a and structure data of RAD51 protein;

[0028] Site determination unit: Select the spatial structure of the miR-320a secondary structure and the RAD51 protein complex, and determine the binding site of the miR-320a secondary structure and the RAD51 protein complex as the binding site of the targeted drug;

[0029] Drug screening unit: A computer-aided drug screening method is used to obtain candidate drugs that target the binding site, wherein the candidate drugs are drugs that promote radiosensitivity of non-small cell lung cancer.

[0030] Furthermore, the method for determining the binding site of the miR-320a secondary structure and the RAD51 protein complex includes:

[0031] Construct n truncated expression mutants of RAD51, where n is a natural number greater than or equal to 1;

[0032] Based on the n truncated expression mutants, dual-luciferase reporter analysis was performed to detect the luciferase activity of the n truncated expression mutants after co-transfection with miR-320a, and the key region of the miR-320a secondary structure and RAD51 protein complex binding was located.

[0033] Single-point mutations were performed on each base in the key region, and the changes in luciferase activity after mutation were detected one by one. The key complementary base region obtained is the binding site of the miR-320a secondary structure and the RAD51 protein complex.

[0034] Optionally, the computer-aided drug screening method includes:

[0035] Obtain the secondary structure of miR-320a and the binding site of the RAD51 protein complex;

[0036] Based on the spatial structure of the miR-320a secondary structure and the binding site of the RAD51 protein complex, small molecule compounds with similar structures were screened from molecular databases.

[0037] The small molecule compounds selected from screening are molecularly docked with the RAD51 protein to calculate the affinity or binding energy of the small molecule compounds to the RAD51 protein receptor, and a score is obtained for each small molecule compound. Candidate drugs are obtained by sorting the small molecule compounds according to the scores.

[0038] Optionally, the small molecule compound is selected from siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic small molecule compound;

[0039] Optionally, the method further includes:

[0040] Obtain a system expressing RAD51;

[0041] The effectiveness of the small molecule compounds was verified by treating the RAD51-expressing system with the small molecule compounds obtained from the screening.

[0042] Optionally, the system expressing RAD51 includes a cell system expressing RAD51, a subcellular system expressing RAD51, a tissue system expressing RAD51, a solution system expressing RAD51, an organ system expressing RAD51, or an animal system expressing RAD51.

[0043] Optionally, small molecule compounds that inhibit RAD51 expression can be screened as candidate drugs.

[0044] A third aspect of the present invention provides a computer-aided screening device based on miR-320a and RAD51 for screening drugs that promote radiosensitivity in non-small cell lung cancer, the device comprising:

[0045] The invention includes a memory and a processor, wherein the memory stores program instructions; and the processor invokes the program instructions, which, when executed, implement the computer-aided screening method for screening drugs that enhance radiosensitivity in non-small cell lung cancer based on miR-320a and RAD51, as described in the first aspect of the invention.

[0046] A fourth aspect of the present invention provides a computer-readable storage medium storing a computer program that, when executed by a processor, implements the computer-aided screening method for screening drugs that promote radiosensitivity in non-small cell lung cancer based on miR-320a and RAD51, as described in the first aspect of the present invention.

[0047] The fifth aspect of the present invention provides a screening method for drugs that promote radiosensitivity in non-small cell lung cancer based on miR-320a and RAD51, as described in the first aspect of the present invention, and the drugs that promote radiosensitivity in non-small cell lung cancer are obtained by screening.

[0048] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0049] This invention is the first to creatively discover that miR-320a promotes radiosensitivity in non-small cell lung cancer by negatively regulating RAD51. Based on this groundbreaking discovery, this invention provides a computer-aided screening method, system, device, and computer-readable storage medium for drugs that promote radiosensitivity in non-small cell lung cancer, based on miR-320a and RAD51. This invention provides an efficient, accurate, and rapid screening method for the research and development of related drugs that promote radiosensitivity in non-small cell lung cancer, and provides a reference for the research field of radiosensitivity in non-small cell lung cancer, with broad application prospects. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 This invention provides a schematic flowchart of a computer-aided screening method based on miR-320a and RAD51 to screen drugs that enhance radiosensitivity in non-small cell lung cancer.

[0052] Figure 2 This invention provides a schematic diagram of a computer-aided screening system based on miR-320a and RAD51 to screen drugs that enhance radiosensitivity in non-small cell lung cancer.

[0053] Figure 3 This invention provides a schematic diagram of a computer-aided screening device based on miR-320a and RAD51 to screen drugs that enhance radiosensitivity in non-small cell lung cancer.

[0054] Figure 4The expression of miR-320a in normal and NSCLC patient tissues and the effect of miR-320a on the radiosensitivity of NSCLC were examined in the TCGA database. Figure A shows that miR-320a expression in cancer tissues was significantly lower than in adjacent normal tissues (p<0.05); Figure B shows that there was no statistically significant difference in mOS between NSCLC patients with high and low miR-320a expression (p>0.05); Figure C shows that miR-320a was upregulated in radiation-sensitive NSCLC tissues.

[0055] Figure 5 RAD51 is a direct target of miR-320a. Figure A: Wayne plot shows that RAD51 is predicted to be a miR-320a-related target gene using TargetScan, miRDB, and miRTarBase databases. Figure B: miR-320a significantly reduced luciferase activity in wild-type or mutant RAD51-3'UTR cells (p<0.05).

[0056] Figure 6 RAD51 promotes radiotherapy resistance in NSCLC cells. Figure A shows that RAD51 expression was significantly upregulated in human lung cancer tissues compared to adjacent normal tissues (P<0.05). Figure B shows that the median overall survival of NSCLC patients with high RAD51 expression was worse than that of patients with low expression (P<0.05). Figure C shows that RAD51 was upregulated in radiotherapy-resistant NSCLC tissues. Figure D shows the expression level of RAD51 in H1299, A549, and H1975 cells using q-PCR and U6 normalized quantification. Figures E, F, and G compare the survival, proliferation, and invasiveness of H1299 and H1975 cell lines after radiation irradiation using CCK-8 assays, clonal assays, and scratch assays.

[0057] Figure 7 RAD51 positively regulates GPX4 expression. Figure A: Western blot results show that GPX4 expression in high-RAD51 NSCLC cells is higher than that in low-RAD51 cells (p<0.05); Figure B: Western blot results show that GPX4 expression in the H1299 cell model increases with increasing irradiation dose (0, 2, 4 Gy), while GPX4 expression decreases in the H1975 cell model (P<0.05).

[0058] Figure 8The expression of miR-320a and RAD51 showed a negative correlation. Figure A shows the expression levels of miR-320a and RAD51 in lung cancer cell lines (A549, H1299, H1975) and the normal lung epithelial cell line Beas-2B, as detected by PCR. Figure B shows the expression level of RAD51 in lung cancer cell lines (A549, H1299, H1975) and the normal lung epithelial cell line Beas-2B, as detected by Western spectroscopy.

[0059] Figure 9 Low expression of miR-320a reduces the radiosensitivity of lung cancer cells. Figure A shows the effect of PCR on the expression levels of miR-320a and RAD51 in A549 cells, as detected by the miR-320a inhibitor. Figure B shows the effect of radiotherapy on lung cancer cell proliferation after miR-320a expression is reduced, as detected by the colony formation assay. Figure C shows the effect of radiotherapy on lung cancer cell migration after miR-320a expression is reduced, as detected by the scratch assay. Figure D shows the changes in RAD51 expression in A549 cells after miR-320a expression is reduced, as detected by Western spectroscopy.

[0060] Figure 10 High expression of miR-320a enhances the radiosensitivity of lung cancer cells. Figure A shows the effect of PCR on the expression levels of miR-320a and RAD51 in H1299 cells; Figure B shows the effect of radiotherapy on lung cancer cell proliferation after increased miR-320a expression as detected by a colony formation assay; Figure C shows the effect of radiotherapy on lung cancer cell migration after increased miR-320a expression as detected by a scratch assay; and Figure D shows the changes in RAD51 expression in H1299 cells after increased miR-320a expression as detected by Western spectroscopy.

[0061] Figure 11 The following figures illustrate the use of lentiviruses to construct RAD51 knockdown cell lines: Figure A shows the expression level of RAD51 in A549 cells after RAD51 knockdown lentivirus infection; Figure B shows the expression level of RAD51 in A549 cells after lentivirus infection using Western spectroscopy; Figure C shows the effect of miR-320a inhibitor on the expression levels of miR-320a and RAD51 in A549-shC and A549-shRAND51 cells using PCR; and Figure D shows the effect of miR-320a inhibitor on the expression level of RAD51 in A549-shC and A549-shRAND51 cells using Western spectroscopy.

[0062] Figure 12The role of RAD51 in reversing miR-320a-mediated radiosensitivity in lung cancer cells. Figure A shows the effect of radiotherapy on changes in miR-320a levels induced by RAD51 knockout after colony formation assay, which was used to detect changes in lung cancer cell proliferation. Figure B shows the effect of radiotherapy on changes in miR-320a levels induced by RAD51 knockout after scratch assay, which was used to detect changes in lung cancer cell migration.

[0063] Figure 13 The effects of miR-320a and RAD51 on ferroptosis in lung cancer cells. Figure A: Flow cytometry analysis of the effects of miR-320a knockdown and / or RAD51 knockdown on ferroptosis in lung cancer cells; Figure B: Western spectroscopy analysis of the effects of miR-320a knockdown and / or RAD51 knockdown on GPX4 expression in lung cancer cells. Detailed Implementation

[0064] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The reagents and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0065] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. In some processes described in the specification, claims, and accompanying drawings of the present invention, multiple operations appearing in a specific order are included. However, it should be clearly understood that these operations may not be executed in the order they appear herein, or may be executed in parallel. The operation numbers, such as S101, S102, S103, etc., are merely used to distinguish different operations, and the numbers themselves do not represent any execution order. Furthermore, these processes may include more or fewer operations, and these operations may be executed sequentially or in parallel.

[0066] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0067] Figure 1 This is a schematic flowchart of a computer-aided screening method based on miR-320a and RAD51 to improve the radiosensitivity of non-small cell lung cancer, provided by an embodiment of the present invention. Specifically, the method includes:

[0068] S101: Obtain secondary structure data of miR-320a and RAD51 protein structure data;

[0069] In one embodiment, the RAD51 protein is a protein found in eukaryotes, homologous to RecA in prokaryotes, and is a highly conserved protein with little variation from yeast to humans. The human RAD51 protein contains 339 amino acids and plays a major role in homologous recombination, participating in the process of searching for homologous sites to pair with DNA. The RAD51 (RAD51 recombinase [Homo sapiens (human)]) has a Gene ID of 5888 in NCBI, and detailed information about this gene (including sequence information) can be obtained through a routine search at https: / / www.ncbi.nlm.nih.gov / gene / . In another embodiment, the miR-320a (hsa-miR-320a-3p) can be obtained through a routine search in miRBase (http: / / www.mirbase.org / ). Currently, there are no studies or reports on the application of RAD51 and miR-320a in screening drugs that promote radiosensitivity in non-small cell lung cancer.

[0070] In one embodiment, radiotherapy refers to radiation therapy, an important means of treating diseases (especially malignant tumors) using ionizing radiation (such as X-rays, gamma rays, proton beams, etc.). In a specific embodiment of the present invention, radiotherapy refers to X-ray irradiation.

[0071] In one embodiment, enhancing the radiosensitivity of non-small cell lung cancer refers to enhancing the response of non-small cell lung cancer tumor cells to radiotherapy (radiotherapy), making tumor cells more likely to be killed or inhibited when receiving the same dose of radiotherapy, thereby improving the radiotherapy effect, reducing the risk of recurrence, and minimizing damage to normal tissues.

[0072] In one embodiment, the experimental materials and methods involved in this invention are as follows:

[0073] (1) Clinical Samples: With the approval of the Ethics Committee of the Affiliated Zhongshan Hospital of Dalian University, 60 pathological slides from patients diagnosed with non-small cell lung cancer (TNM staging unspecified) between January 2016 and December 2021 were screened. These patients had not received any treatment before specimen collection and only received radiotherapy after sampling. Imaging data before and after radiotherapy were used to assess the efficacy of treatment. All fresh samples were rapidly frozen in liquid nitrogen and stored at -80°C until use.

[0074] (2) Cell lines and cell culture: Human bronchial epithelioid cell line (16HBE), human embryonic kidney 293 cell line (HEK293), and cancer-causing human alveolar basal epithelial cell line (A549) were purchased from the Institute of Zoology, Plant and Animal Chemistry and Life Sciences, Chinese Academy of Sciences, and the Shanghai Institutes for Biological Sciences. Human non-small cell lung cancer cell lines (H1299, H1975) were purchased from the American Type Culture Collection (ATCC). The cell lines were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) at 37°C and 5% CO2, supplemented with 10% bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin to ensure optimal cell culture conditions.

[0075] (3) Quantitative Real-Time PCR (qRT-PCR): Total RNA was extracted from cells using a TRIzol purification kit (Invitrogen, Carlsbad, CA, USA), and the concentration and purity of all RNA samples were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Germany). SweScript All-in-One First Strand cDNA Synthesis SuperMix for qPCR (Servicebio, China) was used to transcribe the isolated RNA into cDNA. qRT-PCR was performed using 2x SYBRGreen RT-PCR Master Mix (ROX-free) (Servicebio, China) and a quantitative real-time PCR instrument (Heal Force). -ΔΔCT The relative expression levels of miR-320a and RAD51 were calculated using the method.

[0076] (4) Western blot analysis: Cells were lysed in lysis buffer to obtain the desired protein samples. Proteins were separated by 10% sodium dodecyl sulfate polysaccharide gel electrophoresis (SDS-PAGE) (Servicebio, China) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was then incubated for 1 hour in a TBST solution containing 5% skim milk powder (Solarbio, China). Following this step, the membrane was incubated overnight at 4°C with a specific primary antibody, washed with TBST, and then incubated at 37°C with the corresponding secondary antibody for 45 minutes. Finally, after washing with TBST, the membrane was prepared for chemiluminescence and visualized using a chemiluminescence instrument (CLINX). The film was scanned, and the gray values ​​of the target bars were analyzed using the ImageJ gel image processing system.

[0077] (5) Plasmid transfection: miR-320a mimics, miR-320a inhibitors, and their corresponding controls were purchased from RiboBio, pLenti. Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cells were collected for analysis 24 hours after transfection.

[0078] (6) Immunohistochemical (IHC) staining: Tissue sections were dehydrogenated by immersion in minocycline ethylenediaminetetraacetic acid (EDTA) and rehydrated using a series of graded ethanol solutions. Sections were then immersed in 0.3% peroxidase methanol solution for 30 minutes. During antigen extraction, sections were pretreated with citrate buffer in a microwave oven at 100°C for 15 minutes. Sections were then washed three times with phosphate-buffered saline (PBS) (Thermo Scientific, Germany), co-incubated with primary antibody at 4°C, stained with 3,3'-diaminobenzidine, and then stained with hematoxylin in reverse. The percentage of immunoreactive cells and relative staining intensity were automatically calculated using Aipathwell (Servicebio).

[0079] (7) Dual-luciferase reporter assay: HEK-293FT cells were cultured in 24-well plates (1×10⁻⁶ cells / wells). 5 Cells were cultured in wells and transfected with RAD51-3'UTR-wt or RAD51-3'UTR-mt and hsa-miR-320a-3pmimics or mimics-NC using Lipofectamine 2000 (Invitrogen). After 24 hours, luciferase activity was measured using a dual-luciferase assay kit (Beyotime, China) and normalized to dual-luciferase activity.

[0080] (8) Colony formation assay: Cells were cultured in 6-well plates (1000 cells per well), then irradiated with 0, 2, 4, 6, and 8 Gy of X-rays and incubated in an incubator for 14 days. Cells were fixed with polymethyl methacrylate, stained with crystal violet, and counted. Cell viability at different irradiation doses was evaluated using the results of 0 Gy irradiation as a control.

[0081] (9) Cell scratch assay: Cells were seeded in 6-well plates (1×10⁻⁶ cells / well). 5 Cells were collected (number of cells / well) and irradiated with X-rays at doses of 0, 2, 4, 6, and 8 Gy, respectively. After culturing for 24 hours, the cells were streaked using a pipette tip perpendicular to the well and the marking line, and then placed in culture medium for another 24 hours. Images were taken at 0 and 24 hours and analyzed using ImageJ.

[0082] (10) CCK-8 assay: Cells were seeded in 96-well plates (2×10⁶ cells / wells). 3 Cells were collected (cells / well) and irradiated with X-rays at doses of 0, 2, 4, 6, and 8 Gy, respectively. 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 4 hours. The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA), and the data were recorded.

[0083] (11) Data Analysis: All experimental data were statistically analyzed using R code and GraphPad Prism version 9. The TCGA database was used for gene expression and survival analysis. The TargetScan, miRDB, and miTarBase databases were used to predict the target genes of miR-320a. Kaplan-Meier curves were used for survival analysis. The statistical significance of differences between groups was calculated using Student's t-test. P < 0.05 was considered statistically significant and was indicated by an asterisk (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

[0084] In one embodiment, the present invention demonstrates that miR-320a is downregulated and has a radiosensitizing effect in human non-small cell lung cancer (NSCLC) tissues. Specifically, as follows:

[0085] To determine the role of miR-320a in the occurrence and development of non-small cell lung cancer (NSCLC), we analyzed the miRNA and gene expression profiles of 502 NSCLC samples and 52 adjacent normal lung tissue samples from the TCGA database. The results showed that miR-320a expression was significantly downregulated in NSCLC tissues compared to adjacent normal tissues (P<0.05). Figure 4A). Furthermore, our survival analysis of non-small cell lung cancer patients, grouped according to high and low miR-320a expression levels, showed no statistically significant difference in median overall survival (mOS) between patients with high and low miR-320a expression (p>0.05). Figure 4 B).

[0086] To investigate the relationship between miR-320a and radiosensitivity, we grouped a clinical sample of 60 patients from the Affiliated Zhongshan Hospital of Dalian University based on post-radiotherapy assessments. Patients with complete remission (CR) and partial remission (PR) were assigned to the radiosensitivity group (n=23) according to RECIST 1.1 criteria, while those with stable disease (SD) and progressive disease (PD) were assigned to the radiotolerance group (n=37). The expression level of miR-320a in NSCLC tissues of each group was measured. The results showed that, compared with the radiosensitivity group, the expression of miR-320a was significantly downregulated in the radiotolerance group. Figure 4 C). This result indicates that miR-320a leads to increased sensitivity of NSCLC cells to radiotherapy.

[0087] S102: Select the spatial structure of the miR-320a secondary structure and the RAD51 protein complex, and determine the binding site of the miR-320a secondary structure and the RAD51 protein complex as the binding site of the targeted drug.

[0088] In some embodiments, the method for determining the binding site of the miR-320a secondary structure and the RAD51 protein complex includes:

[0089] Construct n truncated expression mutants of RAD51, where n is a natural number greater than or equal to 1;

[0090] Based on the n truncated expression mutants, dual-luciferase reporter analysis was performed to detect the luciferase activity of the n truncated expression mutants after co-transfection with miR-320a, and the key region of the miR-320a secondary structure and RAD51 protein complex binding was located.

[0091] Single-point mutations were performed on each base in the key region, and the changes in luciferase activity after each mutation were detected. The resulting key complementary base region is the binding site of the miR-320a secondary structure and the RAD51 protein complex.

[0092] In one embodiment, the present invention demonstrates that RAD51 is a direct target of miR-320a. Specifically, as follows:

[0093] To explore the role of miR-320a in NSCLC cell proliferation and radiosensitivity, we used the TargetScan, miRDB, and miRTarBase databases to predict miR-320a-related target genes, and identified RAD51 as a potential target of miR-320a. Figure 5 A).

[0094] To verify this hypothesis, we performed a dual-luciferase reporter gene assay using miR-320a and RAD51. Transfection of HEK 293FT cells with wild-type or mutant RAD51 showed that miR-320a significantly reduced luciferase activity in cells transfected with RAD51-3'UTR (both wild-type and mutant), with statistically significant differences (P < 0.05). Figure 5 B). This indicates that the hsa-miR-320a-3p mimic has an inhibitory effect on the target sequence RAD51-3'UTR.

[0095] In one embodiment, the present invention demonstrates that RAD51 promotes the resistance of NSCLC cells to radiotherapy. Specifically, as follows:

[0096] Based on the above findings, we searched the TCGA database and analyzed 1006 NSCLC samples and 107 adjacent normal lung tissue samples. We found that RAD51 expression was significantly upregulated in non-small cell lung cancer tissues compared to normal tissues (P<0.05). Figure 6 A). Furthermore, we found that RAD51 expression was negatively correlated with survival in NSCLC patients (P<0.05, Figure 6 B).

[0097] Next, we used immunohistochemistry to detect the expression of RAD51 in 60 clinically collected sections. The results showed that the staining intensity and positive rate of RAD51 in the radiotherapy-tolerant group were higher than those in the radiotherapy-sensitive group. Figure 6 C). Furthermore, we investigated the expression of RAD51 in A549, H1299, and H1975 cells using qRT-PCR. The results showed that RAD51 expression was significantly higher in H1299 cells compared to A549 and H1975 cells. Figure 6D). Subsequently, we investigated the relationship between RAD51 and radiosensitivity using two cell lines, H1299 and H1975, using colony formation, cell scratch, and CCK-8 assays. These cells were exposed to different radiation doses (0, 2, 4, 6, 8 Gy). In the colony formation assay, we did not obtain the expected results. The results showed that the proliferative capacity of both cell lines decreased with increasing radiation dose, but there was no significant difference between them. However, the results of cell scratch and CCK-8 assays indicated that, under the same radiation conditions, H1299 cells had stronger migration and proliferation capabilities than H1975. These results suggest that RAD51 reduces the cytotoxic effect of radiation on NSCLC cells after exposure to different radiation doses. Figure 6 E, F, G).

[0098] In one embodiment, the present invention demonstrates that RAD51 positively regulates GPX4 to inhibit ferroptosis in NSCLC cells and enhance their radioresistance. Specifically, as follows:

[0099] To analyze the mechanism by which RAD51 affects the radiosensitivity of NSCLC, we first transfected H1299 cells with a RAD51 inhibitor (inhibitor B02) to generate H1299 cells with low RAD51 expression. Simultaneously, we transfected H1975 cells with RAD51 to generate H1975 cells with high RAD51 expression. We exposed H1975 and H1299 cells to different radiation doses (0, 2, 4, 6, 8 Gy) and analyzed GPX4 expression. The results showed that in the H1299 cell model, GPX4 expression increased with increasing radiation dose (0, 2, 4 Gy), while it decreased in the H1975 cell model (P < 0.05). Figure 7 A). The results show that the trend of GPX4 expression changed in both H1979 and H1299 cells after the radiation dose exceeded 4 Gy. Therefore, in this study, we used 4 Gy as the most suitable radiation dose to affect GPX4 expression. Subsequently, both cell models were exposed to the same radiation dose (4 Gy), and GPX4 expression was analyzed by Western blotting. The results showed that GPX4 expression was higher in non-small cell lung cancer cells with higher Rad51 expression than in NSCLC cells with lower Rad51 expression (P<0.05). Figure 7 B). These results indicate that RAD51 can influence the radiosensitivity of NSCLC cells by actively regulating GPX4 expression and inhibiting ferroptosis.

[0100] In one embodiment, the present invention demonstrates a negative correlation between miR-320a and RAD51 expression. Specifically, as follows:

[0101] To investigate the relationship between miR-320a and RAD51 and the development and progression of lung cancer, this study first examined the expression of miR-320a and RAD51 in lung cancer cell lines (A549, H1299, H1975) and the normal lung epithelial cell line Beas-2B. The results are as follows: Figure 8 As shown, miR-320a was expressed most highly in Beas-2B cells and less so in tumor cells; while RAD51 was expressed least highly in Beas-2B cells and more so in tumor cells. This indicates a negative correlation between miR-320a and RAD51 expression, suggesting that RAD51 may promote lung cancer development, while miR-320a may inhibit it. Furthermore, based on the expression patterns of miR-320a and RAD51 in lung cancer cell lines, A549 and H1299 were selected for further investigation.

[0102] In one embodiment, the present invention demonstrates that miR-320a enhances the sensitivity of NSCLC cells to radiotherapy. Specifically, as follows:

[0103] To investigate the relationship between miR-320a and radiosensitivity, this study used miR-320a inhibitor (5'-UCGCCCUCUCAACCCAGCUUUU-3' ligated to the pLVX-IRES-Puro restriction site XbaI / XhoI) and miR-320amimics (5'-AAAAGCUGGGUUGAGAGGGCGA-3' vector pLVX-miRNA site EcoRI / XhoI) to alter the level of miR-320a in lung cancer cells and then examined its effect on the radiosensitivity of NSCLC cells. First, after transfecting A549 cells with the miR-320a inhibitor, cell colony formation assays and scratch assays were used to detect the effect of miR-320a reduction on the radiosensitivity of A549 cells. The results are as follows: Figure 9 As shown in Figure A, transfection of A549 cells with the miR-320a inhibitor significantly reduced the expression level of miR-320a. Furthermore, at the same radiotherapy dose, the reduced miR-320a expression significantly enhanced the clonogenic and migration abilities of tumor cells. Figure 9 (B and 9C).

[0104] Furthermore, after transfecting miR-320a mimics into H1299 cells, the effect of miR-320a on reducing the radiosensitivity of H1299 cells was detected using cell colony formation assays and scratch assays. The results are as follows: Figure 10As shown in Figure A, after transfecting H1299 cells with miR-320a mimics, the expression level of miR-320a was significantly increased. Furthermore, at the same radiotherapy dose, the increased expression level of miR-320a significantly inhibited the clonogenic and migration abilities of tumor cells. Figure 10 B and 10D).

[0105] The above experimental results indicate that miR-320a is closely related to the radiosensitivity of lung cancer cells. Increasing miR-320a enhances the radiosensitivity of lung cancer cells, while decreasing miR-320a inhibits the radiosensitivity of lung cancer cells.

[0106] In addition, we also examined the expression of RAD51 in lung cancer cells after changes in miR-320a expression levels. The study found that a decrease in miR-320a expression levels significantly increased the expression level of RAD51 in A549 cells. Figure 9 A and 9D); while after the expression level of miR-320a increased, the expression level of RAD51 in H1299 cells decreased significantly (A and 9D); Figure 9 (A and 9D). This indicates that there is indeed a negative correlation between miR-320a and RAD51 expression, and that RAD51 has a potential role in promoting tumor progression.

[0107] In one embodiment, the present invention demonstrates the effect of RAD51 antagonizing miR-320a in enhancing the radiosensitivity of lung cancer cells. Specifically, as follows:

[0108] Based on the aforementioned negative correlation between miR-320a and RAD51 expression, we further explored the relationship between RAD51 and radiosensitivity. In this study, we constructed a RAD51 knockdown cell line (A549-shRAND51) using lentiviral infection and examined its effect on miR-320a's enhancement of radiosensitivity in lung cancer cells. First, we successfully constructed the RAD51 knockdown cell line (A549-shRAND51) and the control cell line (A549-shC) using lentiviral infection. Figure 11 A and 11B). Based on this, after transfecting A549-shC and A549-shRAND51 cells with miR-320a inhibitor ( Figure 11 The effects of RAD51 on the radiosensitivity of miR-320a lung cancer cells were examined using cell colony formation assays and scratch assays (C and 11D).

[0109] The results are as follows Figure 12As shown in A and 12B, at the same radiotherapy dose, inhibiting miR-320a expression enhanced the clonogenic and migration abilities of tumor cells; however, further inhibition of RAD51 expression in tumor cells significantly reduced these abilities at the same radiotherapy dose. These results indicate that knocking down RAD51 can significantly reverse the decrease in radiosensitivity of lung cancer cells caused by reduced miR-320a expression.

[0110] In summary, miR-320a affects the sensitivity of lung cancer cells to radiotherapy by regulating the expression of RAD51, meaning that miR-320a can enhance the sensitivity of lung cancer cells to radiotherapy by inhibiting the expression of RAD51.

[0111] In one embodiment, the present invention demonstrates the effect of miR-320a regulating RAD51 on ferroptosis in lung cancer cells. Specifically, as follows:

[0112] To investigate the molecular mechanism by which miR-320a regulates RAD51 expression and affects the radiosensitivity of lung cancer cells, we first examined its effect on ferroptosis in lung cancer cells. The results are as follows: Figure 13 As shown in Figure A, flow cytometry analysis revealed that inhibiting miR-320a expression significantly reduced Lipid ROS levels in A549 cells; this phenomenon was reversed by knocking down RAD51. Correspondingly, Western blotting showed that GPX4 expression levels in A549 cells increased with miR-320a inhibition and decreased with RAD51 inhibition. Figure 13 B). The above results indicate that miR-320a affects ferroptosis by regulating RAD51.

[0113] Based on the above experimental results, this invention demonstrates that miR-320a promotes the radiosensitivity of NSCLC by negatively regulating RAD51. This proves the feasibility and effectiveness of a computer-aided screening method based on miR-320a and RAD51 to screen drugs that promote radiosensitivity in non-small cell lung cancer. The screened drugs can inhibit the expression of RAD51 protein, thereby promoting the radiosensitivity of non-small cell lung cancer.

[0114] In one embodiment, the method for determining the binding site of the miR-320a secondary structure and the RAD51 protein complex includes: a truncation mutation experiment and a single-base mutation scan. The truncation mutation experiment involves dividing the RAD51 3'UTR into multiple truncated fragments (e.g., 5' truncation, 3' truncation, or deletion of the middle region), constructing WT truncated vectors for each fragment, detecting the luciferase activity after co-transfection with miR-320a, and locating the minimal binding region (i.e., the key region). The single-base mutation scan involves performing a single-point mutation on each base of the predicted binding site, detecting the change in luciferase activity after each mutation, and determining the key complementary base (i.e., the binding site of the miR-320a secondary structure and the RAD51 protein complex).

[0115] In some embodiments, the truncation mutation assay refers to obtaining truncated mutants by deleting a portion of the gene or protein coding sequence (such as the 5' end, 3' end, or internal regions) for studying the structure-function relationship of genes / proteins. Specific experimental methods include: (1) Constructing truncated mutants: Amplifying truncated fragments of the target gene using PCR, or deleting specific regions using gene editing technology (such as CRISPR). Ligating to an expression vector (such as a plasmid) and transferring it into cells or an in vitro expression system (such as E. coli). (2) Functional detection: Detecting the enzyme activity and binding capacity of the truncated protein through in vitro experiments (such as ELISA, pull-down). Observing changes in cell phenotype (such as proliferation, apoptosis, migration) or subcellular localization (such as fluorescence microscopy) through cell experiments. Primarily used to analyze the ligand-binding region of receptor proteins, study the DNA-binding domain of transcription factors, and verify truncated mutations found in cancer.

[0116] In some embodiments, single-nucleotide mutation scanning refers to the systematic introduction of a single-base mutation (e.g., A→T, C→G) into a target gene or DNA sequence, and the effect of each mutation on function is detected one by one or in batches to screen key sites. Specific experimental methods include: (1) Site-directed mutagenesis construction: introducing a single-base mutation at a specific site through PCR-mediated site-directed mutagenesis technology (e.g., QuikChange kit) or gene synthesis. Constructing a plasmid library containing a single mutation (e.g., each plasmid carries a mutation at a different site). (2) High-throughput screening: using a reporter gene system, such as inserting the mutated sequence into a luciferase reporter vector, and reflecting function (e.g., promoter activity, miRNA binding ability) through changes in fluorescence intensity. Using yeast / bacterial screening methods to screen for mutations that affect survival (e.g., functional sites of essential genes) using auxotrophic strains or resistance markers. Combining the CRISPR-Cas9 system to detect the mutant phenotype at the single-cell level. It is mainly used to scan for single-base mutations in miRNA seed regions, verify their binding specificity to target gene mRNA (such as dual-luciferase reporter assay), identify conserved amino acids in mutated kinase active sites (such as lysine in the ATP binding pocket), confirm catalytic function, and analyze the impact of mutations at key sites in the viral genome on infectivity or drug sensitivity (such as mutations in the SARS-CoV-2 spike protein).

[0117] In some embodiments, molecular interaction mechanisms can be systematically resolved at the domain and single-base levels by combining truncated mutation experiments and single-base mutation scanning.

[0118] S103: A candidate drug targeting the binding site is obtained by using a computer-aided drug screening method, wherein the candidate drug is a drug that promotes the radiosensitivity of non-small cell lung cancer.

[0119] In some embodiments, the computer-aided drug screening method includes:

[0120] Obtain the secondary structure of miR-320a and the binding site of the RAD51 protein complex;

[0121] Based on the spatial structure of the miR-320a secondary structure and the binding site of the RAD51 protein complex, small molecule compounds with similar structures were screened from molecular databases.

[0122] The small molecule compounds selected through screening are molecularly docked with the RAD51 protein to calculate the affinity or binding energy of the small molecule compounds to the RAD51 protein receptor, and a score is obtained for each small molecule compound. Candidate drugs are obtained by sorting the small molecule compounds according to the scores.

[0123] In some embodiments, the small molecule compound is selected from siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic small molecule compound.

[0124] In some embodiments, the method further includes:

[0125] Obtain a system expressing RAD51;

[0126] The effectiveness of the small molecule compounds was verified by treating the RAD51-expressing system with the small molecule compounds obtained from the screening.

[0127] Optionally, the system expressing RAD51 includes a cell system expressing RAD51, a subcellular system expressing RAD51, a tissue system expressing RAD51, a solution system expressing RAD51, an organ system expressing RAD51, or an animal system expressing RAD51.

[0128] Optionally, small molecule compounds that inhibit RAD51 expression can be screened as candidate drugs.

[0129] In one embodiment, the animals involved in the animal system expressing RAD51 include, but are not limited to, mice, rats, guinea pigs, rabbits, pigs, chickens, pigeons, monkeys, and dogs.

[0130] In some embodiments, the small molecule compounds include siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog, or inorganic small molecule compounds. It should be noted that the present invention does not impose any particular limitation on the specific type of the small molecule compounds; any small molecule compound that may be used in the computer-aided drug screening method provided by the present invention and may produce the effect of promoting radiosensitivity in non-small cell lung cancer falls within the protection scope of the present invention.

[0131] In one embodiment, in molecular docking, affinity refers to the tightness with which a molecule binds to a receptor. High affinity means a more stable binding, while low affinity indicates a less stable binding. Affinity is typically calculated, for example, by calculating the binding free energy (ΔG) or the binding constant (Kd).

[0132] In molecular docking, affinity depends on the interactions between the molecule and the acceptor, including hydrogen bonds, van der Waals forces, and electrostatic interactions. These interactions collectively determine the binding mode between the molecule and the acceptor, thus affecting affinity.

[0133] To assess affinity, scoring systems or methods are typically used to quantify the interaction between molecules and receptors. These scoring methods are based on different algorithms and physical models and can reflect the binding energy, interaction type, and affinity between molecules and receptors.

[0134] In one embodiment, the mode of screening drugs using computer-aided drug screening technology includes one or more of the following: protein-nucleic acid docking, protein-small molecule docking, and protein-protein docking.

[0135] In one embodiment, the protein-nucleic acid docking refers to a computational simulation process that uses specific algorithms and procedures to dock the structures of proteins and nucleic acids (e.g., miRNAs) together. This process can be used to study the interactions between proteins and nucleic acids, as well as their potential biological functions.

[0136] In protein-nucleic acid docking, software such as NAflex is commonly used for computational simulation. NAflex is a software specifically developed for nucleic acid structure prediction and design, capable of accurate modeling and docking of DNA or RNA molecules. NAflex employs a fragment-based scoring method, enabling fast and accurate docking. It considers various complex interactions, such as hydrogen bonds, ionic bonds, and hydrophobic interactions, and can precisely adjust the side-chain conformation during docking. The basic algorithm of NAflex consists of two stages. The first stage is a low-precision stage, which mainly searches for a coarse match between the protein and nucleic acid. The second stage is a high-precision stage, which considers all side-chain conformations and calculates more accurate interaction energies. In the low-precision stage, NAflex randomly translates and rotates the nucleic acid molecule and performs a certain number of rigid body movements before calculating the interaction energy. After outputting the lowest conformation, it enters the high-precision stage. In the high-precision stage, the program performs further optimization and adjustments to achieve more accurate docking.

[0137] In one embodiment, the protein-small molecule docking refers to a computational simulation process that uses specific algorithms and procedures to dock the structures of proteins and small molecules (such as drug molecules). This process can be used to study the interactions between proteins and small molecules, as well as their potential biological functions.

[0138] In protein-small molecule docking, computational simulations are typically performed using software such as DOCK. DOCK is a highly automated drug design software capable of docking small molecule ligands with biomolecular receptors. It employs a fragment-based scoring method, enabling fast and accurate docking. The basic algorithm of DOCK consists of two stages. The first stage is a low-precision stage, primarily searching for a coarse match between the small molecule ligand and the biomolecular receptor. The second stage is a high-precision stage, which considers all side-chain conformations and calculates more precise interaction energies. In the low-precision stage, DOCK randomly translates and rotates the small molecule ligand and performs a certain number of rigid body movements before calculating the interaction energy. After outputting the lowest conformation, it proceeds to the high-precision stage. In the high-precision stage, the program performs further optimization and adjustments to achieve even more accurate docking.

[0139] In one embodiment, protein-protein docking refers to a computational simulation process that uses specific algorithms and procedures to dock the structures of two proteins together. This process can be used to study protein-protein interactions and potential biological functions.

[0140] RosettaDock is a commonly used docking software for protein-protein docking. It employs a fragment-based scoring method to achieve fast and accurate protein docking. This software can precisely adjust the side-chain conformations during docking and considers various complex interactions, such as hydrogen bonds, ionic bonds, and hydrophobic interactions. RosettaDock's basic algorithm consists of two stages. The first stage is a low-precision stage, which mainly searches for the degree of skeletal shape compatibility between the two proteins. The second stage is a high-precision stage, which considers all side-chain conformations and calculates more precise interaction energies. In the low-precision stage, the program randomly translates and rotates a component of a protein molecule and performs a certain number of rigid body movements, then calculates the interaction energy. After outputting the lowest conformation, it enters the high-precision stage. In the high-precision stage, the program performs 50 MCMPCCcycles: rearranging the conformations and minimizing the interaction energy, using this as the initial starting conformation.

[0141] In one embodiment, the virtual drug screening method provided in this invention is an important method in the field of targeted drug design. Its process is roughly as follows:

[0142] First, based on the known structures of drug-target protein complexes, the binding modes of the drug and target protein are studied to identify key amino acid residues. This step helps to understand how the drug interacts with the target protein and provides a basis for subsequent virtual screening.

[0143] Next, computer-aided drug design methods are used to virtually screen a large number of small molecule compounds. During this process, the binding affinity of the small molecule compounds to the target protein is predicted and evaluated. Typically, small molecules with low binding energies and good shape complementarity are selected as potential drug candidates.

[0144] Finally, the selected candidate drugs were further experimentally validated to confirm their interaction with the target protein and their biological activity.

[0145] In one embodiment, in drug virtual screening, the binding site refers to the region on the target protein that binds to a small molecule compound. To perform receptor-based virtual screening, it is first necessary to determine the binding site of the target protein. This is typically achieved through analysis and study of the target protein's structure.

[0146] Once the binding sites are identified, computer-aided drug design methods can be used to dock compounds from a small molecule library with the target protein. During docking, the computer simulates the interaction between the compound and the binding site and evaluates their binding affinity. Based on the docking results, small molecule compounds with strong binding affinity to the target protein can be screened as potential drug candidates. The determination of binding sites in virtual drug screening is crucial to the success of drug design. Therefore, multiple factors need to be considered when determining binding sites, such as the structural characteristics of the target protein and the binding modes of known ligands. Simultaneously, the binding sites need to be appropriately processed and optimized during virtual screening to improve the accuracy and efficiency of the screening.

[0147] In one embodiment, computer-aided drug screening is a technique that uses computer-aided drug design methods to screen drugs. It can help researchers quickly screen a large number of small molecule compounds for candidate drugs that have strong binding affinity to target proteins and potential pharmacological effects.

[0148] In one embodiment, molecular docking is a method for drug design based on the characteristics of the receptor and the interaction between the receptor and drug molecules. It is a theoretical simulation method that primarily studies intermolecular interactions (such as ligand-receptor interactions) and predicts their binding modes and affinities. This method is widely used in the early stages of drug development to help researchers quickly screen compounds with potential pharmacological effects.

[0149] Molecular docking methods primarily focus on spatial matching and energy matching. Spatial matching refers to the geometric complementarity between the drug molecule and the receptor protein, while energy matching refers to the minimization of the interaction between the drug molecule and the receptor protein. Geometric matching calculations typically employ methods such as grid-based computation and fragment growth, while energy calculations utilize methods such as simulated annealing and genetic algorithms.

[0150] Based on the degree and method of simplification, molecular docking methods can be divided into rigid docking, semi-flexible docking, and flexible docking. In rigid docking, the conformation of the docking molecules remains unchanged during the calculation; only their spatial position and orientation are altered. Semi-flexible docking allows for partial conformational changes during the calculation. Flexible docking allows for even more conformational changes.

[0151] In one embodiment, the molecular libraries used in virtual drug screening mainly include the following:

[0152] ZINC: Contains over 250 million commercially available compounds for virtual screening of small molecules.

[0153] PubChem: Contains bioactive substances for virtual screening of small molecules.

[0154] DrugBank contains drugs and small molecules for drug design and discovery.

[0155] ChEMBL: Contains small molecules for drug discovery and chemical genomics research.

[0156] ChemDB contains a large number of known small molecules for chemical genomics research and drug discovery.

[0157] HMDB: Contains a large number of known small molecules used for chemical genomics research and drug discovery.

[0158] BindingDB contains a large number of known small molecules for chemical genomics research and drug discovery.

[0159] SMPDB contains a large number of known small molecules for chemical genomics research and drug discovery.

[0160] In addition, there are some commercial databases such as ChemDiv, Enamine, Lifechemicals, Specs, Chembridge, Maybridge, Microsource, Vitas-M, and Interbioscreen, which are also frequently used for virtual drug screening.

[0161] Figure 2 This is a schematic diagram of a computer-aided screening system based on miR-320a and RAD51 to improve the radiosensitivity of non-small cell lung cancer, provided in an embodiment of the present invention. Specifically, the system includes:

[0162] Data acquisition unit: Acquires secondary structure data of miR-320a and structure data of RAD51 protein;

[0163] Site determination unit: Select the spatial structure of the miR-320a secondary structure and the RAD51 protein complex, and determine the binding site of the miR-320a secondary structure and the RAD51 protein complex as the binding site of the targeted drug;

[0164] Drug screening unit: A computer-aided drug screening method is used to obtain candidate drugs that target the binding site, wherein the candidate drugs are drugs that promote radiosensitivity of non-small cell lung cancer.

[0165] In some embodiments, the method for determining the binding site of the miR-320a secondary structure and the RAD51 protein complex includes:

[0166] Construct n truncated expression mutants of RAD51, where n is a natural number greater than or equal to 1;

[0167] Based on the n truncated expression mutants, dual-luciferase reporter analysis was performed to detect the luciferase activity of the n truncated expression mutants after co-transfection with miR-320a, and the key region of the miR-320a secondary structure and RAD51 protein complex binding was located.

[0168] Single-point mutations were performed on each base in the key region, and the changes in luciferase activity after mutation were detected one by one. The key complementary base region obtained is the binding site of the miR-320a secondary structure and the RAD51 protein complex.

[0169] Optionally, the computer-aided drug screening method includes:

[0170] Obtain the secondary structure of miR-320a and the binding site of the RAD51 protein complex;

[0171] Based on the spatial structure of the miR-320a secondary structure and the binding site of the RAD51 protein complex, small molecule compounds with similar structures were screened from molecular databases.

[0172] The small molecule compounds selected from screening are molecularly docked with the RAD51 protein to calculate the affinity or binding energy of the small molecule compounds to the RAD51 protein receptor, and a score is obtained for each small molecule compound. Candidate drugs are obtained by sorting the small molecule compounds according to the scores.

[0173] Optionally, the small molecule compound is selected from siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic small molecule compound;

[0174] Optionally, the method further includes:

[0175] Obtain a system expressing RAD51;

[0176] The effectiveness of the small molecule compounds was verified by treating the RAD51-expressing system with the small molecule compounds obtained from the screening.

[0177] Optionally, the system expressing RAD51 includes a cell system expressing RAD51, a subcellular system expressing RAD51, a tissue system expressing RAD51, a solution system expressing RAD51, an organ system expressing RAD51, or an animal system expressing RAD51.

[0178] Optionally, small molecule compounds that inhibit RAD51 expression can be screened as candidate drugs.

[0179] Figure 3 This is a schematic diagram of a computer-aided screening device based on miR-320a and RAD51 to enhance the radiosensitivity of non-small cell lung cancer, provided in an embodiment of the present invention. Specifically, the device includes:

[0180] The system includes a memory for storing program instructions and a processor for invoking the program instructions, which, when executed, implement the previously described computer-aided screening method for drugs that enhance radiosensitivity in non-small cell lung cancer based on miR-320a and RAD51.

[0181] This invention also provides a computer-readable storage medium storing a computer program that, when executed by a processor, implements the aforementioned computer-aided screening method for drugs that enhance radiosensitivity in non-small cell lung cancer based on miR-320a and RAD51.

[0182] This invention also provides a drug for promoting radiosensitivity in non-small cell lung cancer, obtained by screening using the computer-aided screening method based on miR-320a and RAD51 as described above.

[0183] The verification results of this verification embodiment show that assigning inherent weights to indications can moderately improve the performance of this method compared to the default settings.

[0184] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working processes of the systems, devices, and units described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here.

[0185] In the several embodiments provided in this application, it should be understood that the disclosed systems, apparatuses, and methods can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative; for instance, the division of units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be an indirect coupling or communication connection between apparatuses or units through some interfaces, and may be electrical, mechanical, or other forms.

[0186] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0187] Furthermore, the functional units in the various embodiments of the present invention can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit.

[0188] Those skilled in the art will understand that all or part of the steps in the various methods of the above embodiments can be implemented by a program instructing related hardware. The program can be stored in a computer-readable storage medium, which may include: read-only memory (ROM), random access memory (RAM), disk or optical disk, etc.

[0189] Those skilled in the art will understand that all or part of the steps in the methods of the above embodiments can be implemented by a program instructing related hardware. The program can be stored in a computer-readable storage medium, such as a read-only memory, a disk, or an optical disk.

[0190] The computer device provided by the present invention has been described in detail above. For those skilled in the art, there will be changes in the specific implementation and application scope based on the ideas of the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A computer-aided screening method based on miR-320a and RAD51 to screen drugs that improve radiosensitivity in non-small cell lung cancer, characterized in that, The method includes: Obtain secondary structure data of miR-320a and structure data of RAD51 protein; The spatial structure of the miR-320a secondary structure and the RAD51 protein complex was selected, and the binding site of the miR-320a secondary structure and the RAD51 protein complex was determined as the binding site of the targeted drug. A computer-aided drug screening method was used to obtain candidate drugs that target the binding site, wherein the candidate drugs are drugs that promote radiosensitivity in non-small cell lung cancer. The method for determining the binding site of the miR-320a secondary structure and the RAD51 protein complex includes: Construct n truncated expression mutants of RAD51, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants, dual-luciferase reporter analysis was performed to detect the luciferase activity of the n truncated expression mutants after co-transfection with miR-320a, and the key region of the miR-320a secondary structure and RAD51 protein complex binding was located. Single-point mutations were performed on each base in the key region, and the changes in luciferase activity after mutation were detected one by one. The key complementary base region obtained is the binding site of the miR-320a secondary structure and the RAD51 protein complex. The computer-aided drug screening method includes: Obtain the secondary structure of miR-320a and the binding site of the RAD51 protein complex; Based on the spatial structure of the miR-320a secondary structure and the binding site of the RAD51 protein complex, small molecule compounds with similar structures were screened from molecular databases. The small molecule compounds selected from screening are molecularly docked with the RAD51 protein to calculate the affinity or binding energy of the small molecule compounds to the RAD51 protein receptor, and a score is obtained for each small molecule compound. Candidate drugs are obtained by sorting the small molecule compounds according to the scores. The method further includes: Obtain a system expressing RAD51; The effectiveness of the small molecule compounds was verified by treating the RAD51-expressing system with the small molecule compounds obtained from the screening. The system expressing RAD51 includes a cell system expressing RAD51, a subcellular system expressing RAD51, a tissue system expressing RAD51, a solution system expressing RAD51, an organ system expressing RAD51, or an animal system expressing RAD51. Small molecule compounds that inhibit RAD51 expression were screened as candidate drugs.

2. The method for screening drugs that promote radiosensitivity in non-small cell lung cancer based on miR-320a and RAD51 according to claim 1, characterized in that, The small molecule compounds are selected from siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic small molecule compounds.

3. A computer-aided screening system based on miR-320a and RAD51 for drugs that enhance radiosensitivity in non-small cell lung cancer, characterized in that, The system includes: Data acquisition unit: Acquires secondary structure data of miR-320a and structure data of RAD51 protein; Site determination unit: Select the spatial structure of the miR-320a secondary structure and the RAD51 protein complex, and determine the binding site of the miR-320a secondary structure and the RAD51 protein complex as the binding site of the targeted drug; Drug screening unit: A computer-aided drug screening method is used to obtain candidate drugs that target the binding site, wherein the candidate drugs are drugs that promote radiosensitivity of non-small cell lung cancer. The method for determining the binding site of the miR-320a secondary structure and the RAD51 protein complex includes: Construct n truncated expression mutants of RAD51, where n is a natural number greater than or equal to 1; Based on the n truncated expression mutants, dual-luciferase reporter analysis was performed to detect the luciferase activity of the n truncated expression mutants after co-transfection with miR-320a, and the key region of the miR-320a secondary structure and RAD51 protein complex binding was located. Single-point mutations were performed on each base in the key region, and the changes in luciferase activity after mutation were detected one by one. The key complementary base region obtained is the binding site of the miR-320a secondary structure and the RAD51 protein complex. The computer-aided drug screening method includes: Obtain the secondary structure of miR-320a and the binding site of the RAD51 protein complex; Based on the spatial structure of the miR-320a secondary structure and the binding site of the RAD51 protein complex, small molecule compounds with similar structures were screened from molecular databases. The small molecule compounds selected from screening are molecularly docked with the RAD51 protein to calculate the affinity or binding energy of the small molecule compounds to the RAD51 protein receptor, and a score is obtained for each small molecule compound. Candidate drugs are obtained by sorting the small molecule compounds according to the scores. The method further includes: Obtain a system expressing RAD51; The effectiveness of the small molecule compounds was verified by treating the RAD51-expressing system with the small molecule compounds obtained from the screening. The system expressing RAD51 includes a cell system expressing RAD51, a subcellular system expressing RAD51, a tissue system expressing RAD51, a solution system expressing RAD51, an organ system expressing RAD51, or an animal system expressing RAD51. Small molecule compounds that inhibit RAD51 expression were screened as candidate drugs.

4. The computer-aided screening system for drugs that promote radiosensitivity in non-small cell lung cancer based on miR-320a and RAD51 according to claim 3, characterized in that, The small molecule compounds are selected from siRNA, shRNA, dsRNA, microRNA, antisense nucleic acid, antibody, polypeptide, protein analog, peptide analog or inorganic small molecule compounds.

5. A computer-aided screening device based on miR-320a and RAD51 to screen drugs that enhance radiosensitivity in non-small cell lung cancer, characterized in that, The device includes: A memory and a processor, wherein the memory is used to store program instructions; and the processor is used to invoke the program instructions, which, when executed, implement the computer-aided screening method for screening drugs based on miR-320a and RAD51 to enhance radiosensitivity in non-small cell lung cancer as described in claim 1 or 2.

6. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores a computer program that, when executed by a processor, implements the computer-aided screening method for screening drugs based on miR-320a and RAD51 to enhance radiosensitivity in non-small cell lung cancer, as described in claim 1 or 2.

7. A screening method for drugs that promote radiosensitivity in non-small cell lung cancer based on miR-320a and RAD51 according to claim 1 or 2, wherein the drugs that promote radiosensitivity in non-small cell lung cancer are obtained by screening.

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