A nucleic acid aptamer targeting IGF2BP2 protein and its application

Abstract

This invention provides a nucleic acid aptamer that targets and binds to the IGF2BP2 protein and its application, belonging to the field of pharmaceutical technology. This invention is the first to design a highly specific nucleic acid aptamer targeting the RNA recognition protein IGF2BP2, obtaining a nucleic acid aptamer that can target and specifically bind to the IGF2BP2 protein (see SEQ ID No. 1-2). This nucleic acid aptamer can effectively inhibit the activity of tumor cells, tumor stem cells, and breast cancer brain metastases, breast cancer lung metastases, breast cancer liver metastases, breast cancer kidney metastases, and breast cancer bone metastases, providing a new drug component for the treatment of malignant and refractory tumors (especially glioblastoma and distant metastatic breast cancer). Furthermore, it was discovered that modification with N6-methyldeoxyadenosine monophosphate can further enhance the specificity of the nucleic acid aptamer and strengthen its efficacy in inhibiting tumor cell activity.

Description

Technical Field

[0001] This invention relates to a nucleic acid aptamer that targets and binds to the IGF2BP2 protein and its application, belonging to the field of pharmaceutical technology. Background Technology

[0002] Cancer, the second leading cause of death worldwide, has become a major public health problem posing a serious threat to human life and health. Currently, the prevention, treatment, and control of recurrence and metastasis of malignant tumors in clinical practice mainly rely on comprehensive treatment plans, specifically including five major categories: surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Surgical treatment reduces the risk of local recurrence by precisely removing tumor tissue and marginal cancer cells, but it is difficult to address potential micrometastases. Radiotherapy uses high-energy rays to kill residual cancer cells and is mostly used for controlling local tumors, with limited effectiveness against systemic metastases. Chemotherapy, based on cytotoxic drugs, suffers from low clinical efficacy, strong toxic side effects, and is prone to damaging normal tissues. Targeted therapy targets specific molecular targets of tumor cells, using small molecule drugs or monoclonal antibodies for precise intervention, effectively inhibiting tumor growth and spread, but problems such as drug resistance and limited target coverage limit its long-term efficacy. Immunotherapy, by activating the body's own immune system to recognize and eliminate tumor cells, provides a new direction for cancer treatment, but it also has limitations such as low clinical efficacy and a tendency to trigger immune-related adverse reactions. In summary, the five existing treatment methods all revolve around inhibiting tumor growth and enhancing the body's anti-tumor immunity, but there is still considerable room for improvement in their treatment effects and patient prognosis, and they are unable to fundamentally solve the core problems of cancer recurrence, metastasis, and drug resistance.

[0003] Studies have confirmed that tumor stem cells are a small group of special cells in tumor tissues with self-renewal capacity and multi-lineage differentiation potential. They are the main source of tumor cell heterogeneity and the core culprits in maintaining tumor growth, causing recurrence and metastasis, and developing treatment resistance. Therefore, effectively eliminating tumor stem cells is a key breakthrough for achieving radical cancer cure. However, current routine clinical treatments (such as chemotherapy, radiotherapy, and targeted therapy) mainly target ordinary tumor cells and lack effective therapies that can specifically eliminate tumor stem cells. At the same time, existing preclinical studies often target key pathways necessary for the maintenance of normal stem cells, such as Notch and Wnt / β-catenin, which can easily damage normal tissue stem cells and inevitably produce serious side effects. Therefore, discovering new specific targets for tumor stem cells and developing highly effective and low-toxicity therapeutic drugs based on these targets has become an urgent and important issue in the field of cancer research.

[0004] Glioblastoma is the most malignant brain tumor. 70%–80% of patients have a disease course of 3–6 months, and only 10% have a course exceeding one year. Glioblastoma stem cells are a crucial factor leading to glioblastoma progression and recurrence. Glioblastoma stem cells are a cell group with self-renewal and multi-lineage differentiation potential, serving as the "seeds" for glioblastoma development, invasion, radiotherapy and chemotherapy resistance, recurrence, and drug resistance. Current methods for preventing and treating glioblastoma recurrence and metastasis rely on indirect methods such as inhibiting cancer cell growth or enhancing anti-tumor immunity, which have weak targeting and poor selectivity towards the stem cells that lead to tumor metastasis, recurrence, and drug resistance.

[0005] Breast cancer remains a common malignant tumor in women. Late-stage recurrence and metastasis primarily affect the bones, lungs, liver, and brain, with a clear molecular subtype preference. HR-positive subtypes are more prone to bone metastasis, while HER2-positive and triple-negative subtypes are more likely to metastasize to internal organs and the brain. The overall 5-year survival rate for distant metastatic breast cancer is only 10%-20%. Poor drug penetration in drug-resistant breast cancer and brain metastatic breast cancer, as well as the lack of specific targets in triple-negative breast cancer, remain core clinical challenges.

[0006] IGF2BP2, acting as a 6mA reader, is widely highly expressed in glioblastoma (GBM) and is a key driver of its invasion, metastasis, and treatment resistance. It promotes EMT, angiogenesis, autophagy regulation, and temozolomide (TMZ) resistance by recognizing 6mA-modified and stabilizing target mRNAs (such as VEGFA, HIF1A, MMP14, MGMT, and DANCR). It also globally regulates alternative splicing and immune-related pathways. Its high expression is significantly associated with advanced GBM, in situ invasion, and poor prognosis. At the level of tumor metastasis, IGF2BP2 stabilizes metastasis-related transcripts and activates PI3K in various solid tumors. Akt / MAPK / NF The IGF2BP2 pathway, remodeling of the metastatic microenvironment, and exosome-mediated signal transduction promote distant colonization and organ-specific metastasis (such as lung / bone metastasis in breast cancer). IGF2BP2 has become a popular drug target, but to date, no drugs targeting the IGF2BP2 protein have been approved globally, and no nucleic acid aptamer drugs have entered the research and development or clinical application stage. Related research is still in its infancy, providing a clear entry point and research value for this study. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a nucleic acid aptamer that targets and binds specifically to the IGF2BP2 protein and its applications. This invention is the first to design a highly specific nucleic acid aptamer targeting the RNA recognition protein IGF2BP2, obtaining a nucleic acid aptamer that can target and specifically bind to the IGF2BP2 protein. This nucleic acid aptamer can effectively inhibit the activity of tumor cells, tumor stem cells, and breast cancer brain metastases, breast cancer lung metastases, breast cancer liver metastases, breast cancer kidney metastases, and breast cancer bone metastases, providing a new drug component for the treatment of malignant and refractory tumors (especially glioblastoma and distant metastatic breast cancer). Furthermore, it was discovered that modification with N6-methyldeoxyadenosine monophosphate can further enhance the specificity of the nucleic acid aptamer and strengthen its efficacy in inhibiting tumor cell activity.

[0008] To address the aforementioned technical problems, the present invention aims to provide a nucleic acid aptamer that targets and binds to the IGF2BP2 protein. The nucleic acid aptamer targets the IGF2BP2 protein (Gene ID: 10644), and the sequence of the nucleic acid aptamer includes: the sequence shown in SEQ ID No. 1 or a nucleotide sequence that has at least 70% identity with the sequence in SEQ ID No. 1.

[0009] Further, the nucleic acid aptamer includes methylation modification, which includes N6-methyldeoxyadenosine (6mA) modification. Preferably, the N6-methyldeoxyadenosine (6mA) is the adenine (A) at position 15 from the 5′-3′ direction in the sequence shown in SEQ ID No. 1. Specifically, the adenine (A) at position 15 from the 5′-3′ direction in the sequence shown in SEQ ID No. 1 is modified with N6-methyldeoxyadenosine (6mA) to obtain the sequence shown in SEQ ID No. 2.

[0010] The present invention also discloses the use of the above-mentioned nucleic acid aptamer that targets and binds to IGF2BP2 protein in the preparation of drugs that have an inhibitory effect on malignant tumor cells.

[0011] Preferably, the malignant tumor is a tumor dependent on the IGF2BP2 protein, and the tumor is one or more of glioblastoma, lung cancer, liver cancer, and breast cancer.

[0012] Furthermore, the nucleic acid aptamer that targets and binds to the IGF2BP2 protein can enter tumor cells with the assistance of transfection reagents or biological carriers. The tumor cells involved include one or more of the following: human glioblastoma cells (LN229, U-87), human glioblastoma stem cells (GSC0722), human breast cancer cells (MDA-MB-231), human breast cancer stem cells (BCSC0208), human breast cancer brain metastases (MDA-MB-231-BM2), human breast cancer lung metastases (MDA-MB-231-LuM), human breast cancer liver metastases (MDA-MB-231-LiM), human breast cancer kidney metastases (MDA-MB-231-KiM), human breast cancer bone metastases (MDA-MB-231-BoM), human lung cancer cells (A549), human liver cancer cells (HepG2), mouse breast cancer brain metastases (4T1-BM2), mouse breast cancer lung metastases (4T1-LuM), mouse breast cancer liver metastases (4T1-LiM), mouse breast cancer kidney metastases (4T1-KiM), and mouse breast cancer bone metastases (4T1-BoM).

[0013] On the other hand, the present invention also provides a composition for treating malignant tumors (especially glioblastoma, distant metastatic breast cancer), the composition comprising the aforementioned nucleic acid aptamer that targets and binds to the IGF2BP2 protein and excipients.

[0014] The dosage form of the composition can be injection, powder, emulsion, or suspension, and can be prepared by general methods. The excipients can be one or more of the following: suitable solvents, cosolvents, solubilizers, propellants, emulsifiers, colorants, disintegrants, fillers, lubricants, binders, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, integrators, penetration enhancers, pH adjusters, buffers, release inhibitors, transfection reagents, sustained-release agents, and biological carriers (such as exosomes and liposomes).

[0015] Preferably, the concentration of the nucleic acid aptamer targeting and binding to the IGF2BP2 protein is 0.1 nM-1000 nM, more preferably 1 nM-500 nM; and more preferably 3 nM-200 nM.

[0016] Furthermore, the present invention also provides the use of the nucleic acid aptamer or the composition thereof in regulating the activity of IGF2BP2 protein.

[0017] On the other hand, the present invention also provides an IGF2BP2 protein inhibitor, wherein the IGF2BP2 protein inhibitor comprises the aforementioned nucleic acid aptamer.

[0018] This invention has the following beneficial effects: For the first time, this invention designs a highly specific nucleic acid aptamer targeting the RNA recognition protein IGF2BP2, obtaining a nucleic acid aptamer that can target and specifically bind to the IGF2BP2 protein. This nucleic acid aptamer can effectively inhibit tumor cells (LN229 cells, U-87 cells, A549 cells, HepG2 cells, MDA-MB-231 cells), tumor stem cells (GSC0722 cells, BCSC0208), and breast cancer brain metastases (MDA-MB-231-BM2, 4T1-BM2), and breast cancer lung metastases (MDA-MB-231-LuM, 4T1-LuM). The study investigated the activity of MDA-MB-231-LiM, 4T1-LiM, MDA-MB-231-KiM, 4T1-KiM, and MDA-MB-231-BoM, and 4T1-Li-BoM, providing a novel drug component for the treatment of malignant tumors (especially glioblastoma and distant metastatic breast cancer). Furthermore, it was discovered that N6-methyldeoxyadenosine modification further enhances the specificity of the nucleic acid aptamer and strengthens its efficacy in inhibiting tumor cell activity; thereby preventing tumor metastasis, recurrence, and drug resistance, offering a new approach to the treatment of malignant tumors. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of a nucleic acid aptamer. Figure 2 The results of pull-down experiments on IGF2BP2 nucleic acid aptamers and IGF2BP2 protein; Figure 3 The effect of 200 nM IGF2BP2 nucleic acid aptamer on the viability of LN229 cells was determined by CCK8 assay. Figure 4 To detect the effect of 200 nM IGF2BP2 nucleic acid aptamer on the colony-forming ability of LN229 cells in a plate colony assay; Left: Representative plot; Right: Experimental statistical plot; Figure 5 To investigate the effect of 200 nM IGF2BP2 aptamer on the infiltration ability of LN229 cells using Transwell assays; Left: Representative plot; Right: Experimental statistical plot; Scale bar in the plot represents 50 μm; Figure 6 The effect of 70 nM IGF2BP2 nucleic acid aptamer on the cell viability of various tumor cells was detected by CCK8 assay. Figure 7The effect of 70 nM IGF2BP2 nucleic acid aptamer on the cell viability of stem cells GSC0722 (left) and BCSC0208 (right) was determined by CCK8 assay. Figure 8 The effect of 70 nM IGF2BP2 nucleic acid aptamer on tumor spheroid formation in glioma stem cells GSC0722 and breast cancer stem cells BCSC0208; the scale bar in the figure represents 50 μm. Figure 9 The effects of 70 nM IGF2BP2 aptamer on the cell viability of various breast cancer metastatic cell lines were determined using the CCK8 assay: human breast cancer brain metastases (MDA-MB-231-BM2), human breast cancer lung metastases (MDA-MB-231-LuM), human breast cancer liver metastases (MDA-MB-231-LiM), human breast cancer kidney metastases (MDA-MB-231-KiM), human breast cancer bone metastases (MDA-MB-231-BoM), and mouse breast cancer brain metastases (4T1-BM2), mouse breast cancer lung metastases (4T1-LuM), mouse breast cancer liver metastases (4T1-LiM), mouse breast cancer kidney metastases (4T1-KiM), and mouse breast cancer bone metastases (4T1-BoM). Detailed Implementation

[0020] To more clearly illustrate the overall concept of the present invention, detailed descriptions of embodiments are provided below with reference to the accompanying drawings. It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise specified, reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are all commercially available conventional products. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed.

[0021] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of molecular biology, biochemistry, analytical chemistry, cell culture, recombinant DNA technology, medicine, pharmacy, and related areas. Specifically, they can be performed according to *Molecular Cloning: A Laboratory Manual (Fourth Edition)*. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. The main reagents and consumables used in the examples are shown in Table 1.

[0022] Table 1. Main reagents and consumables used in the examples

[0023] The cell lines involved in the examples are as follows: human glioblastoma cells LN229 were purchased from the American Type Culture Collection (ATCC). Human hepatocellular carcinoma cells HepG2, human breast cancer cells MDA-MB-231, human non-small cell lung cancer cells A549, and human glioblastoma cells U-87 were purchased from the China Center for Type Culture Collection (CCTCC). Glioblastoma stem cells GSC0722 were first isolated from glioblastoma tissue by Professor Fang Runping of Shandong University and published in an article by Professor Ju Jianhua et al. (Org Lett. 2025 Jan 10;27(1):476-481. doi: 10.1021 / acs.orglett.4c04491.). Breast cancer stem cells BCSC0208 were obtained through in vitro screening and enrichment using a tumor sphere assay. Human breast cancer brain metastases (MDA-MB-231-BM2), human breast cancer lung metastases (MDA-MB-231-LuM), human breast cancer liver metastases (MDA-MB-231-LiM), human breast cancer kidney metastases (MDA-MB-231-KiM), and human breast cancer bone metastases (MDA-MB-231-BoM) were obtained from primary cells isolated and cultured after distant metastasis in a constructed nude mouse orthotopic breast cancer model. Mouse breast cancer brain metastases (4T1-BM2), mouse breast cancer lung metastases (4T1-LuM), mouse breast cancer liver metastases (4T1-LiM), mouse breast cancer kidney metastases (4T1-KiM), and mouse breast cancer bone metastases (4T1-BoM) were obtained from primary cells isolated and cultured after distant metastasis in a constructed BALB / c mouse orthotopic breast cancer model.

[0024] The method for preparing the culture medium required in this embodiment of the invention is as follows: EGF (epidermal growth factor) and bFGF (basic fibroblast growth factor) solutions: Dissolve 1 mg of recombinant human bFGF and 1 mg of recombinant human EGF in 10 mL of sterile PBS, respectively, and dispense 100 μL / tube and freeze at -80°C.

[0025] DMEM complete medium: DMEM basic (1×) high glucose medium supplemented with 10% FBS, 1% penicillin and streptomycin and 1% non-essential amino acids is used as the culture medium for adherent cells such as LN229, U-87, HepG2, MDA-MB-231, and A549.

[0026] DMEM / F12 complete medium: DMEM / F12 medium was supplemented with B-27 additive (50×), 20 ng / mL human bFGF and 20 ng / mL EGF to serve as a suspension cell culture medium for GSC0722 and BCSC0208.

[0027] Example 1: Binding of IGF2BP2 aptamer to IGF2BP2 protein In this embodiment, the IGF2BP2 nucleic acid aptamer was designed. The specific DNA nucleic acid aptamer sequence is shown in Table 2 and... Figure 1 As shown, it was synthesized by Suzhou Genewiz Biotechnology Co., Ltd.

[0028] Table 2. Nucleic acid aptamer sequences targeting IGF2BP2

[0029] This embodiment uses pull-down experiments to detect the binding ability of a series of doses (0.3 pmol, 0.9 pmol, 2.7 pmol) of the IGF2BP2 aptamer (BP2aptDNA-1) and a series of doses (0.3 pmol, 0.9 pmol, 2.7 pmol) of the 6 mA-modified IGF2BP2 aptamer (BP2aptDNA-1-6 mA) to the RNA recognition protein IGF2BP2. The method is as follows (taking BP2aptDNA-1 as an example): (1) Denaturation and renaturation of IGF2BP2 aptamer: The above nucleic acid aptamers were dissolved in nuclease-free water (final concentration 100 μM) and placed in a PCR instrument for denaturation and annealing using the procedure in Table 3.

[0030] Table 3: PCR Procedure

[0031] The denatured and renatured IGF2BP2 aptamers were aliquoted and stored at 4°C for long-term preservation.

[0032] (2) Protein binding to nucleic acid aptamer: The purified recombinant protein (IGF2BP2, Active Motif 81990) was thawed on ice and diluted with binding buffer (25 mM Tris-HCl pH7.4, 150 mM NaCl, 5% glycerol, 0.1% Triton X-100) to a concentration of 5 pmol. After mixing, an equal amount of protein was placed in an EP tube and different doses (0.3 pmol, 0.9 pmol, 2.7 pmol) of IGF2BP2 nucleic acid aptamer with biotin modification at the 5′ end (BP2aptDNA-1) were added. The tubes were placed in a rotary mixer at 4°C and incubated at 15 rpm for 3 h. (3) Streptavidin magnetic beads pull-down: After washing the streptavidin magnetic beads 3 times with binding buffer, add 10 μL of streptavidin magnetic beads to each EP tube in (2), place them in a disc rotary mixer at 4℃, and incubate at 15 rpm for 1 h. (4) Protein elution: Use a handheld centrifuge to briefly centrifuge the EP tubes, place them on a magnetic rack, and discard the supernatant after the magnetic beads are collected. Wash the magnetic beads 6 times with binding buffer. Add 40 μL of binding buffer and 10 μL of 5× loading buffer (Invitrogen, AM8546G) to each tube and boil at 100℃ for 10 min. (5) Western Blot detection of target protein: Protein samples were separated by SDS-PAGE gel electrophoresis. Proteins on the gel were transferred to a PVDF membrane by constant current transfer at 200 mA for 2.5 h. The PVDF membrane was blocked by incubation with 5% BSA (bovine serum albumin) at room temperature for 1 h. IGF2BP2 specific primary antibody (Wuhan Sanying Biotechnology Co., Ltd., 11601-1, 1:1000) was incubated with the PVDF membrane overnight on a shaker at 4°C. Unbound primary antibody was washed away with TBST. Horseradish peroxidase (HRP) labeled secondary antibody (Beyotime, A0208, 1:1000) was incubated with the PVDF membrane on a shaker at room temperature for 1 h. Unbound secondary antibody was washed away with TBST. Chemiluminescent substrate ECL was added for development.

[0033] In this embodiment, the above-described pull-down assay was used to detect the binding affinity of a series of doses of the nucleic acid aptamer BP2aptDNA-1 and the 6mA-modified nucleic acid aptamer BP2aptDNA-1-6mA to the RNA recognition protein IGF2BP2. The results are as follows: Figure 2As shown in the figure, the results demonstrate that this embodiment is the first to discover that the above-mentioned DNA nucleic acid aptamer can specifically bind to the IGF2BP2 protein, and that the binding ability of the aptamer to IGF2BP2 can be further improved after 6mA modification.

[0034] The results above indicate that the nucleic acid aptamers BP2aptDNA-1 and BP2aptDNA-1-6mA have a strong IGF2BP2 binding ability.

[0035] Example 2: Inhibitory effect of IGF2BP2 aptamer on human glioblastoma LN229 cells In this embodiment, the inhibitory effect of 200 nM IGF2BP2 nucleic acid aptamer on the function of human glioblastoma LN229 cells was determined by CCK-8 (Cell Counting Kit-8), plate colony formation, and transwell infiltration assays.

[0036] 1. CCK-8 Experiment The principle of CCK-8 assay for cell viability is mainly based on the relationship between cell metabolic activity and cell number. Cell viability is assessed by measuring the cell's ability to reduce the CCK-8 reagent. This example investigated the inhibitory effect of 200nMIGF2BP2 nucleic acid aptamers (BP2aptDNA-1, BP2aptDNA-1-6mA) on the viability of human glioblastoma LN229 cells. The method is as follows: (1) Cell seeding: Collect human glioblastoma LN229 cells with a confluence of 60%-80% (approximately 4 × 10⁶ cells in a 10cm culture dish). 6 (Number of cells) were passaged, the old culture medium was discarded, and 2 mL of PBS was added to wash away any remaining old culture medium. 1 mL of 0.25% trypsin was added, and after the cells became rounded, they were discarded. 1 mL of fresh DMEM complete culture medium was added to peel the cells from the bottom of the dish. The cells were mixed well, and 20 μL of the cell suspension was mixed with an equal volume of 0.4% trypan blue. 10 μL of the mixture was added to a cell counter for counting. The cells were diluted to 20,000 cells / well and evenly added to a 24-well plate. The plate was then returned to the incubator for further culture.

[0037] (2) Denaturation and renaturation of IGF2BP2 aptamer: Place the nucleic acid aptamers in a PCR instrument and denature and renature them using the procedure in Table 3; then store the denatured and renatured IGF2BP2 nucleic acid aptamers at 4°C for long-term storage.

[0038] (3) Transfection: After culturing the inoculated cells for 12-16 hours, aptamer transfection was performed. The IGF2BP2 aptamer was introduced into tumor cells using a transfection method. Transfection was performed using the jetPRIME® DNA / siRNA transfection reagent, as detailed in Table 4.

[0039] Table 4 Composition of the Nucleic Acid Aptamer Transfection System

[0040] The IGF2BP2 aptamer stock solution concentration is 100 μM. Take a 1.5 mL sterile EP tube and add jetPRIME according to the volumes shown in Table 4 above. ® Add IGF2BP2 nucleic acid aptamer to buffer, mix well by pipetting, and add jetPRIME. ® Reagent was mixed by pipetting and incubated at room temperature for 15 min. The mixture was then transferred dropwise to a 24-plate and replaced with fresh DMEM complete medium after 4 h.

[0041] (4) Cell plating: 24 h after transfection, the old culture medium was aspirated and 500 μL of PBS was added to wash away any residual old culture medium. 500 μL of 0.25% trypsin was added, and once the cells became rounded, the old culture medium was aspirated and 1 mL of fresh DMEM complete culture medium was added to detach the cells from the bottom of the dish. After mixing the cells, 100 μL of cell suspension (approximately 1 × 10⁶ cells / mL) was taken from each cell suspension. 3 (1 cell) was transferred to a 96-well plate.

[0042] (5) CCK-8 assay for tumor cell viability: 10 μL of CCK-8 reagent was added to the corresponding wells of a 96-well plate at 0, 1, 2, 3 and 5 days after transfection. After incubation at 37℃ for 3.5 h, the absorbance at 450 nm and 600 nm (reference) wavelengths was measured using a microplate reader.

[0043] (6) Data Analysis: The inhibitory effect of 200 nM IGF2BP2 aptamer on LN229 tumor cells is as follows: Figure 3 As shown, when an equal amount of transfection reagent without nucleic acid aptamers was used as a blank control, IGF2BP2 nucleic acid aptamers at 200 nM significantly inhibited the cell viability of LN229 tumor cells, and the inhibitory effect became more pronounced over time.

[0044] 2. Plate cloning experiment The plate colony formation assay is a classic experimental method for detecting cell proliferation capacity and assessing cell colony formation potential in vitro, and is widely used in tumor cell biology research. This example examines the inhibitory effects of 200 nMIGF2BP2 nucleic acid aptamers (BP2aptDNA-1 and BP2aptDNA-1-6mA) on the proliferation and colony formation capacity of human glioblastoma LN229 cells, as follows: (1) Experimental preparation Select glioma LN229 cells in the logarithmic growth phase after transfection (transfection method as above), prepare DMEM complete medium in advance (containing 10% fetal bovine serum, 1% penicillin and antibiotic, 1% non-essential amino acids), 4% paraformaldehyde fixative, and 0.25% crystal violet staining solution, and prepare 6-well cell culture plates, trypsin, centrifuge tubes, pipettes and other experimental equipment. Adjust the incubator to 37℃, 5% CO2 and saturated humidity in advance. All reagents and equipment are autoclaved before use.

[0045] (2) Cell treatment and inoculation First, cells in the logarithmic growth phase were digested with 0.25% trypsin. Digestion was terminated by adding an appropriate amount of DMEM complete medium. The cell suspension was gently pipetted to ensure even cell dispersion and avoid cell clumps. Cells were then accurately counted using a cell counting chamber and seeded into 6-well plates at a density of 1 × 10⁶ cells per well. 3 Each group of cells was divided into three replicates to reduce experimental error. After inoculation, the culture plate was gently shaken to ensure that the cells were evenly distributed at the bottom of the wells.

[0046] (3) Cell culture Place the seeded 6-well plate into a 37°C incubator for static culture. Change the DMEM complete medium every 3 days during the culture period, being gentle to avoid blowing up the adherent cells. Observe the cell growth status at the same time and check for contamination in a timely manner. Continue culturing for 14 days, or terminate the culture when most individual clones contain more than 50 cells and the clone diameter reaches 0.3-1.0 mm.

[0047] (4) Fixation and staining After culture is complete, discard the culture medium from each well and gently rinse the cells 2-3 times with PBS buffer to remove residual culture medium and dead cells. Then, add 1 mL of 4% paraformaldehyde fixative to each well and fix for 20 min at room temperature. After fixation, rinse again 2-3 times with PBS buffer to remove the fixative. After rinsing, add 1 mL of 0.25% crystal violet staining solution to each well and stain for 30 min at room temperature. After staining, rinse slowly with distilled water until the background color is removed and the clones are clearly stained. Allow the culture plate to air dry.

[0048] (5) Counting and Data Analysis Microscopic observation and photography were performed on the colonies in the entire 6-well plate and individual wells. The number of colonies with ≥50 cells per well was counted, and the colony formation rate was calculated as (colony formation rate (%) = (number of colonies formed / number of seeded cells) × 100%. All experimental data were analyzed using SPSS statistical software. Quantitative data are expressed as mean ± standard deviation. One-way ANOVA was used for overall differences among multiple groups, and the SNK-q test was used for further pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant. The statistical differences in cell colony formation rates among groups were used to evaluate the regulatory effect of nucleic acid aptamers on the proliferation and in vitro colony formation ability of LN229 glioma cells.

[0049] The results of the inhibitory effect of 200 nM IGF2BP2 aptamer on clonogenesis of LN229 tumor cells are as follows: Figure 4 As shown, when an equal amount of transfection reagent without nucleic acid aptamers was used as a blank control, IGF2BP2 nucleic acid aptamers (BP2aptDNA-1 and BP2aptDNA-1-6mA) at 200 nM significantly inhibited the formation of LN229 tumor cell clones.

[0050] 3. Transwell testing The Transwell infiltration assay utilizes chambers with microporous polycarbonate membranes to construct a two-layer culture system. Its purpose is to detect cell migration and invasion capabilities in vitro: chemokine induction and observation of cell passage through the microporous membrane reflect cell migration ability; coating the membrane with matrix gel to simulate the extracellular matrix further allows for the detection of cell degradation of the matrix and invasion through barriers. This assay is commonly used to assess the metastatic and invasive potential of tumor cells, explore the regulatory effects of genes or drugs on cell motility, and is a frequently used functional assay for in vitro drug screening.

[0051] This embodiment investigated the effects of 200nMIGF2BP2 nucleic acid aptamers (BP2aptDNA-1 and BP2aptDNA-1-6mA) on the invasive ability of human glioblastoma LN229 cells. The methods are as follows: (1) Experimental preparation Prepare in advance the following: Transwell chambers for 24-well plates, Matrigel (for invasion experiments only), serum-free DMEM medium (DMEM high-glucose medium supplemented with 1% antibiotics and 1% non-essential amino acids), complete DMEM medium (containing 10% FBS, 1% antibiotics and 1% non-essential amino acids), 0.25% trypsin, PBS buffer, 4% paraformaldehyde fixative, and 0.1% crystal violet staining solution. Select cells in the logarithmic growth phase after transfection with a confluence of 70%-80%. Perform serum-free starvation treatment on the cells for 12-24 hours before the experiment to ensure the chemotactic activity of the cells during the experiment.

[0052] (2) Transwell chamber pretreatment Thaw the Matrigel gel overnight at 4°C, then dilute it 1:4 with DMEM serum-free medium on ice. Add 100 μL of the diluted gel to the upper chamber and incubate at 37°C for 2 h until solidified. Add 200 μL of DMEM serum-free medium to hydrate for 30 min, then aspirate the liquid.

[0053] (3) Cell inoculation Cells were digested with 0.25% trypsin, centrifuged, resuspended in DMEM serum-free medium, and counted at a density of 1×10⁻⁶. 6 cells / mL.

[0054] Lower chamber: Add 600-800 μL of DMEM complete medium to each well for chemotaxis induction.

[0055] Upper chamber: Add 200 μL of cell suspension (approximately 1.5 × 10⁻⁶ cells / mL). 5 (cells), avoid bubbles.

[0056] Incubate at 37℃ and 5% CO2 for 24-48 h.

[0057] (4) Fixation and staining Remove the chamber and wash gently with PBS 1-2 times. Gently wipe away unmigrated cells from the upper chamber with a damp cotton swab (wipe away Matrigel cells during invasion experiments). Fix with 4% paraformaldehyde at room temperature for 20 min. Wash twice with PBS, then stain with 0.1% crystal violet for 20 min. Wash away any excess stain with PBS and air dry.

[0058] (5) Observation and quantification Take photos of three random fields of view under a microscope and count the number of cells that have penetrated the membrane.

[0059] The results of the inhibitory effect of 200 nM IGF2BP2 aptamer on the invasion of LN229 tumor cells are as follows: Figure 5As shown, when an equal amount of transfection reagent without nucleic acid aptamers was used as a blank control, IGF2BP2 nucleic acid aptamers (BP2aptDNA-1 and BP2aptDNA-1-6mA) at 200 nM both inhibited the invasive ability of LN229 tumor cells.

[0060] Example 3: Selective inhibitory effect of low concentration (70 nM) IGF2BP2 aptamer on human glioblastoma stem cells GSC0722 and breast cancer stem cells BCSC0208 Furthermore, we used the CCK8 assay to detect the effects of low concentrations (70 nM) of the IGF2BP2 aptamer BP2aptDNA-1-6mA on the viability of human malignant tumor cells, including human glioblastoma cells (LN229, U-87), human lung cancer cells (A549), human liver cancer cells (HepG2), human breast cancer cells (MDA-MB-231), as well as human glioblastoma stem cells (GSC0722) and breast cancer stem cells (BCSC0208). The experimental method is the same as the CCK8 assay described in Example 2, and DMEM / F12 complete medium was used as the culture medium for GSC0722 and BCSC0208 suspension cells.

[0061] The results are as follows Figure 6 As shown, when the transfection reagent without nucleic acid aptamers was used as a blank control, 48 hours after transfection, the low concentration (70 nM) of IGF2BP2 nucleic acid aptamer BP2aptDNA-1-6mA showed weak inhibitory activity against the viability of various tumor cells, but the inhibitory activity against tumor stem cells GSC0722 and BCSC0208 was the most significant. This indicates that IGF2BP2 nucleic acid aptamer BP2aptDNA-1-6mA has a strong selective inhibitory effect on tumor stem cells, and after modification with 6mA, it can exert a stronger inhibitory effect at a lower concentration.

[0062] Given that IGF2BP2 plays a central role in maintaining the function of tumor stem cells, the fact that BP2aptDNA-1-6mA can significantly inhibit tumor stem cells at low concentrations is presumably closely related to its strong binding affinity to IGF2BP2. Furthermore, the CCK8 assay was used to further verify the inhibitory effect of low concentrations (70 nM) of IGF2BP2 aptamers BP2aptDNA-1 and BP2aptDNA-1-6mA on the viability of human glioblastoma stem cells (GSC0722) and breast cancer stem cells (BCSC0208). The experimental method follows the CCK8 assay method described in Example 2, and DMEM-F12 complete medium was used as the culture medium for GSC0722 and BCSC0208 suspension cells.

[0063] The results are as follows Figure 7As shown, when the transfection reagent without nucleic acid aptamers served as a blank control, the low concentration (70 nM) of IGF2BP2 nucleic acid aptamer significantly inhibited the viability of GSC0722 cells (left image) and BCSC0208 cells (right image). Furthermore, tumor microsphere experiments also confirmed the inhibitory effect of 70 nM IGF2BP2 nucleic acid aptamers BP2aptDNA-1 and BP2aptDNA-1-6mA on the proliferation of human glioblastoma stem cells (GSC0722) and breast cancer stem cells (BCSC0208). Figure 8 ).

[0064] Example 4: Inhibitory effect of low concentration (70 nM) IGF2BP2 aptamer on distant metastatic breast cancer cells Furthermore, we used the CCK8 assay to detect the cell viability of low concentration (70 nM) IGF2BP2 aptamer BP2aptDNA-1-6mA on human breast cancer brain metastases (MDA-MB-231-BM2), human breast cancer lung metastases (MDA-MB-231-LuM), human breast cancer liver metastases (MDA-MB-231-LiM), human breast cancer kidney metastases (MDA-MB-231-KiM), human breast cancer bone metastases (MDA-MB-231-BoM), and mouse breast cancer brain metastases (4T1-BM2), mouse breast cancer lung metastases (4T1-LuM), mouse breast cancer liver metastases (4T1-LiM), mouse breast cancer kidney metastases (4T1-KiM), and mouse breast cancer bone metastases (4T1-BoM).

[0065] The results are as follows Figure 9 As shown, when using a transfection reagent without the nucleic acid aptamer as a blank control, 48 hours after transfection, the low concentration (70 nM) of the IGF2BP2 nucleic acid aptamer BP2aptDNA-1-6mA significantly inhibited the aforementioned distant metastatic breast cancer cells, indicating that the IGF2BP2 nucleic acid aptamer BP2aptDNA-1-6mA has a strong inhibitory effect on distant metastatic breast cancer cells. This study suggests that IGF2BP2 expression is upregulated in distant metastatic breast cancer cells, and the strong inhibitory effect of low concentration BP2aptDNA-1-6mA on distant metastatic breast cancer cells may be due to its strong binding affinity to the upregulated IGF2BP2 expression in the cells.

[0066] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included in the present invention.

Claims

1. A nucleic acid aptamer that targets and binds to the IGF2BP2 protein, characterized in that, The sequence of the nucleic acid aptamer includes: the sequence shown in SEQ ID No. 1 or a nucleotide sequence that has at least 70% identity with the sequence in SEQ ID No.

1.

2. The nucleic acid aptamer as described in claim 1, characterized in that, The nucleic acid aptamer includes a methylated sequence.

3. The nucleic acid aptamer as described in claim 2, characterized in that, The methylation modification includes N6-methyldeoxyadenosine modification.

4. The nucleic acid aptamer as described in claim 3, characterized in that, The adenine at position 15 of the sequence shown in SEQ ID No. 1, counted from 5′ to 3′, was modified with N6-methyldeoxyadenosine to obtain the sequence shown in SEQ ID No.

2.

5. Use of the nucleic acid aptamer according to any one of claims 1-4 in the preparation of a drug having an inhibitory effect on malignant tumor cells; wherein the malignant tumor cells are tumor cells dependent on IGF2BP2 protein.

6. The use as described in claim 5, characterized in that, The nucleic acid aptamer that targets and binds to the IGF2BP2 protein enters tumor cells with the assistance of a transfection reagent or a biological carrier; the tumor cells involved are one or more of the following: glioblastoma cells, glioblastoma stem cells, human lung cancer cells, human liver cancer cells, human breast cancer cells, human breast cancer stem cells, human breast cancer brain metastases, human breast cancer lung metastases, human breast cancer liver metastases, human breast cancer kidney metastases, human breast cancer bone metastases, mouse breast cancer brain metastases, mouse breast cancer lung metastases, mouse breast cancer liver metastases, mouse breast cancer kidney metastases, and mouse breast cancer bone metastases.

7. A composition for treating malignant tumors, the composition comprising a nucleic acid aptamer and excipients that target and bind to the IGF2BP2 protein as described in any one of claims 1-4.

8. The composition of claim 7, characterized in that, The dosage form of the composition is any one of injection, powder, emulsion, suspension, and sustained-release formulation.

9. The use of the nucleic acid aptamer according to any one of claims 1-4 in regulating the activity of IGF2BP2 protein.

10. An IGF2BP2 protein inhibitor, wherein the IGF2BP2 protein inhibitor comprises the nucleic acid aptamer according to any one of claims 1-4.

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