MOE modified oligonucleotide drugs targeting bclaf1 and their use in manec treatment

Abstract

This invention discloses a MOE-modified oligonucleotide drug targeting BCLAF1 and its application in the preparation of drugs for treating mixed glandular neuroendocrine carcinoma (MANEC), belonging to the field of biomedical technology. This invention provides a chemically modified antisense oligonucleotide (BCLAF1-ASO) targeting BCLAF1, which can specifically inhibit the transdifferentiation of adenocarcinoma (AC) components to neuroendocrine carcinoma (NEC) components in MANEC by precisely blocking the HCFC1-BCLAF1-POU3F2 molecular regulatory axis. This invention also reveals the application value of BCLAF1 as a key node driving the malignant progression of MANEC, achieving sensitization of HCFC1-overexpressing MANEC cells to platinum-based chemotherapy regimens, significantly improving treatment efficacy. This invention also optimizes the pharmacokinetic characteristics of ASO, solving the technical challenges of its easy degradation and low bioavailability in the solid tumor microenvironment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and precision medicine technology, specifically relating to an oligonucleotide drug targeting the HCFC1-BCLAF1-POU3F2 regulatory axis, its preparation method and its application in the treatment of mixed glandular neuroendocrine carcinoma (MANEC), particularly relating to an antisense oligonucleotide with chemosensitizing effect after specific chemical modification. Background Technology

[0002] Mixed adenoneuroendocrine carcinoma (MANEC) is a rare and highly malignant tumor composed of both adenocarcinoma (AC) and neuroendocrine carcinoma (NEC) components. Due to its high tumor heterogeneity, MANEC is characterized by its aggressiveness, high early metastasis rate, and extremely poor prognosis, with a 5-year survival rate of only 10%-20%.

[0003] The molecular mechanisms and intervention challenges of MANEC component transformation: Clinical and pathological studies have confirmed that the transformation of AC components into NEC components (i.e., "AC-to-NEC" transdifferentiation) is a core link driving the malignant progression of MANEC and mediating acquired chemotherapy resistance. Although signaling pathways such as Notch and Wnt / β-catenin have been reported to participate in partial differentiation regulation, interventions developed targeting these pathways often suffer from insufficient specificity and high off-target toxicity due to their broad involvement in normal physiological processes. While HCFC1 and BCLAF1 are involved in some tumors, the key role of the HCFC1-BCLAF1-POU3F2 axis in driving MANEC cell component transformation has not yet been revealed with current technology. This results in a lack of precise intervention targets in clinical practice that can specifically block the "AC-to-NEC" transformation process.

[0004] Antisense oligonucleotides (ASOs) specifically block target gene expression through the base complementarity principle, and are considered an ideal weapon for intervening in transcriptional regulation. However, unmodified ASOs are easily degraded by nucleases in plasma and tissues, and in the complex solid tumor microenvironment of MANEC, the cellular uptake efficiency and bioavailability of the drug are extremely low.

[0005] 2'-O-(2-methoxyethyl) (2'-MOE) modification can enhance the pharmacokinetic properties of oligonucleotides. However, for the specific target BCLAF1, there are currently no reports or effective technical solutions on how to precisely design MOE modification sites (e.g., a chimeric structure with a specific ratio of MOE modification and phosphate thioester bond layout) based on the secondary structure of its mRNA and the pathophysiological characteristics of MANEC to induce a synergistic sensitizing effect of HCFC1 overexpressing cells on chemotherapeutic drugs while achieving efficient degradation of BCLAF1 mRNA.

[0006] In summary, the existing technologies have the following significant drawbacks: (1) Lack of specificity in molecular intervention targets: Existing treatment methods fail to precisely intervene in the specific regulatory axis of AC component to NEC component transformation in MANEC, resulting in difficulty in inhibiting the malignant phenotypic transformation of tumors. (2) Lack of efficient inhibition methods for BCLAF1: There are currently no specific drugs available for clinical application for BCLAF1, a core node, especially oligonucleotide drug regimens that can simultaneously take into account stability, targeting, and bioactivity. (3) Gap in intervention of chemotherapy resistance mechanisms: Existing technologies have not yet solved the problem of resistance of MANEC to first-line clinical chemotherapy regimens, and lack a comprehensive treatment strategy that can produce a significant "target-chemotherapy" sensitization synergistic effect. (4) Insufficient pharmacokinetic stability and delivery efficiency: Conventional oligonucleotide drugs have short half-lives in vivo circulation, making it difficult to penetrate into the complex tissue interior of MANEC. It is urgent to optimize their steady-state concentration in the tumor lesion area through specific chemical modification layout. Summary of the Invention

[0007] To address the technical bottlenecks in the clinical treatment of mixed glandular neuroendocrine carcinoma (MANEC), such as the lack of pathologically specific intervention targets, drug resistance to traditional chemotherapy regimens due to tumor heterogeneity, and poor in vivo stability of conventional antisense oligonucleotide (ASO) drugs, this invention aims to: 1. A chemically modified antisense oligonucleotide (BCLAF1-ASO) targeting BCLAF1 is provided, which specifically inhibits the transdifferentiation of adenocarcinoma (AC) components to neuroendocrine carcinoma (NEC) components in MANEC by precisely blocking the HCFC1-BCLAF1-POU3F2 molecular regulatory axis.

[0008] 2. To reveal the application value of BCLAF1 as a key node, and to achieve sensitization of HCFC1-overexpressing MANEC cells to platinum-based chemotherapy regimens, thereby significantly improving the therapeutic effect.

[0009] 3. By using a specific 2'-MOE-PS backbone chimeric modification layout, the pharmacokinetic characteristics of ASO are optimized, solving the technical challenges of its easy degradation and low bioavailability in the solid tumor microenvironment.

[0010] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One of the objectives of this invention is to provide an antisense oligonucleotide, the sequence of which is shown in SEQ ID NO.5.

[0011] Furthermore, the cytosine in the antisense oligonucleotide is 5-methylcytosine.

[0012] Furthermore, the antisense oligonucleotide contains a phosphate thioester backbone modification.

[0013] Furthermore, the antisense oligonucleotide has a gapmer structure.

[0014] Furthermore, the gapmer structure is a 5-11-5 chimeric pattern, wherein the 5 nucleotides at the 5' end and the 5 nucleotides at the 3' end are wing regions, and the 11 nucleotides in the middle are gap regions.

[0015] Furthermore, the nucleotides in the wing region are modified with 2'-O-(2-methoxyethyl).

[0016] Furthermore, the antisense oligonucleotide has the following modified layout: 5'-(MOE-T)(MOE-T)(MOE-5-Me-C)(MOE-A)(MOE-G)ACTTTACCTGC(MOE-T) (MOE-5-Me-C) (MOE-A)(MOE-G)(MOE-G)-3'; in" " indicates modification of the thiophosphate skeleton, (MOE-) indicates 2'-O-(2-methoxyethyl) modification, and (5-Me-C) indicates 5-methylcytosine.

[0017] A second objective of this invention is to provide a pharmaceutical composition comprising the antisense oligonucleotide and a pharmaceutically acceptable carrier or excipient.

[0018] A third objective of this invention is to provide the use of the antisense oligonucleotide or the pharmaceutical composition in the preparation of a medicament for treating mixed glandular neuroendocrine carcinoma.

[0019] Furthermore, the drug can be used in combination with chemotherapy drugs.

[0020] This invention addresses the core challenges in the clinical treatment of mixed glandular neuroendocrine carcinoma (MANEC), including the lack of pathologically specific targets, low chemosensitivity, and a high likelihood of acquired resistance. By providing a BCLAF1-ASO with a specific chemically modified layout, it achieves precise intervention along the HCFC1-BCLAF1-POU3F2 axis, resulting in the following significant beneficial effects: (i) The target is novel and the intervention is precise, blocking the transdifferentiation of MANEC components from the source. 1. Originality of Mechanism Discovery: This invention reveals for the first time that the HCFC1-BCLAF1-POU3F2 axis is a core molecular switch driving the transdifferentiation of adenocarcinoma (AC) components into neuroendocrine carcinoma (NEC) components in MANEC. Compared to widely expressed pathways such as Notch or Wnt, this regulatory axis exhibits high pathological specificity in the malignant progression of MANEC.

[0021] 2. High efficiency and selectivity of targeted inhibition: BCLAF1-ASO uses a specific 21nt sequence to precisely bind to the target mRNA functional site. Experimental data show that its protein inhibition rate in HCFC1-overexpressing cells is as high as 90%, and it exhibits significant target selectivity (with minimal effect on parental cells), effectively avoiding the off-target toxicity common in traditional targeted drugs.

[0022] 3. Thoroughness of signal pathway blocking: The drug of this invention downregulates BCLAF1 expression at the transcriptional level, thereby downregulating the activity of downstream pro-differentiation factor POU3F2, which can significantly reverse the neuroendocrine phenotype of tumors and fundamentally inhibit the malignant progression of tumors.

[0023] (ii) Significantly enhances chemosensitivity and synergistically reverses acquired drug resistance 1. Synergistic Sensitization Effect: The BCLAF1-ASO of this invention exhibits a strong synergistic effect when used in combination with cisplatin / etoposide. The IC50 of cells in the combination group was significantly lower than that in the chemotherapy monotherapy group. Specifically, in the FOLFIRI regimen, IC50 decreased from 40 uM to 15.7 uM; in the FOLFOX regimen, IC50 decreased from 47.73 uM to 17.43 uM, effectively reversing drug resistance induced by HCFC1; and in the EP regimen, IC50 decreased from 55.16 uM to 21.72 uM. In vivo experiments showed that the ASO-BCLAF1 combined with the FOLFOX regimen significantly inhibited the growth of xenografts derived from HCFC1-overexpressing gastric cancer cells, reducing tumor volume and weight, confirming that targeting BCLAF1 can enhance the chemosensitivity of neuroendocrine-differentiated gastric cancer cells to the FOLFOX regimen in vivo.

[0024] (iii) The technology transfer path is clear and has extremely high clinical application value. 1. Mass production feasibility of the process: The mature solid-phase synthesis process of phosphorous amide can be adopted to achieve large-scale customization from milligram level to gram level, and the purity control (≥98%) and quality control process are clear, which fully meets the industrial production requirements of GMP standards.

[0025] 2. Application prospects of precision medicine: This invention provides a theoretical basis and drug approach for "stratified treatment" for MANEC patients with HCFC1 overexpression, a specific drug-resistant group, filling the clinical gap in the field of MANEC where there is a lack of specific adjuvant drugs.

[0026] 3. Universality of the treatment model: The constructed "targeted blockade of transdifferentiation + traditional chemotherapy sensitization" model can be smoothly connected with the existing first-line clinical chemotherapy regimens without changing the existing diagnosis and treatment pathways, and has strong clinical promotion feasibility.

[0027] In summary, this invention has the following innovations and advancements: (1) New target discovery: BCLAF1 is established as a key target for intervening in the transformation of MANEC components for the first time, breaking through the predicament that existing treatments cannot block the transformation of AC to NEC. (2) Structural novelty: A specific 2'-MOE and 5-Me-C chimeric layout was designed, solving the problems of easy degradation and weak penetration of conventional ASO in complex tumor tissues. (3) Breakthrough in clinical strategy: It was found that BCLAF1-ASO can not only inhibit tumor growth, but also restore the sensitivity of HCFC1-overexpressing gastric cancer cells that have developed drug resistance to chemotherapy, which has significant clinical sensitization value. Attached Figure Description

[0028] Figure 1This is the process of verifying the necessity of BCLAF1 function in the rescue experiment in Embodiment 1 of the present invention. (AD) qRT-PCR and Western Blot were used to verify the knockdown efficiency of three shBCLAF1 lines in SNU-1 (AB) and HGC-27 (CD) cells; (E) qRT-PCR was used to detect the mRNA levels of BCLAF1, POU3F2, CGA, and SYP in SNU-1 cells after HCFC1 overexpression and BCLAF1 knockdown; (F) Western Blot was used to detect the protein levels of BCLAF1, POU3F2, CgA, and SYN in SNU-1 cells after HCFC1 overexpression and BCLAF1 knockdown; (G) qRT-PCR was used to detect the mRNA levels of BCLAF1, POU3F2, CGA, and SYP in HGC-27 cells after HCFC1 overexpression and BCLAF1 knockdown; (H) Western Blot was used to detect the protein levels of BCLAF1, POU3F2, CgA, and SYN in HGC-27 cells after HCFC1 overexpression and BCLAF1 knockdown. (I) Immunohistochemical staining results of HCFC1, BCLAF1 and POU3F2 in clinical samples of gastric tumors (200×).

[0029] Figure 2 This invention illustrates the effects of HCFC1 overexpression and ASO-BCLAF1 on the proliferation and chemosensitivity of HGC-27 cells in Example 2. (A) CCK-8 assay of the relative viability of HGC-27 WT and HCFC1OE cells after 48 h of culture; (B) CCK-8 assay of the relative viability of HGC-27 WT and HCFC1OE cells after ASO-BCLAF1 intervention; (CD) Dose-response curves and colony formation assays of cells under the FOLFIRI regimen; (EF) Dose-response curves and colony formation assays of cells under the FOLFOX regimen; (GH) Dose-response curves and colony formation assays of cells under the EP regimen.

[0030] Figure 3 This invention presents the in vivo intervention effect of ASO-BCLAF1 combined with FOLFOX on HGC-27 cell xenografts in nude mice in Example 3. (A) Schematic diagram of the in vivo experimental procedure; (B) Gross morphological photographs of xenografts in each group of nude mice after intervention; (C) Weight change curves of nude mice in each group during intervention; (D) Growth curves of xenografts in each group of nude mice; (E) Weight statistics of xenografts in each group of nude mice after intervention, n=6 / group; (F) HE and immunohistochemical detection of Ki67, HCFC1, BCLAF1, POU3F2, SYN and CGA expression. Detailed Implementation

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0032] The core technical solution of this invention is as follows: 1. Design of antisense oligonucleotide drugs targeting BCLAF1 (1) Target and sequence information Based on the BCLAF1 mRNA (GenBank: NM_014739.3) protein coding sequence, this invention designs an antisense oligonucleotide sequence with highly efficient degradation activity: 5'-TTCAGACTTTACCTGCTCAGG-3' (21nt, i.e. SEQ ID NO.5 below).

[0033] (2) Chemical modification strategy (2'-MOE chimeric structure) The BCLAF1-ASO described in this invention has a full length of 21 nt, and its antisense chain sequence and detailed modification layout are as follows: 5'-(MOE-T) (MOE-T) (MOE-5-Me-C) (MOE-A) (MOE-G) A C T T T A C C T G C (MOE-T) (MOE-5-Me-C) (MOE-A) (MOE-G) (MOE-G)-3' Description of modification features: Gapmer structure: It adopts a "5-11-5" chimeric pattern. The 5' end and the 3' end each have 5 nucleotides as wings, and the middle 11 nucleotides are the gap.

[0034] 2'-MOE modification: All 10 nucleotides in the wing region are modified with 2'-O-(2-methoxyethyl) (2'-MOE) to enhance binding affinity (Tm value) and resist exonucleases.

[0035] 5-Methylation modification: all cytosine in the sequence is 5-methylcytosine (5-Me-C) to reduce immunogenicity and further stabilize the double-stranded structure.

[0036] Backbone modification: All 20 meta phosphate positions in the entire sequence were modified with phosphate thioester (PS) (labeled as...). This endows the drug with excellent resistance to endonucleases.

[0037] Modifier descriptions: (MOE-A)'2, (MOE-5-Me-C)'2, (MOE-G)'3, (MOE-T)'3, '20.

[0038] (3) Physicochemical properties Molecular weight: 7300-7500 Da (optimal value 7412 Da).

[0039] Melting point (Tm): 65-72℃ (ensuring high hybridization stability at physiological temperatures).

[0040] Water solubility: ≥15 mg / mL (pH 7.4 PBS buffer) 2. Preparation process of BCLAF1-ASO This invention employs a solid-phase synthesis method for phosphorous amides, and the key process control points are as follows: The phosphorus amide was synthesized using a solid-phase method. Key steps included: Coupling: PyBOP is used to activate the monomer, and the coupling efficiency of each step needs to be 99%.

[0041] Thioation: A thioating agent is introduced after each round of coupling to ensure complete meta-thioation of phosphate.

[0042] Purification: Purification was performed using RP-HPLC (reversed-phase high-performance liquid chromatography), and high-purity target peaks were collected.

[0043] Quality control: The molecular weight was verified using MALDI-TOF mass spectrometry to ensure product purity of 98.5%.

[0044] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0045] Example 1: Knocking down BCLAF1 can achieve precise blocking of the HCFC1-BCLAF1-POU3F2 axis. Table 1 Main experimental reagents

[0046] Table 2. Main experimental instruments and their sources

[0047] 1. Lentiviral cell transfection and construction of stable transfected strains (1) Construction of recombinant vector: pLenti-CMV-Puro was selected as the backbone plasmid and the full-length coding sequence of human HCFC1 was directionally inserted into the multiple cloning site (Accession: NM_001440843.1) to construct the overexpression vector (named HCFC1). OE The original empty vector plasmid was used as a control (WT). The recombinant clones were bidirectionally sequenced using the Sanger method, which confirmed that the inserted sequence was highly consistent with the target template.

[0048] (2) Construction of BCLAF1 knockdown lentiviral vector: The shRNA sequence targeting the human BCLAF1 gene was designed and synthesized by Shanghai Jikai Biotechnology Co., Ltd. and inserted into the GV112 lentiviral vector. The target sequence is shown in Table 3 below. At the same time, a nonsense sequence negative control lentivirus (shNC) was constructed.

[0049] Table 3. shRNA target sequences (5'-3') targeting human BCLAF1

[0050] (3) Lentiviral packaging and titer enrichment: When HEK-293T cells are in the logarithmic growth phase (confluence approximately 70%~80%), PEI-mediated three-plasmid co-transfection technology is used to package HCFC1. OE Alternatively, control plasmids and helper packaging elements (pMD2.G, psPAX2) could be introduced into cells. The culture medium was refreshed 6 hours after transfection to reduce cytotoxicity, and the cells were maintained for 72 hours. Subsequently, the virus-rich culture medium was collected, centrifuged at 3000 rpm, clarified by filtering through a 0.45 μm pore membrane, and the virus was enriched by ultracentrifugation sedimentation.

[0051] (4) Screening and enrichment of stable cell lines: HGC-27 and SNU-1 gastric cancer cells were selected as target cells, and lentivirus was introduced at a confluence of 40%–50%. To enhance infection efficiency, 10 μg / mL polybrene was added to the transfection system. 72 hours after transfection, long-term resistance screening was performed using 4 μg / mL puromycin. After approximately 7 days of continuous pressure culture and medium replacement, a cell population with stable expression characteristics was finally obtained.

[0052] (5) Verification of transfection efficiency: To confirm the upregulation effect of HCFC1 expression, verification was performed at both the transcriptional and translational levels. mRNA abundance was measured by qRT-PCR, and protein translation level was analyzed by Western blot to ensure the effectiveness of the constructed stable cell line.

[0053] 2. RNA extraction, reverse transcription, and real-time quantitative PCR experiments (1) Total RNA isolation and purification: Total RNA was extracted from cell samples using the Trizol reagent method. The steps are as follows: ① Remove the cell culture medium and wash the residue with sterile PBS.

[0054] ② Introduce 500 μL of lysis buffer into each well and mechanically pipette to completely disintegrate the cells. Then transfer the extract to a microcentrifuge tube free of nuclease contamination.

[0055] ③ Subsequently, 200 μL of enzyme-free water was added for liquid phase separation. The mixture was stirred vigorously and placed at room temperature for 10 min. The upper aqueous phase rich in RNA was obtained by high-speed centrifugation at 4℃ (12000 rpm).

[0056] ④ Further remove protein and salt impurities by precipitation with isopropanol and washing with anhydrous ethanol. Finally, after the precipitate has dried at room temperature, the RNA particles are reconstituted with an appropriate amount of enzyme-free ultrapure water.

[0057] (2) Qualitative and quantitative quality control of RNA: The concentration and purity of the extract were accurately determined using a NanoDrop ultra-micro spectrophotometer. This invention strictly screens samples with an absorbance (OD) ratio of OD260 / OD280 between 1.8 and 2.0 to ensure that the integrity and biological purity of the RNA meet the requirements of subsequent experiments.

[0058] (3) Reverse transcription synthesis of cDNA: A 20 μL reverse transcription system was constructed using the HiScript IV premixed reaction system. 500 ng template RNA was mixed with 4 μL of 5×HiScript IV qRT SuperMix and then made up with enzyme-free water. First-strand cDNA was synthesized efficiently using a gradient temperature control program in a PCR instrument (37℃ extension for 15 min, 85℃ inactivation for 5 s). The resulting product was immediately stored at -20℃ as a template library for subsequent real-time quantitative detection.

[0059] (4) Real-time quantitative PCR reaction: A 10 μL qPCR reaction system was prepared using the SYBR Green method, as follows: Table 4 Real-time quantitative reaction system

[0060] (5) Real-time quantitative PCR reaction: Add the reaction system to a 96-well PCR plate, gently centrifuge to remove air bubbles, and place it in a qPCR instrument for reaction, following the procedure below: Table 5 Real-time quantitative reaction program

[0061] (6) Primer sequences: GAPDH or ACTB proteins were selected as standardized internal controls at the transcriptional level. To address fluctuations in target gene expression, 2... (-ΔΔCt) Difference analysis was used for quantitative assessment to accurately characterize changes in the relative abundance of genes. Primer sequences were purchased from Origene and are available for access via their website (https: / / www.origene.com / ).

[0062] 3. Total protein component extraction, quantification, and Western blot detection procedure (1) Cell lysis and total protein preparation: After removing the cell culture medium, the cells were washed with PBS buffer. Then, 80 μL of pre-chilled RIPA high-efficiency lysis medium (pre-incorporated with 1% protease inhibitor) was introduced into each well, and static lysis was performed for 30 minutes in an ice bath. Adherent components were removed using a cell scraper, and the homogenate was transferred to centrifuge tubes. The clear protein aqueous phase was precisely aspirated by high-speed sedimentation at 4°C and 12,000 rpm to ensure effective removal of insoluble cell debris and precipitates.

[0063] (2) Protein abundance determination based on BCA method: Protein concentration was determined using the BCA colorimetric reaction system. A 50:1 mixture of solutions A and B was prepared and temporarily stored on ice in the dark. A calibration system consisting of serially diluted standards was established in a 96-well plate, and the test samples were added to the wells at a dilution of 1:25. The colorimetric reagent was added and incubated at 37°C for 30 minutes. Finally, the characteristic absorbance signal at OD value of 570 nm was detected using a microplate reader, and the original protein concentration of each sample was accurately calculated using a regression standard curve.

[0064] (3) Protein denaturation: Based on the quantitative results, extract an equal mass of protein components and mix with 5×SDS-PAGE loading buffer. Place the sample in a 100℃ metal dry bath for denaturation for 10 minutes. Store the denatured protein at -20℃ for subsequent loading.

[0065] (4) SDS-PAGE gel preparation: According to the operating instructions of the SDS-PAGE gel preparation kit, prepare 12.5% ​​separating gel and 5% stacking gel respectively. Slowly pour the prepared gel solution into the gel mold and let it stand at room temperature until the gel is completely solidified.

[0066] (5) The specific components of the electrophoresis buffer and transfer buffer are shown in the table below: Table 6. Components of Electrophoresis Buffer and Transfer Buffer

[0067] (7) Polyacrylamide gel electrophoresis: Place the solidified gel in the electrophoresis apparatus and fill the tank with 1× electrophoresis buffer. Determine the sample loading amount based on the previous protein quantification data, and sequentially introduce protein standards and denatured samples to be tested into the sample wells. Start the constant voltage mode (120V) to drive the electrophoresis until the bromophenol blue dye line reaches the lower edge of the gel, and then stop the current.

[0068] (8) Protein transfer: Remove the gel and cut the appropriate gel fragments according to the target molecular weight. Immerse the PVDF membrane in anhydrous methanol for 30 s to activate it, then equilibrate it with the gel and filter paper in pre-cooled transfer solution for 10 min. Assemble the transfer clamp in the order of "filter paper-PVDF membrane-gel-filter paper", and use rubber rollers to remove air bubbles at the interface. Pour pre-cooled transfer solution into the transfer tank, set a constant current parameter of 400 mA to perform efficient wet transfer, and dynamically optimize the transfer time according to the molecular weight of the target protein.

[0069] (9) Membrane blocking: After the transfer is completed, the PVDF membrane with transferred protein is immersed in the rapid blocking solution and shaken at low speed on a horizontal shaker at room temperature for half an hour to block the excess non-specific protein binding sites on the membrane surface.

[0070] (10) Primary antibody incubation: Take out the blocked PVDF membrane and rinse it, add the diluted primary antibody (HCFC1, CgA, SYN dilution ratio is 1:1000; GAPDH dilution ratio is 1:5000), and then place it on a shaker to hybridize overnight at a constant temperature of 4℃ to ensure that the antibody fully binds to the target protein.

[0071] (11) Secondary antibody incubation: Take out the PVDF membrane incubated with the primary antibody and perform TBST washing (10 min × 3 times), then add HRP-labeled secondary antibody (1:5000 dilution) and incubate for 1 h at room temperature.

[0072] (12) Chemiluminescence development and result analysis: The PVDF membrane incubated with secondary antibody was removed and TBST washing was performed (10 min × 3 times). ECL substrate solution prepared in a 1:1 ratio was uniformly applied to the membrane surface. The membrane was exposed and developed using a chemiluminescence imaging platform to capture characteristic band signals and store images. Finally, optical density analysis was performed using ImageJ software. The relative abundance of each protein was quantitatively assessed using the expression abundance of GAPDH or β-Actin as a homogenization standard.

[0073] To clarify the functional necessity of BCLAF1 in the neuroendocrine differentiation of HCFC1-driven gastric adenocarcinoma cells, this invention first designed and synthesized three shRNA lentiviruses targeting BCLAF1. After transfection into SNU-1 and HGC-27 cells, the knockdown efficiency was verified by qRT-PCR and Western Blot. The results showed that shBCLAF1-3 had the best knockdown efficiency, significantly downregulating the mRNA and protein levels of BCLAF1 (P<0.001). Therefore, this sequence was selected for subsequent rescue experiments. Figure 1 A- Figure 1 D).

[0074] Subsequently, in SNU-1 and HGC-27 cells with stable HCFC1 overexpression, functional rescue experiments were performed by simultaneously knocking down BCLAF1 expression. qRT-PCR results showed that HCFC1 overexpression significantly upregulated the mRNA levels of neuroendocrine differentiation markers CGA and SYP (P<0.01), while simultaneous knockdown of BCLAF1 significantly reversed this effect, resulting in a significant downregulation of CGA and SYP mRNA levels (P<0.05). Figure 1 E, Figure 1 G); Western blot results further confirmed that the upregulation of CgA and SYN protein levels induced by HCFC1 overexpression could be effectively reversed by BCLAF1 knockdown. Figure 1 F, Figure 1 H). The above results indicate that BCLAF1 is a key mediator of HCFC1-driven neuroendocrine differentiation of gastric adenocarcinoma cells, and knockdown of BCLAF1 can effectively block HCFC1-induced neuroendocrine phenotypic transformation.

[0075] To further validate the pathological significance of this transcriptional cascade at the clinical level, this invention detected the protein expression levels of HCFC1, BCLAF1, and POU3F2 in tissue sections from 20 clinical gastric tumor samples using immunohistochemical staining. The results showed that HCFC1, BCLAF1, and POU3F2 all exhibited specific high expression in the NEC region, while showing low or no expression in the AC region, demonstrating a high degree of consistency in their expression patterns. Figure 1 I).

[0076] Example 2: In vitro experiments demonstrated that targeted silencing of BCLAF1 can restore the chemosensitivity of HCFC1-overexpressing gastric cancer cells. 1. Design and synthesis of antisense oligonucleotides The antisense oligonucleotide (ASO) designed targeting the human BCLAF1 gene sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. To enhance its in vivo stability and targeted degradation efficiency, this ASO employs a gapmer structure design: each of the two ends is modified with 2'-O-methoxyethyl (2'-MOE), and the DNA window consisting of 11 deoxyribonucleotides in the middle is responsible for activating RNase H. Furthermore, all cytosines in this sequence are 5-methylated to reduce immunogenicity, and the entire backbone is thiolated.

[0077] Table 7 Antisense Oligonucleotide Sequences

[0078] Specifically: (1) Target and sequence information Based on the BCLAF1 mRNA (GenBank: NM_014739.3) protein coding sequence, this invention designs an antisense oligonucleotide sequence with highly efficient degradation activity: 5'-CCTGAGCAGGTAAAGTCTGAA-3' (21nt, i.e. SEQ ID NO.5).

[0079] (2) Chemical modification strategy (2'-MOE chimeric structure) The BCLAF1-ASO described in this invention has a full length of 21 nt, and its antisense chain sequence and detailed modification layout are as follows: 5'-(MOE-T) (MOE-T) (MOE-5-Me-C) (MOE-A) (MOE-G) A C T T T A C C T G C (MOE-T) (MOE-5-Me-C) (MOE-A) (MOE-G) (MOE-G)-3' Description of modification features: Gapmer structure: It adopts a "5-11-5" chimeric pattern. The 5' end and the 3' end each have 5 nucleotides as wings, and the middle 11 nucleotides are the gap.

[0080] 2'-MOE modification: All 10 nucleotides in the wing region are modified with 2'-O-(2-methoxyethyl) (2'-MOE) to enhance binding affinity (Tm value) and resist exonucleases.

[0081] 5-Methylation modification: all cytosine in the sequence is 5-methylcytosine (5-Me-C) to reduce immunogenicity and further stabilize the double-stranded structure.

[0082] Backbone modification: All 20 meta phosphate positions in the entire sequence were modified with phosphate thioester (PS) (labeled as...). This endows the drug with excellent resistance to endonucleases.

[0083] Modifier descriptions: (MOE-A)'2, (MOE-5-Me-C)'2, (MOE-G)'3, (MOE-T)'3, '20.

[0084] 2. Cell transfection HGC-27 cells in logarithmic growth phase were harvested at a concentration of 3 × 10⁻⁶. 3 / wells were inoculated into 96-well plates, 1×10 3 Cells were seeded in 24-well plates and transfected with ASO according to the Lipomaster 3000 transfection reagent instructions when the cell adhesion and confluence reached 40%-50%. The ASO concentration gradient was 0-600 nM. After 6 h of transfection, the medium was replaced with fresh complete medium, and the cells were cultured for another 48 h before being used for subsequent experiments.

[0085] 3. Cell viability assay and chemotherapy sensitivity (IC50) analysis (1) HGC-27 cells (WT group and HCFC1) were placed in the WT group and HCFC1 group. OE (Group) 3×10 3 / Well density was introduced into 96-well plates. After the cells entered the adherent growth phase, gene silencing intervention with 20 nM ASO was performed according to the ASO transfection procedure. Eight technical replicates were set up for each group, and background control wells without cells were reserved to eliminate interference from the culture medium.

[0086] (2) After transfection, the cells were cultured for another 24 h and then treated with different concentrations of chemotherapy drugs: FOLFIRI (SN-38 + 5-FU, concentration gradient 0-200 μmol / L), FOLFOX (oxaliplatin + 5-FU, concentration gradient 0-200 μmol / L), EP (etoposide + cisplatin, concentration gradient 0-200 μmol / L), and the control group was given an equal volume of complete culture medium containing DMSO.

[0087] (3) After the drug has been in effect for 72 h, the medium is changed, and then 10 μL of CCK-8 reagent is added to each well. The incubator is continued for 1 h, and the absorbance value (OD value) at 450 nm wavelength is detected by microplate reader.

[0088] (4) Calculate the relative cell viability and proliferation inhibition rate: Relative cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100% Proliferation inhibition rate (%) = 100% - Relative cell viability (%).

[0089] (5) Using GraphPad Prism 9.0 software, dose-response curves were plotted using a nonlinear regression four-parameter model, and the IC50 of each chemotherapy regimen on cells was calculated.

[0090] 4. Plate colony formation experiment (1) Cell seeding and ASO intervention: HGC-27 (WT and HCFC1) were seeded and ASO was applied to cells. OE Cells (group 1) were seeded at a low density of 500 cells / well in 24-well plates. After complete cell adhesion, 20 nM ASO was introduced strictly according to the transfection protocol. To maintain the sustained effect of gene silencing, dynamic transfection was performed every 3 days, and three parallel biological replicates were set up for each group of experiments.

[0091] (2) Gradient drug administration: Commonly used clinical chemotherapy regimens, namely FOLFIRI, FOLFOX and EP, were introduced into the culture system, and a series of concentration gradients (0-200 μmol / L) were set up; the control group was given the same volume of conventional complete culture medium. During the drug treatment, freshly prepared culture medium containing the corresponding drug concentration was replaced every 3 days to simulate a continuous pharmacological stress environment.

[0092] (3) Fixation and staining: After 14 days of continuous culture, the supernatant was removed and the cells were gently rinsed with PBS buffer. Cell colonies were fixed in situ with 4% PFA (30 min), followed by room temperature staining with 0.1% crystal violet. After slowly washing away excess background color with running water and air drying at room temperature, clear and visible clonal spots were formed.

[0093] (4) Image acquisition: Each group of clones was photographed using a microscopic imaging system, and clones with a diameter exceeding 75 μm were strictly defined as valid clones. The clone formation rate of each experimental group was calculated by counting the total number of valid clones.

[0094] This invention first examined the effect of HCFC1 overexpression on the proliferation of HGC-27 cells using a CCK-8 assay. The results showed that after 48 h of culture, the relative viability of HGC-27 HCFC1OE cells was significantly higher than that of WT cells. Figure 2(A, P<0.001) confirms that HCFC1 overexpression can significantly promote the proliferation of gastric adenocarcinoma cells.

[0095] To further investigate the effect of HCFC1 overexpression on the chemosensitivity of gastric cancer cells, cells were treated with FOLFIRI, FOLFOX, and EP, respectively, and dose-response curves were plotted and IC50 values ​​were calculated. The results showed that: (1) Under FOLFIRI treatment, the IC50 of HGC-27 WT cells was 10.71 μmol / L, while the IC50 of HCFC1OE cells was significantly increased to 40.00 μmol / L ( Figure 2 C); (2) Under FOLFOX intervention, the IC50 of HGC-27 WT cells was 9.529 μmol / L, and the IC50 of HCFC1OE cells was significantly increased to 47.73 μmol / L ( Figure 2 (E); (3) Under EP intervention, the IC50 of HGC-27 WT cells was 19.43 μmol / L, and the IC50 of HCFC1OE cells was significantly increased to 55.16 μmol / L (E); Figure 2 G).

[0096] The plate colony formation assay further validated the above results. Under the same chemotherapy drug concentration, the number and colony formation rate of HGC-27HCFC1OE cells were significantly higher than those of WT cells. Figure 2 D、 Figure 2 F, Figure 2 H).

[0097] The above results collectively confirm that HCFC1 overexpression not only promotes the proliferation of gastric adenocarcinoma cells, but also mediates significant drug resistance to three first-line clinical chemotherapy regimens: FOLFIRI, FOLFOX, and EP.

[0098] To verify the core role of BCLAF1 in HCFC1-mediated chemotherapeutic resistance, this invention used ASO-BCLAF1 to target and silence BCLAF1 expression in HGC-27 cells, and detected changes in cell proliferation and chemosensitivity. CCK-8 results showed that in HGC-27 WT cells, there was no significant difference in relative cell viability between the ASO-BCLAF1 transfection group and the ASO-NC control group (P>0.05); however, in HGC-27 HCFC1OE cells, compared with the ASO-NC group, ASO-BCLAF1 transfection significantly reduced cell proliferation (P>0.05). Figure 2 B, P<0.01), indicating that BCLAF1 silencing can specifically inhibit the proliferation of HCFC1-overexpressing gastric cancer cells, with no significant effect on wild-type cells.

[0099] Further investigation was conducted to examine the effect of BCLAF1 silencing on chemosensitivity. The results showed that: (1) Under the FOLFIRI regimen, there was no significant difference in IC50 between the ASO-BCLAF1 group and the ASO-NC group in HGC-27 WT cells (11.22 μmol / L vs 10.71 μmol / L); while in HCFC1OE cells, the IC50 of the ASO-NC group was 40.00 μmol / L, and the IC50 of the ASO-BCLAF1 group was significantly higher. 50 Significantly reduced to 15.70 μmol / L ( Figure 2 (C); (2) Under FOLFOX intervention, there was no significant difference in IC50 between the ASO-BCLAF1 group and the ASO-NC group in HGC-27 WT cells (8.571 μmol / L vs 9.529 μmol / L); while in HCFC1OE cells, the IC50 of the ASO-NC group was 47.73 μmol / L, and the IC50 of the ASO-BCLAF1 group was significantly reduced to 17.43 μmol / L (C); Figure 2 E); (3) Under EP intervention, in HGC-27 WT cells, the IC50 of the ASO-BCLAF1 group and the ASO-NC group was significantly different. 50 There was no significant difference (17.39 μmol / L vs 19.43 μmol / L); however, in HCFC1OE cells, the IC50 of the ASO-NC group was 55.16 μmol / L, while the IC50 of the ASO-BCLAF1 group was significantly reduced to 21.72 μmol / L. Figure 2 G).

[0100] The results of the plate colony formation assay were consistent with the IC50 assay results: in HCFC1OE cells, the number and rate of colonies formed in the ASO-BCLAF1 combined with chemotherapy group were significantly lower than those in the ASO-NC combined with chemotherapy group; however, in WT cells, there was no significant difference in colony formation ability between the ASO-BCLAF1 and ASO-NC groups. Figure 2 D、 Figure 2 F, Figure 2 H).

[0101] The above results collectively confirm that targeted silencing of BCLAF1 can specifically reverse chemotherapy resistance in gastric cancer cells mediated by HCFC1 overexpression, restoring their sensitivity to three chemotherapy regimens: FOLFIRI, FOLFOX, and EP.

[0102] Example 3: In vivo targeting of BCLAF1 combined with FOLFOX significantly inhibited the growth of neuroendocrine-differentiated gastric cancer xenografts. 1. Construction and in vivo intervention of nude mouse xenograft tumor model (1) Model construction: HGC-27 WT and HCFC1 in the logarithmic growth phase were selected. OE Cells were resuspended in sterile PBS and the cell concentration was adjusted to 1×10⁻⁶. 8 per mL, at 100 μL / animal (1×10⁻⁶) 7 A dose of (number of cells) was administered subcutaneously to the right back of nude mice.

[0103] (2) Experimental grouping and intervention: The condition of nude mice was observed daily, and the long diameter (a) and short diameter (b) of the tumor were measured with vernier calipers. The results were calculated according to the formula V=a×b. 2 / 2 Calculate tumor volume. When the tumor volume reaches 100 mm... 3 On day 0, the nude mice were randomly divided into 8 groups of 6 mice each, as follows: ①WT+ASO-NC group: ASO-NC (10 mg / kg) was injected subcutaneously around the tumor + normal saline was injected intraperitoneally; ②WT+ASO-BCLAF1 group: ASO-BCLAF1 (10 mg / kg) was injected subcutaneously around the tumor, plus normal saline was injected intraperitoneally; ③WT+ASO-NC+FOLFOX group: ASO-NC (10 mg / kg) was injected subcutaneously around the tumor, followed by FOLFOX injected intraperitoneally. ④WT+ASO-BCLAF1+FOLFOX group: ASO-BCLAF1 (10 mg / kg) was injected subcutaneously around the tumor, followed by FOLFOX injection intraperitoneally; ⑤HCFC1 OE +ASO-NC group: ASO-NC (10 mg / kg) was injected subcutaneously around the tumor + normal saline was injected intraperitoneally; ⑥HCFC1 OE +ASO-BCLAF1 group: ASO-BCLAF1 (10 mg / kg) was injected subcutaneously around the tumor + normal saline was injected intraperitoneally; ⑦HCFC1 OE +ASO-NC+FOLFOX group: ASO-NC (10 mg / kg) was injected subcutaneously around the tumor, followed by FOLFOX injected intraperitoneally. ⑧HCFC1 OE +ASO-BCLAF1+FOLFOX group: ASO-BCLAF1 (10 mg / kg) was injected subcutaneously around the tumor, followed by FOLFOX via intraperitoneal injection.

[0104] (3) Dosing regimen: ASO was administered once every 3 days for a total of 6 times; the FOLFOX regimen was administered with 5-FU 50 mg / kg, oxaliplatin 5 mg / kg and leucovorin 20 mg / kg. 5-FU and leucovorin were dissolved in sterile saline, and oxaliplatin was dissolved in 5% glucose injection. The 5-FU and leucovorin were injected intraperitoneally once every 3 days for a total of 6 times; the control group was injected with an equal volume of sterile saline.

[0105] (4) Indicator monitoring: During the intervention period, the weight of nude mice and the long and short diameters of the tumor were measured every 3 days, and the tumor growth curve and weight change curve were plotted. After the intervention (Day 18), the nude mice were euthanized by cervical dislocation after anesthesia with 1.5% isoflurane. After the tumor tissue was completely removed, the tumor was weighed by electronic balance and photographed for record.

[0106] 2. Paraffin embedding, sectioning, and IHC staining of tumor tissue (1) Paraffin embedding and section preparation: Tumor samples fixed with 4% PFA were sequentially dehydrated with ethanol, cleared with xylene, and infiltrated with paraffin to complete tissue embedding and modular construction. Continuous tissue sections with a thickness of 4 μm were cut using a paraffin microtome and attached to a high-cleanliness, non-detachable glass slide. The sections were then baked at 60℃ for 2 hours and stored at room temperature in a sealed container for later use.

[0107] (2) IHC staining procedure: ① Dewaxing and hydration: The sectioned samples were eluted twice with xylene solution for 15 min each time to completely remove paraffin, and then the tissue was rehydrated by a descending gradient of ethanol solutions (100%-75%, 5 min each time). ② Antigen retrieval: To fully expose the antigenic epitopes masked by formaldehyde cross-linking, the slides were placed in citrate buffer (pH 6.0) and subjected to autoclaving for 2 min. After the ambient temperature naturally cooled to room temperature, the slides were washed with PBS buffer by shaking (5 min × 3 times). ③ Inactivation of endogenous peroxidase: Add endogenous peroxidase blocking solution evenly to the surface of the tissue section and incubate at room temperature in the dark for 15 min to block the activity of endogenous peroxidase in the tissue. After incubation, wash with PBS buffer by shaking (5 min × 3 times). ④ Serum blocking: Add 5% normal goat serum blocking solution, evenly cover the tissue surface, and block at room temperature for 1 hour; ⑤ Immunohistochemistry: Add primary antibodies at specific dilution ratios (HCFC1, CgA, and SYN are all prepared at a ratio of 1:200; Ki-67 is prepared at a ratio of 1:2000), and incubate overnight in a humidified chamber at 4°C. The next day, add HRP-labeled specific secondary antibodies (incubate at room temperature for 1 h) to complete antigen-secondary antibody binding; ⑥ DAB colorimetric reaction: Add freshly prepared DAB colorimetric solution and monitor the reaction process dynamically under a microscope in real time. Stop the reaction immediately after the characteristic brownish-red particle deposition appears; ⑦ Hematoxylin counterstaining and blueing: Hematoxylin was used to perform contrast counterstaining on cell nuclei, and the nuclei were differentiated by hydrochloric acid alcohol to optimize contrast; ⑧ Dehydration, clearing and mounting: The sections then underwent ascending gradient ethanol dehydration (75%, 85%, 95%, 100%, 2 min each time) and xylene clearing treatment (15 min × 2 times), and finally permanent mounting was completed using neutral resin; ⑨ Image acquisition and quantitative analysis: Images were captured using an upright digital scanning microscope, and grayscale and morphological analysis were performed using Image-ProPlus software to quantitatively determine the expression ratio of positive cells.

[0108] The FOLFOX regimen is a first-line standard chemotherapy regimen for the clinical treatment of gastric cancer. Therefore, this invention uses a nude mouse xenograft tumor model to verify in vivo the antitumor effect and chemosensitizing effect of ASO-BCLAF1 combined with the FOLFOX regimen. The in vivo experimental procedure is as follows: Figure 3 A.

[0109] During the intervention period, there was no significant difference in body weight among the groups of nude mice. The ASO-BCLAF1 intervention group showed normal mental status, food and water intake, and no drug-related toxic reactions such as diarrhea, rash, or sudden weight loss occurred. Figure 3 (C) This confirms that ASO-BCLAF1 has good in vivo safety.

[0110] Tumor growth curves showed that the tumor growth rate in the HCFC1OE+ASO-NC group was significantly faster than that in the WT+ASO-NC group (P<0.001), confirming that HCFC1 overexpression can significantly promote tumorigenesis and growth of gastric cancer cells in vivo, consistent with in vitro experimental results. Compared with the HCFC1OE+ASO-NC group, the tumor growth rate in the HCFC1OE+ASO-BCLAF1 group was significantly slower, and the tumor volume was significantly reduced after 18 days of intervention (P<0.001); while in WT cell-derived tumors, there was no significant difference in tumor growth between the ASO-BCLAF1 group and the ASO-NC group (P>0.05).

[0111] Tumor growth rates in all experimental groups treated with combined FOLFOX chemotherapy were significantly slower than in the control group without chemotherapy (P<0.001). Specifically, the tumor growth rate in the HCFC1OE+ASO-NC+FOLFOX group was significantly faster than that in the WT+ASO-NC+FOLFOX group (P<0.001), and the tumor volume was significantly higher after 18 days of intervention, confirming that HCFC1 overexpression can mediate resistance to the FOLFOX regimen in gastric cancer cells. However, the tumor growth rate and endpoint tumor volume in the HCFC1OE+ASO-BCLAF1+FOLFOX group were significantly lower than those in the HCFC1OE+ASO-NC+FOLFOX group (P<0.001). Figure 3 B. Figure 3 D).

[0112] The tumor weight statistics were consistent with the tumor growth curves: the tumor weight in the HCFC1OE+ASO-NC group was significantly higher than that in the WT+ASO-NC group (P<0.001); the tumor weight in the HCFC1OE+ASO-BCLAF1+FOLFOX group was significantly lower than that in the HCFC1OE+ASO-NC+FOLFOX group (P<0.001); however, among tumors derived from WT cells, there was no significant difference in tumor weight between the ASO-BCLAF1 combined with FOLFOX group and the ASO-NC combined with FOLFOX group (P>0.05). Figure 3 E).

[0113] Immunohistochemical results showed that the proportion of Ki67-positive tumors in the HCFC1OE+ASO-NC group was significantly higher than that in the WT+ASO-NC group (P<0.001); the proportion of Ki67-positive tumors in the HCFC1OE+ASO-BCLAF1+FOLFOX group was significantly lower than that in the HCFC1OE+ASO-NC+FOLFOX group (P<0.001); while in WT cell-derived tumors, there was no significant difference in the proportion of Ki67-positive tumors between the ASO-BCLAF1 combined with FOLFOX group and the ASO-NC combined with FOLFOX group (P>0.05). Figure 3 E). In addition, ASO-BCLAF1 significantly reduced the positive rates of BCLAF1, POU3F2, CGA, and SYN in the HCFC1 overexpression group.

[0114] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An antisense oligonucleotide, characterized in that, Its sequence is shown in SEQ ID NO.

5.

2. The antisense oligonucleotide according to claim 1, characterized in that, The cytosine in the antisense oligonucleotide is 5-methylcytosine.

3. The antisense oligonucleotide according to claim 1, characterized in that, The antisense oligonucleotide contains a phosphate thioester backbone modification.

4. The antisense oligonucleotide according to claim 1, characterized in that, The antisense oligonucleotide has a gapmer structure.

5. The antisense oligonucleotide according to claim 4, characterized in that, The gapmer structure is a 5-11-5 chimeric pattern, where the 5 nucleotides at the 5' end and the 5 nucleotides at the 3' end are wing regions, and the 11 nucleotides in the middle are gap regions.

6. The antisense oligonucleotide according to claim 5, characterized in that, The nucleotides in the wing region are modified with 2'-O-(2-methoxyethyl).

7. The antisense oligonucleotide according to claim 1, characterized in that, The antisense oligonucleotide has the following modified configuration: 5'-(MOE-T)(MOE-T)(MOE-5-Me-C)(MOE-A)(MOE-G)ACTTTACCTGC(MOE-T) (MOE-5-Me-C) (MOE-A)(MOE-G)(MOE-G)-3'; in" " indicates modification of the thiophosphate skeleton, (MOE-) indicates 2'-O-(2-methoxyethyl) modification, and (5-Me-C) indicates 5-methylcytosine.

8. A pharmaceutical composition, characterized in that, It includes the antisense oligonucleotide as described in any one of claims 1-7, and a pharmaceutically acceptable carrier or excipient.

9. Use of the antisense oligonucleotide of any one of claims 1-7 or the pharmaceutical composition of claim 8 in the preparation of a medicament for treating mixed glandular neuroendocrine carcinoma.

10. The application according to claim 9, characterized in that, The drug can be used in combination with chemotherapy drugs.

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