Adaptor targeting sting agonist and application thereof in reversing colorectal cancer 5-fu drug resistance

Abstract

The application discloses an aptamer targeted STING agonist and application thereof in reversing 5-FU drug resistance of colorectal cancer, and belongs to the technical field of biological medicine. The aptamer targeted STING agonist "Apt-dsDNA" provided by the application can target PTK7 protein on the surface of colorectal cancer cells, and has high tumor enrichment degree. In a 5-FU drug resistant colorectal cancer cell line, treatment makes IC50 decrease from 1356.0 muM to 418.8 muM, and the sensitivity of the colorectal cancer cells to 5-fluorouracil is increased by 3.2 times. In a HCT8R / 5-FU nude mouse subcutaneous transplantation model, the tumor inhibition rate of the "Apt-dsDNA" combined with 5-FU is greater than or equal to 80%, and the "Apt-dsDNA" has good in-vivo safety.

Description

Technical Field

[0001] This invention relates to an aptamer-targeted STING agonist and its application in reversing 5-FU resistance in colorectal cancer, belonging to the field of biomedical technology, specifically involving tumor immunotherapy, nucleic acid drug delivery, and tumor chemotherapy sensitization. Background Technology

[0002] The cGAS-STING signaling pathway is a core component of the innate immune system, playing a crucial role in anti-tumor immune responses. STING (Stimulator of Interferon Genes), as the pivotal molecule in this pathway, can induce the production of type I interferon and pro-inflammatory cytokines, activate dendritic cells, and promote T cell infiltration, thereby enhancing anti-tumor immune effects. In recent years, STING agonists have become a research hotspot in tumor immunotherapy, showing promising therapeutic prospects in various solid tumors such as colorectal cancer, melanoma, and lymphoma.

[0003] Currently, commonly used STING agonists in clinical research mainly include cyclic dinucleotides (such as cGAMP, c-di-GMP, c-di-AMP) and their derivatives, and small molecule compounds (such as DMXAA, ADU-S100, MK-1454). These agonists are mostly administered systemically and lack tumor targeting, leading to the following problems: Systemic toxicity: Non-targeted administration activates systemic immune cells, triggering serious adverse reactions such as cytokine storms and hepatotoxicity, limiting the dosage; Low tumor enrichment: The drug concentration in the tumor microenvironment is insufficient, making it difficult to reach the effective activation threshold; Off-target effects: Activation of the STING pathway in normal tissues, leading to autoimmune-like reactions.

[0004] In addition, 5-fluorouracil (5-FU) is a cornerstone drug for chemotherapy in colorectal cancer (CRC), but about 50% of metastatic CRC patients have primary or acquired resistance. The mechanisms of 5-FU resistance are complex and diverse, mainly including: (1) Abnormal metabolic pathways: overexpression or gene amplification of thymidine synthase (TS), high expression of dihydropyrimidine dehydrogenase (DPD) leading to rapid degradation of 5-FU and reduced FdUMP synthesis; (2) Enhanced DNA damage repair: MMR defects (such as MLH1 promoter hypermethylation) lead to the inability to recognize 5-FU-induced DNA mismatches, resulting in cell escape and apoptosis; (3) Cell apoptosis resistance: p53 mutation, Bcl-2 upregulation, Bax downregulation, and GSK3β-mediated apoptosis inhibition; (4) Immunosuppression of the tumor microenvironment: 5-FU treatment upregulates PD-L1 expression, inducing immune escape; CAF secretory factors activate the JAK / STAT and PI3K / AKT pathways to protect cancer cells; (5) Multidrug resistance (MDR): overexpression of drug efflux pumps such as ABCC5, ABCC10, and ABCB1 reduces the effective concentration of 5-FU in cells.

[0005] Currently, the main strategies for overcoming 5-FU resistance include: (1) combination of TS inhibitors: such as pemetrexed and raltitrexed, but cross-resistance exists; (2) DPD inhibitors: such as Gimeracil, which only partially improve metabolic disorders; (3) immune checkpoint inhibitors: such as anti-PD-1 / PD-L1 antibodies, which are only effective against MSI-H type CRC (accounting for about 15%); (4) epigenetic regulators: such as 5-azacytidine, which have limited clinical efficacy and high toxicity. However, none of the above strategies have systematically solved the problem of 5-FU resistance, and there is a lack of specific targeted delivery systems for colorectal cancer.

[0006] Nucleic acid aptamers are a class of single-stranded oligonucleotide molecules obtained through SELEX technology that can specifically recognize target molecules. However, existing aptamer drug delivery systems have the following shortcomings: limited aptamer selection: most tumor-targeting aptamers target cell membrane surface receptors (such as EGFR, HER2), but there is insufficient screening of aptamers for colorectal cancer-specific markers; single drug loading method: most are physical loading or simple coupling, resulting in poor controllability of drug release; (3) low functional integration: the targeting module and the pharmacodynamic module function independently, lacking synergistic design. Summary of the Invention

[0007] To address the shortcomings of the prior art, this invention provides an aptamer-targeted modified STING agonist and its application in reversing 5-FU resistance in colorectal cancer. The aim is to achieve the following through aptamer-mediated active targeted delivery: (1) specific recognition and drug enrichment of colorectal cancer cells; (2) efficient local activation of the STING pathway in tumors; (3) reversal of 5-FU resistance in colorectal cancer and restoration of chemosensitivity; and (4) reduction of systemic toxicity and improvement of the therapeutic window.

[0008] The first technical solution provided by the present invention is a double-stranded DNA, wherein the double-stranded DNA is a DNA double-stranded structure composed of a tumor-targeting aptamer and a STING agonist, and the targeting marker of the tumor-targeting aptamer is the PTK7 protein on the surface of tumor cells.

[0009] In some embodiments, the double-stranded DNA is formed by annealing and hybridizing single-stranded DNA as shown in SEQ ID NO. 1~2, respectively.

[0010] The second technical solution provided by the present invention is a composition containing the double-stranded DNA described in the first technical solution.

[0011] The third technical solution provided by the present invention is a combination drug, which includes the double-stranded DNA and 5-fluorouracil described in the first technical solution.

[0012] The fourth technical solution provided by the present invention is a 5-FU resistance sensitizer for colorectal cancer, wherein the 5-FU resistance sensitizer for colorectal cancer contains the double-stranded DNA described in the first technical solution, or the composition described in the second technical solution, or the combination drug described in the third technical solution.

[0013] The fifth technical solution provided by the present invention is the use of the double-stranded DNA described in the first technical solution, or the composition described in the second technical solution, or the combination drug described in the third technical solution in the preparation of a drug for alleviating, reducing and / or treating colorectal cancer.

[0014] In some embodiments, the pharmaceutical product contains a pharmaceutically acceptable carrier or excipient.

[0015] In some embodiments, the pharmaceutically acceptable carrier includes liposomes, microcells, metal particles, or polymer particles.

[0016] In some implementations, the application includes at least one of the following functions: (1) Inhibits the proliferation and migration of colorectal cancer cells; (2) Enhances the sensitivity of colorectal cancer cells to 5-fluorouracil; (3) Inhibit the growth of individual colorectal tumors.

[0017] The sixth technical solution provided by the present invention is the use of the double-stranded DNA described in the first technical solution, or the composition described in the second technical solution, in the preparation of a medicine for alleviating, reducing and / or reversing 5-fluorouracil resistance in colorectal cancer.

[0018] In some embodiments, the pharmaceutical product contains a pharmaceutically acceptable carrier or excipient.

[0019] In some embodiments, the pharmaceutically acceptable carrier includes liposomes, microcells, metal particles, or polymer particles.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Targeting colorectal cancer cells Targeting the PTK7 protein on the surface of colorectal cancer cells, resulting in high tumor enrichment. 2. In vitro chemotherapy sensitization effect In the 5-FU-resistant colorectal cancer cell line (HCT8 / 5-FU-R), "Apt-dsDNA" treatment resulted in increased intravascular coagulation (ICP-C). 50 The value decreased from 1356.0 to 331.6, increasing the sensitivity of rectal cancer cells to 5-fluorouracil by 3.2 times.

[0021] 3. In vivo anti-tumor effect In the HCT8R / 5-FU nude mouse subcutaneous transplantation model, the tumor inhibition rate of the "Apt-dsDNA" combined with 5-FU group was ≥80%. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the structure of the present invention.

[0023] Figure 2 The figure shows the results of an acrylamide gel electrophoresis experiment to verify the construction of this invention.

[0024] Figure 3 The figure shows the results of the anti-nuclease stability study of the present invention.

[0025] Figure 4 This is a flow cytometry result of the uptake of drug-resistant colorectal cancer cells and endothelial cells according to the present invention.

[0026] Figure 5 This is a diagram showing the results of cell uptake observed in the immunofluorescence confocal assay of this invention.

[0027] Figure 6 The figure shows the experimental results of CCK-8 assay for enhancing the resistance of drug-resistant colorectal cancer cells to 5-FU, as presented in this invention.

[0028] Figure 7 This is a diagram showing the experimental results of plate cloning according to the present invention.

[0029] Figure 8 The figure shows the qPCR experimental results for mechanism verification of the present invention.

[0030] Figure 9 The WB experimental results are shown in the figure for verifying the mechanism of this invention.

[0031] Figure 10 The figure shows the results of the cell immunofluorescence experiment used to verify the mechanism of this invention.

[0032] Figure 11 This is a schematic diagram illustrating the in vivo antitumor effects and safety of the present invention. Detailed Implementation

[0033] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0034] Test method: 1. Acrylamide gel electrophoresis experiment A vertical slab electrophoresis system was used. A 20% separating gel (T=20%, C=3.3%) was prepared and poured to 2 cm from the top. 200 μL of deionized water was added for liquid sealing, and polymerization was carried out at room temperature for 30 min. The aqueous layer was decanted, and a 5% stacking gel was poured in. A 1.5 mm / 10-well sample comb was inserted, and polymerization was carried out for 20 min. 1× Tris-glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3) was injected into the electrophoresis tank. 10 μL of the sample to be tested was mixed with 10 μL of 2× loading buffer, and 15 μL was loaded. A negative control and molecular weight standard (5 μL) were also included. Electrophoresis was carried out at a constant voltage of 120 V for approximately 90 min (bromophenol blue to 1 cm from the bottom). The gel was fixed with fixative (methanol:acetic acid:water = 5:1:5) for 15 min, stained with 0.1% Coomassie Brilliant Blue R-250 for 30 min, and destained three times with destaining solution (3:1:6) for 10 min each time. Images are recorded using a gel imaging system, and ImageJ is used to analyze the grayscale values ​​of the bands.

[0035] 2. Flow cytometry experiment Logarithmic growth phase HCT8R cells and control HUVEC cells were collected and subjected to 5×10⁻⁶ cells. 5 Cells were seeded per well in 6-well plates. After adhesion, the medium was replaced with fresh medium containing 500 nM Cy5-labeled Apt-dsDNA, and incubated at 37°C in the dark for 0, 2, and 4 h. After incubation, cells were collected by trypsin digestion, washed three times with PBS, and resuspended in PBS. Cy5 fluorescence intensity (excitation / emission wavelength: 635 / 670 nm) was detected using a BD FACSAria III flow cytometer. Ten cells were collected from each sample.4 Cells. Mean fluorescence intensity (MFI) was analyzed using FlowJo software, and statistical significance was calculated using GraphPad Prism.

[0036] 3. CCK-8 method The CCK-8 assay is based on the colorimetric reaction of the water-soluble tetrazolium salt WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt). In the presence of the electron carrier 1-methoxy-5-methylphenazineonium sulfate dimethyl ester (1-Methoxy PMS), WST-8 can be reduced by dehydrogenases in the mitochondria of living cells to a highly water-soluble orange-yellow formazan product. The amount of formazan product generated is directly proportional to the number of living cells, the intensity of the color is directly proportional to the degree of cell proliferation, and inversely proportional to cytotoxicity. The absorbance (OD value) at 450 nm can be measured by an ELISA reader to indirectly reflect the number of living cells.

[0037] 4. Plate clone test HCT8R cells were seeded at a density of 500 cells / well in 6-well plates and allowed to adhere overnight. The cells were then treated with the following methods: blank control, 5-FU (4 μg / mL), Apt-dsDNA (500 nM), or a combination of 5-FU and Apt-dsDNA for 24 hours. The drug-containing medium was discarded and replaced with fresh complete medium, and the cells were cultured for 10–14 days. After visible colonies appeared, the medium was discarded, the cells were washed twice with PBS, fixed with methanol for 15 min, and stained with 0.1% crystal violet for 30 min. The number of colonies containing more than 50 cells was counted under a microscope, and the colony formation rate was calculated.

[0038] 5. Real-time quantitative PCR Total RNA was extracted from cells using the TRIzol method, and RNA concentration and purity were determined using NanoDrop 2000. cDNA was synthesized by reverse transcription using the PrimeScript RT Reagent Kit and amplified using SYBR Green Premix on a CFX Connect real-time quantitative PCR instrument. GAPDH was used as an internal control gene, and 2... - The relative expression level of the target gene was calculated using the ΔΔCt method.

[0039] 6. Western blot for protein immunoblotting Cells were lysed with RIPA lysis buffer (containing protease and phosphatase inhibitors) to extract total protein, which was quantified using the BCA method. Equal volumes of protein samples were separated by 10% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% skim milk for 1 h, anti-STING, p-STING, TBK1, p-TBK1, and GAPDH primary antibodies (all diluted 1:2000) were added, and the membranes were incubated overnight at 4°C. After washing, HRP-labeled secondary antibody was added and incubated at room temperature for 1 h. ECL chemiluminescence was used for color development, and images were acquired using an imaging system. The band grayscale values ​​were analyzed using ImageJ software.

[0040] 7. Immunofluorescence staining Cell slides were fixed with 4% paraformaldehyde for 15 min, permeated with 0.1% Triton X-100 for 10 min, and blocked with 5% goat serum for 1 h. Then, anti-STING and anti-TBK1 primary antibodies (both diluted 1:200) were added and incubated overnight at 4°C. The next day, Alexa Fluor 488 and 594-labeled fluorescent secondary antibodies were added and incubated at room temperature for 1 h. Cell nuclei were counterstained with DAPI. Observation and image acquisition were performed using a laser confocal microscope.

[0041] 8. Animal experiments SPF-grade BALB / c nude mice (5 weeks old) were acclimatized and subcutaneously inoculated with HCT8R cell suspension (1×10⁻⁶ cells) in the right axilla. 7 (Number of animals per 100 μL PBS). When the tumor volume reached approximately 100 mm3, the animals were randomly divided into 4 groups (5 animals per group): (1) Control group (PBS); (2) 5-FU monotherapy group (intraperitoneal injection); (3) Apt-dsDNA monotherapy group (tail vein injection); (4) Combined administration group (Apt-dsDNA tail vein injection + 5-FU intraperitoneal injection). Administered once every other day for a total of 4 times. The long and short diameters of the tumor were measured every 2 days, and the tumor volume was calculated using the following formula: Tumor volume (V) = Long diameter × Short diameter 2 / 2 After the drug administration was completed, the animals were euthanized, the tumors and major organs (heart, liver, spleen, lungs and kidneys) were removed, weighed and photographed, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and subjected to hematoxylin-eosin staining, immunohistochemistry (Ki-67) and TUNEL apoptosis detection.

[0042] Raw materials used in the examples: 1. The base sequences involved in the following examples were all customized by Sangon Biotech (Shanghai) Co., Ltd.

[0043] 2. The complete culture medium was RPMI Medium 1640 basal medium (GIBCO), containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin antibiotics (White Shark), phosphate-buffered saline (PBS, Sewell Biotech), and 0.25% trypsin-EDTA digestion solution (White Shark).

[0044] 3. CCK-8 cell proliferation assay kit (Beyotime).

[0045] 4. Human colorectal cancer 5-fluorouracil resistant cells (HCT8R / 5-FU) (Zhejiang Meisen Cell Technology Co., Ltd.)

[0046] 5. Crystal Violet (Azure).

[0047] 6. TRIzol reagent (Invitrogen, USA).

[0048] 7. SYBR Green Premix (Takara, Japan).

[0049] 8. BALB / c-nude athymic nude mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.)

[0050] Example 1: Preparation and Characterization of Apt-dsDNA This invention constructs an aptamer-functionalized double-stranded DNA (Apt-dsDNA) nucleic acid structure, which simultaneously possesses tumor-targeting ability and STING pathway activation activity. For example... Figure 1 As shown, this structure uses linear double-stranded DNA as its core (which can be recognized by cyclic guanosine monophosphate-adenosine synthase cGAS) and connects Sgc8 aptamers at both ends, enabling it to specifically bind to protein tyrosine kinase 7 (PTK7), which is highly expressed on the surface of colorectal cancer cells. The double-stranded DNA backbone is formed by the annealing hybridization of two complementary oligonucleotide chains (A chain and B chain), and the Sgc8 aptamer binds to a pre-defined overhanging sequence.

[0051] The specific nucleotide sequences of chains A and B are as follows: Chain A: 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGAAAAATACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA-3'; B-Chain: 5'-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGAGGGGTGTAGATCATGTACAGATCAGTCATAGATCACTAGTAGATCTGTA-3'.

[0052] The specific preparation method is as follows: Single-stranded DNA was synthesized by Sangon Biotech Co., Ltd., and both single-stranded DNAs were purified by high-performance liquid chromatography (HPLC). After obtaining the oligonucleotide powder product, it was centrifuged briefly at 12,000 rpm for 1 minute, and dissolved and mixed with 1×TE buffer in an equimolar ratio. The two oligonucleotide chains were annealed at 95℃ for 5 minutes, and then the temperature was slowly reduced to 25℃ at 0.1℃ every 8 seconds. The assembly effect was verified by non-denaturing polyacrylamide gel electrophoresis (PAGE). Figure 2 The electrophoretic migration rate of the double-stranded DNA complex slows down, and characteristic bands appear, indicating successful assembly.

[0053] To evaluate in vitro physiological stability, aptamer-functionalized double-stranded DNA was placed in a system containing 10% fetal bovine serum (FBS) and incubated at 37°C for a maximum of 8 hours. Results are as follows: Figure 3 As shown, the double-stranded DNA backbone has good stability, with about 80% of the structure remaining intact after 4 hours of incubation.

[0054] Example 2: Apt-dsDNA was successfully taken up by cells and showed targeting properties to tumor cells. The experiment used Cy5 fluorescently labeled Apt-dsDNA to investigate the internalization effect in PTK7-positive 5-fluorouracil-resistant HCT8R colorectal cancer cells. Flow cytometry results showed ( Figure 4 The intracellular fluorescence intensity gradually increased with incubation time, reaching a 4.8-fold increase after 4 hours of incubation compared to the initial value (p<0.001), indicating excellent cellular uptake efficiency. Furthermore, in HEUVC endothelial cells, the fluorescence intensity remained unchanged after 0, 2, and 4 hours of incubation, indicating that the aptamer Sgc8 has targeting properties for tumor cells. Laser confocal microscopy results ( Figure 5 This further confirms the conclusion, showing that the Cy5 fluorescence signal is distributed in a punctate pattern in the cytoplasm, mainly located inside the cell.

[0055] In summary, this invention successfully prepared an Apt-dsDNA double-stranded nucleic acid structure with good stability, tumor targeting, and efficient internalization by colorectal cancer cells.

[0056] Example 3: Effect of "Apt-dsDNA" on the proliferation of HCT8R / 5-FU cells 1. Cell Culture and Inoculation HCT8R / 5-FU cells in the logarithmic growth phase were observed under an inverted microscope to confirm uniform cell morphology, plumpness, clear edges, and 80%-90% confluence. The original culture medium was discarded, and the cells were washed twice with PBS. 0.25% trypsin-EDTA digestion solution was added, and the cells were incubated at 37°C for 3 min. When the cells became rounded and the intercellular spaces increased, serum-containing complete culture medium was added to terminate the digestion. The cells were collected by gentle pipetting, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in complete culture medium and counted using a hemocytometer. After thoroughly mixing the cell suspension, the cells were divided into two groups at 2 × 10⁻⁶ cells / mL. 4 / wells were seeded into 96-well plates (an equal amount of PBS or culture medium was added to the edge wells to prevent edge effects), and the 96-well plates were pre-cultured in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere.

[0057] 2. Sample processing After the cells have adhered, discard the original culture medium. The experimental groups are set up as follows: Blank control group: containing only complete culture medium and no cells; Negative control group (Control / NC): Contains cells, with an equal volume of complete culture medium, but without the test sample; Experimental group: containing cells, after adding "Apt-dsDNA" and reacting for 24 hours, the cells were aspirated. Five concentration gradients of 5-FU were prepared, with five replicates for each concentration, and the total volume of each well was maintained at 200 μL. The 96-well plate was then returned to a 37°C, 5% CO2 incubator for another 24 hours.

[0058] 3. CCK-8 colorimetric reaction After incubation, discard the culture medium in each well and add 100 μL of fresh complete culture medium containing 10% CCK-8 solution to each well. Avoid generating air bubbles during sample addition (air bubbles will interfere with OD value readings). Incubate the culture plate in a 37°C, 5% CO2 incubator for another 2 hours.

[0059] 4. Absorbance Measurement After incubation, the 96-well plate was placed in a microplate reader. The absorbance (OD) of each well was measured at 450 nm using the microplate reader. 450 Record the OD value of each well.

[0060] In this embodiment, the "Apt-dsDNA" treatment group IC 50 The concentration decreased from 1356.0 μM to 418.8 μM, representing a 3.2-fold increase in sensitization. CCK-8 assay confirmed that "Apt-dsDNA" significantly inhibited cell proliferation and enhanced the sensitivity of drug-resistant colorectal cancer cells HCT8R / 5-FU to 5-FU. Figure 6 ) Example 3: Inhibitory effect of Apt-dsDNA on HCT8R cell proliferation Plate colony formation experiment Logarithmic growth phase HCT8R cells were seeded at a density of 500 cells / well in 6-well plates and allowed to adhere overnight before being divided into four groups: (1) blank control group; (2) 5-FU monotherapy group (4 μg / mL); (3) Apt-dsDNA monotherapy group (500 nM); (4) combined drug therapy group (5-FU 4 μg / mL + Apt-dsDNA 500 nM). After 48 h of drug treatment, each group was replaced with fresh complete culture medium and cultured for 10–14 days. After the formation of visible colonies, the cells were washed with PBS, fixed with methanol, stained with 0.1% crystal violet, and the number of colonies containing more than 50 cells was counted under a microscope.

[0061] The results are as follows Figure 7 As shown, the average number of colonies in the blank control group was 78, in the 5-FU monotherapy group it was 53, and in the Apt-dsDNA monotherapy group it was 54. There were no statistically significant differences between the monotherapy groups and the control group, indicating that HCT8R cells are resistant to 5-FU, and that Apt-dsDNA monotherapy has limited inhibitory effect on colony formation. However, the average number of colonies in the combination therapy group was significantly reduced to 18, a statistically significant difference compared to the 5-FU monotherapy group. P<0.01). The above results indicate that Apt-dsDNA can enhance the inhibitory effect of 5-FU on the clonogenic ability of HCT8R cells and effectively reduce the proliferation ability of tumor cells.

[0062] Example 4: Activation of the cGAS-STING signaling pathway in HCT8R cells by Apt-dsDNA To clarify whether Apt-dsDNA can effectively initiate the cGAS-STING signaling cascade in 5-fluorouracil-resistant colorectal cancer HCT8R cells, this example systematically validates the results at three levels: transcriptional level, protein translation and phosphorylation modification, and subcellular localization of core signaling proteins.

[0063] 1. Real-time quantitative PCR detection: HCT8R cells in logarithmic growth phase were treated with 500 nM Apt-dsDNA for 24 h, with cells treated with an equal amount of PBS serving as a blank control. Total RNA was extracted, reverse transcribed, and then subjected to real-time quantitative PCR using the SYBR Green method to detect the relative mRNA expression levels of STING and TBK1 genes. GAPDH was used as an internal reference gene. - Calculated using the ΔΔCt method.

[0064] The results are as follows Figure 8 As shown, compared with the blank control group, the relative expression level of STING mRNA in the Apt-dsDNA treatment group increased by approximately 9.5-fold (P<0.01), and the relative expression level of TBK1 mRNA increased by approximately 18.2-fold (P<0.01). These results indicate that Apt-dsDNA can significantly upregulate the gene expression of core effector molecules in the cGAS-STING pathway at the transcriptional level.

[0065] 2. Protein immunoblotting detection HCT8R cells were treated with 500 nM Apt-dsDNA for 24 h, and total protein was extracted. After quantification by BCA method, the protein was separated by SDS-PAGE and transferred to nitrocellulose membrane. Immunoblotting was performed with anti-STING, p-STING, TBK1, p-TBK1 and GAPDH antibodies, and the gray values ​​of the bands were analyzed by ImageJ software.

[0066] The results are as follows Figure 9 As shown, compared with the blank control group, the p-STING protein level in the Apt-dsDNA treatment group increased to 1.70 times that of the control group (P<0.001), while the total STING protein level did not change significantly (1.05 times, not statistically significant). The total TBK1 protein level increased to 1.60 times that of the control group (P<0.01), and the p-TBK1 phosphorylation level increased to 3.20 times that of the control group (P<0.01). P<0.01). After correction to their respective total protein levels, the phosphorylation ratios of STING and TBK1 increased to 1.80-fold (P<0.05) and 2.10-fold (P<0.05) of the control group, respectively. P<0.01). The above results indicate that Apt-dsDNA mainly activates the cGAS-STING signaling pathway by promoting phosphorylation modification of STING and TBK1 proteins.

[0067] 3. Immunofluorescence staining detection HCT8R cells were seeded onto cell slides and treated with 500 nM Apt-dsDNA for 24 h. After fixation with 4% paraformaldehyde, permeabilization with 0.1% Triton X-100, and blocking with 5% goat serum, anti-STING and anti-TBK1 primary antibodies (both diluted 1:200) were added and incubated overnight at 4°C. The next day, Alexa Fluor 488 and 594 labeled fluorescent secondary antibodies were added and incubated in the dark for 1 h. The cell nuclei were counterstained with DAPI, and the cells were observed and images were acquired under a laser confocal microscope.

[0068] The results are as follows Figure 10As shown, in the blank control group cells, STING (green fluorescence) and TBK1 (red fluorescence) were diffusely distributed in the cytoplasm with weak fluorescence signals. In the Apt-dsDNA treated group cells, the fluorescence intensity of STING and TBK1 was significantly enhanced, and the two showed obvious punctate co-aggregation in the cytoplasm (yellow overlapping signal), indicating that Apt-dsDNA can promote the formation of a signaling complex of STING and TBK1 in the cytoplasm, further confirming the activation of the cGAS-STING signaling pathway at the subcellular level.

[0069] Example 5: Verification of the in vivo antitumor efficacy and safety of "Apt-dsDNA" in a BALB / c nude mouse subcutaneous xenograft model Phase 1 Model Construction: Single vaccination of 1×10⁶ cells in an SPF environment. 7 Cells were injected subcutaneously into the right axilla of 20 five-week-old nude mice, and tumor volume was dynamically monitored until the average volume reached 100 mm. 3 The subjects were randomly divided into 4 groups (n=5): Group A: saline blank control, Group B: Apt-dsDNA single drug, Group C: 5-FU single drug, and Group D: nucleic acid sequence + 5-FU combined group. The day was defined as D0 to ensure that there was no statistically significant difference at the baseline of each group.

[0070] Phase II Intervention Validation: Group A (Blank Control): PBS only. Four intravenous injections were administered, each at a dose of 10 mL / kg, for a total injection volume of 150 µL. Group B (Apt-dsDNA): Injection was via tail vein, starting Day 0, every 2 days for a total of 4 doses over 8 consecutive days, at a dose of 10 mg / kg dissolved in sterile PBS, for a final volume of 150 µL. Group C (5-FU): 5-fluorouracil, a first-line clinical chemotherapy drug, was administered via intraperitoneal injection, starting Day 3, every 2 days for a total of 4 doses over 8 consecutive days, at a dose of 25 mg / kg dissolved in sterile PBS, for a final volume of 150 µL. Group D (Nucleic Acid + 5-FU): 5-FU was administered via intraperitoneal injection, starting Day 0, every 2 days for a total of 4 doses over 8 consecutive days, at a dose of 25 mg / kg dissolved in sterile PBS, for a final volume of 150 µL. The nucleic acid was administered via tail vein injection, once every 2 days for a total of 4 doses over 8 consecutive days, with each dose being 10 mg / kg, dissolved in sterile PBS to a final volume of 150 µL. In the combination group, the two drugs were administered alternately every other day to reduce cumulative local irritation. Body weight was recorded every other day, and the long and short diameters of the tumor were measured with calipers. At the experimental endpoint (D15), the patient was euthanized by cervical dislocation, the tumor was completely dissected, weighed, and major organs were harvested.

[0071] The experimental results are shown in Figure 11The tumor inhibition rate of the "Apt-dsDNA" combined with 5-FU group was ≥80%, and the in vivo safety profile was assessed as good. Among all groups, the combined treatment group had the smallest terminal tumor volume, indicating superior in vivo antitumor efficacy. No significant weight fluctuations were observed between groups during treatment, suggesting good treatment tolerance. H&E staining of tumor tissue showed significant apoptotic morphological features in the combined treatment group, including cell shrinkage, nuclear pyknosis, and increased cytoplasmic eosinophilicity. Ki-67 immunohistochemical staining showed a significantly lower proportion of proliferating cells in the combined treatment group compared to the control group. TUNEL apoptosis assay showed a significant increase in apoptotic cells within the tumors of the combined treatment group. Furthermore, HE staining of major organs (heart, liver, spleen, lungs, and kidneys) showed no significant damage, indicating good in vivo safety.

[0072] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A double-stranded DNA, characterized in that, The double-stranded DNA is a DNA double-stranded structure composed of a tumor-targeting aptamer and a STING agonist. The targeting marker of the tumor-targeting aptamer is the PTK7 protein on the surface of tumor cells. The double-stranded DNA is formed by annealing and hybridizing single-stranded DNA as shown in SEQ ID NO.1~2 respectively.

2. A composition, characterized in that, The composition contains the double-stranded DNA as described in claim 1.

3. A combination drug, characterized in that, The combination drugs include the double-stranded DNA and 5-fluorouracil as described in claim 1.

4. A 5-FU resistance sensitizer for colorectal cancer, characterized in that, The colorectal cancer 5-FU resistance sensitizer contains the double-stranded DNA of claim 1, or the composition of claim 2, or the combination drug of claim 3.

5. The use of the double-stranded DNA of claim 1, or the composition of claim 2, or the combination drug of claim 3 in the preparation of a medicament for alleviating, reducing and / or treating colorectal cancer.

6. The application according to claim 5, characterized in that, The drug contains a pharmaceutically acceptable carrier or excipient.

7. The application according to claim 6, characterized in that, Pharmaceutically acceptable carriers include liposomes, microcells, metal particles, or polymer particles.

8. The application according to claim 5, characterized in that, The application includes at least one of the following functions: (1) Inhibits the proliferation of colorectal cancer cells; (2) Enhances the sensitivity of colorectal cancer cells to 5-fluorouracil; (3) Inhibit the growth of individual colorectal tumors.

9. The use of the double-stranded DNA of claim 1, or the composition of claim 2, or the combination drug of claim 3 in the preparation of a medicament for alleviating, reducing and / or reversing 5-fluorouracil resistance in colorectal cancer.

10. The application according to claim 9, characterized in that, The drug contains a pharmaceutically acceptable carrier or excipient, wherein the pharmaceutically acceptable carrier includes liposomes, microcells, metal particles, or polymer particles.

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