MYEOV expression inhibitor, pharmaceutical composition and application of MYEOV expression inhibitor in preparation of pancreatic cancer gemcitabine drug resistance resisting drugs

Abstract

The invention discloses an MYEOV expression inhibitor, a pharmaceutical composition and application of the MYEOV expression inhibitor and the pharmaceutical composition in preparation of pancreatic cancer gemcitabine resistance drugs, and relates to the technical field of biology, in particular to the MYEOV expression inhibitor which is shRNA, and the nucleotide sequence of the shRNA is shown as SEQ ID NO.1. The invention further discloses an application of the MYEOV expression inhibitor and the pharmaceutical composition in preparation of pancreatic cancer gemcitabine resistance drugs. By knocking out or inhibiting the expression of MYEOV, the drug resistance of pancreatic cancer to a chemotherapeutic drug gemcitabine can be weakened.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a MYEOV expression inhibitor, a pharmaceutical composition, and its use in the preparation of a gemcitabine-resistant drug for pancreatic cancer. Background Technology

[0002] The global annual incidence of pancreatic cancer is approximately 13.1 per 100,000, with a five-year survival rate of only about 13%. Due to its highly aggressive nature, lack of typical early symptoms, and insidious lesions, most patients are diagnosed at an advanced stage with metastasis, thus losing the opportunity for radical surgery. Radical surgery is only suitable for 15-20% of early-stage patients, but the recurrence rate is extremely high. Chemotherapy is a core treatment method for all stages of pancreatic cancer, including neoadjuvant, adjuvant, advanced, and palliative care, with the core goal of controlling tumor progression, alleviating clinical symptoms, reducing the risk of postoperative recurrence, and prolonging overall survival. However, its efficacy is severely limited by tumor heterogeneity and drug resistance mechanisms.

[0003] Gemcitabine is a nucleoside analogue antimetabolite that has long been the dominant first-line standard of care in systemic chemotherapy for pancreatic cancer. However, drug resistance has limited its efficacy, resulting in limited improvement in patient prognosis. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, where gemcitabine resistance limits its efficacy and thus the improvement of patient prognosis is not significant, the present invention provides a MYEOV expression inhibitor, a pharmaceutical composition, and its use in the preparation of an anti-gemcitabine-resistant drug for pancreatic cancer, thereby solving the defect that gemcitabine resistance limits its efficacy and thus the improvement of patient prognosis is not significant.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A MYEOV expression inhibitor, wherein the MYEOV expression inhibitor is shRNA, and the nucleotide sequence of the shRNA is shown in SEQ ID NO. 1.

[0006] Optionally, MYEOV expression inhibitors can be knocked down and overexpressed in PANC-1 / GR and CFPAC-1 / GR cells.

[0007] The present invention also provides a pharmaceutical composition comprising a MYEOV expression inhibitor and gemcitabine, wherein the MYEOV expression inhibitor is shRNA, and the nucleotide sequence of the shRNA is shown in SEQ ID NO. 1.

[0008] Optionally, the pharmaceutical composition may include pharmaceutically acceptable excipients or carriers.

[0009] Optionally, the excipients include one or more of suspending agents, suspending aids, thickeners, colorants, and antioxidants.

[0010] Optionally, the excipients may also include one or more of the following: pH adjuster, osmotic pressure adjuster, thickener, wetting agent, and coating material.

[0011] Optionally, the pharmaceutical composition may be an oral formulation or an injectable formulation.

[0012] Optionally, the oral preparation may be granules, tablets, capsules, powders, syrups, oral liquids, or tinctures.

[0013] Optionally, the injectable formulation is an intravenous injection formulation, an intramuscular injection formulation, or an injectable powder.

[0014] This invention discloses the use of a MYEOV expression inhibitor in the preparation of a gemcitabine-resistant drug for pancreatic cancer. The MYEOV expression inhibitor is shRNA, and the nucleotide sequence of the shRNA is shown in SEQ ID NO. 1.

[0015] The beneficial effects of the present invention include at least the following: The MYEOV expression inhibitor of the present invention can weaken the resistance of pancreatic cancer to the chemotherapy drug gemcitabine by knocking out or inhibiting the expression of MYEOV; (2) The present invention can overcome the resistance of pancreatic cancer to chemotherapy drugs by synthesizing MYEOV inhibitors or knockout reagents and using them in combination with gemcitabine when treating pancreatic cancer. It has important guiding significance and broad prospects in clinical application. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the Western blot detection results of MYEOV protein expression in Example 1 of the present invention; Figure 2 This is a statistical result graph showing the MYEOV mRNA level in pancreatic cancer cells after lentivirus knockdown and overexpression of MYEOV in Examples 1, 2, Comparative Examples 1 and 2 of the present invention. Figure 3 This is a schematic diagram showing the results of MYEOV protein levels in pancreatic cancer cells after lentivirus knockdown and overexpression of MYEOV in Examples 1, 2, 1, and 2 of the present invention. Figure 4a These are colony formation diagrams of PANC-1 / GR and CFPAC-1 / GR cells with MYEOV knockdown, overexpression, and control group cells in the colony formation experiments of Examples 1, 2, Comparative Examples 1 and 2 of the present invention. Figure 4bThis is a statistical analysis chart of the colony formation rate of PANC-1 / GR cells with MYEOV knockdown, overexpression, and control group cells in the colony formation experiments of Examples 1, 2, Comparative Examples 1 and 2 of the present invention; Figure 4c This is a statistical analysis of the colony formation rate of CFPAC-1 / GR cells with MYEOV knockdown, overexpression, and control group cells in the colony formation experiments of Examples 1, 2, Comparative Examples 1 and 2 of the present invention. Figure 5a These are fluorescence images of the PANC-1 / GR cell control group and Edu-positive cells after knockdown and overexpression of MYEOV in Examples 1, 2, 1, and 2 of this invention. Figure 5b This is a statistical analysis of the PANC-1 / GR cell control group and the Edu-positive cell rate after knockdown and overexpression of MYEOV in Examples 1, 2, Comparative Examples 1 and 2 of the present invention. Figure 6a These are fluorescence images of CFPAC-1 / GR cell control groups and Edu-positive cells after knockdown and overexpression of MYEOV in Examples 1, 2, Comparative Examples 1 and 2 of the present invention. Figure 6b This is a statistical analysis of the CFPAC-1 / GR cell control group and the Edu-positive cell rate after knockdown and overexpression of MYEOV in Examples 1, 2, Comparative Examples 1 and 2 of the present invention. Figure 7 This is a flowchart of the nude mouse tumor model experiments of Embodiment 1, Embodiment 2, Comparative Example 1 and Comparative Example 2 of the present invention; Figure 8 These are comparative images of nude mice and schematic diagrams of tumor size from the nude mouse animal tumor model experiments of Examples 1, 2, Comparative Examples 1 and 2 of the present invention; Figure 9 This is a statistical analysis chart of tumor volume in nude mouse animal tumor model experiments of Examples 1 and 2, and Comparative Examples 1 and 2 of the present invention; Figure 10 This is a statistical analysis chart of tumor weight in nude mouse animal tumor model experiments of Examples 1 and 2, and Comparative Examples 1 and 2 of the present invention; Figure 11 This is a statistical analysis chart of the weight of nude mice in the nude mouse tumor model experiments of Examples 1 and 2, and Comparative Examples 1 and 2 of the present invention. Detailed Implementation

[0017] The present invention will now be further described with reference to the accompanying drawings and specific embodiments.

[0018] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used in this invention are conventional in the art and can be obtained through conventional commercial means; unless otherwise specified, the methods used in this invention are conventional methods in the art.

[0019] In this invention, "sh" refers to short hairpin RNA.

[0020] In this invention, OE refers to over-expression.

[0021] In this invention, WT is an abbreviation for Wild-Type. For example, PANC-1 is a classic human pancreatic cancer cell line, commonly used in pancreatic cancer research.

[0022] PANC-1 / WT represents PANC-1 cells in their natural state, without any gene editing or modification. Their gene sequence and biological characteristics remain unchanged from the original cell line.

[0023] CFPAC-1 / WT represents CFPAC-1 cells in their natural state, without gene editing or modification. Their gene sequence and biological characteristics remain unchanged from the original cell line.

[0024] In this invention, DMEM (Dulbecco's Modified Eagle Medium) is a basic cell culture medium.

[0025] In this invention, PBS (phosphate-buffered saline) is a commonly used buffer in biological experiments for washing cells or diluting samples. PBS can be a product of Thermo Fisher Scientific China.

[0026] The RIPA lysis buffer (Radio Immunoprecipitation Assay Lysis Buffer) used in this invention is a commonly used rapid cell / tissue lysis buffer, mainly used to extract soluble proteins from animal cells or tissues, and is suitable for Western Blot (WB), immunoprecipitation (IP), and co-immunoprecipitation (Co-IP).

[0027] In this invention, Western blotting refers to the protein blotting method (or immunoblotting method).

[0028] In this invention, ATCC (American Type Culture Collection) is a globally renowned biological resource center, whose full name is the American Type Culture Collection.

[0029] In this invention, ACTB is the gene encoding β-actin, located on human chromosome 7, and the protein it encodes has a molecular weight of approximately 42-43 kDa. As a housekeeping gene, ACTB is relatively stably expressed in most cells and is often used as an internal control in experiments (such as Western blotting and qPCR).

[0030] In this invention, the loading buffer is the sample loading buffer.

[0031] PVDF membrane is a polyvinylidene fluoride membrane.

[0032] In this invention, CFPAC-1 / GR is a gemcitabine (GEM) resistant cell line derived from the human pancreatic cancer cell line CFPAC-1.

[0033] EdU (5-ethynyl-2'-deoxyuridine) is a novel thymidine deoxynucleoside analog that can replace thymidine and be incorporated into newly synthesized DNA during DNA replication.

[0034] Its acetylenic group can undergo a covalent reaction with fluorescent or biotin-labeled azide to form a stable triazole ring, thereby allowing cell proliferation to be detected by fluorescence.

[0035] Hoechst 33342 solution is a commonly used reagent for staining cell nuclei.

[0036] Preparation of staining solution: 100× stock solution: Dilute with PBS to 10-50 μM.

[0037] Live cell staining: Add directly to the culture medium.

[0038] Staining procedure: Adherent cells: Cover the sample and incubate at room temperature for 3-5 minutes.

[0039] Suspension cells: Add 3 times the volume of staining solution, mix well and incubate.

[0040] Washing: Wash 2-3 times with PBS.

[0041] Click reaction solution is a commonly used chemical reagent in biomarking and drug development, mainly used to detect cell proliferation or synthesize specific compounds.

[0042] The drug-resistant pancreatic cancer cell line PANC-1 / GR is a drug-resistant subpopulation obtained by inducing the parental cell line PANC-1 with gemcitabine (GEM).

[0043] The BCA (Bicinchoninic Acid Assay) method used in this invention is a protein quantification method widely used in the field of biochemistry.

[0044] In this invention, TBST (Tris-Buffered Saline with Tween) is a commonly used washing buffer in biochemical experiments, mainly used for membrane washing steps in experiments such as Western blot and immunohistochemistry. Its core components include a Tris-HCl buffer system, sodium chloride (NaCl), and Tween-20.

[0045] In this invention, SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) was used. ACTB is the gene name for β-actin, which encodes a protein that is an important component of the cytoskeleton.

[0046] SDS-PAGE can be performed using products from Changzhou Boyi Biotechnology Co., Ltd.

[0047] EP tubing is a precision tubing commonly used in laboratories and industries.

[0048] EP tubes can be products of Sangon Biotech (Shanghai) Co., Ltd.

[0049] The wet transfer method in this invention is a classic method for transferring proteins from a gel to a membrane in Western blotting experiments, and it is particularly suitable for the transfer of high molecular weight proteins (>100 kDa). Specific operations include: 1) Prepare the transfer apparatus: Immerse the transfer clamp, sponge pad, filter paper, and membrane in transfer buffer (containing methanol) for 10 minutes; 2) Activate the PVDF membrane: Pre-activate with methanol for 30 seconds, then place it in the transfer solution for later use; 3) Assemble the "sandwich": Stack the cathode plate, sponge pad, filter paper, gel, PVDF membrane, filter paper, sponge pad, and anode plate in sequence; 4) Transfer conditions: Adjust the voltage, current and time according to the target molecular weight. Low molecular weight proteins can be transferred quickly.

[0050] In this invention, sh-MYEOV, OE-MYEOV lentiviral particles and control lentiviral particles were provided by Shanghai Geneplus Technology Co., Ltd.

[0051] A. Regarding the construction of a gemcitabine-resistant pancreatic cancer cell model: 1) Cell Culture: The human pancreatic cancer cells (PANC-1 and CFPAC-1) used were purchased from ATCC in the United States. Gemcitabine-resistant pancreatic cancer cells (PANC-1 / GR and CFPAC-1 / GR) were induced and established by the applicant's laboratory. PANC-1 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-dextrin antibiotics, and CFPAC-1 cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-dextrin antibiotics. The culture conditions were 5% CO2 and 37°C. Gemcitabine-resistant cells were cultured with an additional appropriate concentration of gemcitabine in the medium. 2) Construction of gemcitabine-resistant cells: Human pancreatic cancer cells (PANC-1 and CFPAC-1) were induced with an initial concentration of 0.1 μM. After 24 h of drug administration, the cells were cultured in a drug-free medium until they could grow stably and be passaged in the medium at that concentration. Then, the concentration of gemcitabine was gradually and steadily increased until pancreatic cancer gemcitabine-resistant cell lines resistant to 1 μM drug concentration were obtained, namely PANC-1 / GR and CFPAC-1 / GR cells.

[0052] Example 1: This invention discloses a MYEOV expression inhibitor as shRNA, the nucleotide sequence of which is shown in SEQ ID NO. 1. Specifically, SEQ ID NO. 1 is CTCACCTGGGAGAAGCCTTTA.

[0053] sh-MYEOV cells were obtained by infecting gemcitabine-resistant pancreatic cancer cells (PANC-1 / GR and CFPAC-1 / GR) with lentivirus at doses of 0.25 μM, 0.5 μM, and 1 μM.

[0054] Example 2: This invention discloses a MYEOV expression inhibitor as shRNA, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0055] OE-MYEOV cells were obtained by infecting gemcitabine-resistant pancreatic cancer cells (PANC-1 / GR and CFPAC-1 / GR) with lentivirus at doses of 0.25 μM, 0.5 μM, and 1 μM.

[0056] Comparative Example 1: Comparative Example 1 is the sh control group, which is the blank control group of the sh-MYEOV cell group in Example 1.

[0057] Comparative Example 2: Comparative Example 2 is the OE control group, which is the blank control group of the OE-MYEOV cell group in Example 2.

[0058] Example 3: The present invention also provides a pharmaceutical composition comprising a MYEOV expression inhibitor and gemcitabine, wherein the MYEOV expression inhibitor is shRNA, and the nucleotide sequence of the shRNA is shown in SEQ ID NO. 1.

[0059] The pharmaceutical composition includes pharmaceutically acceptable excipients or carriers.

[0060] The excipients include one or more of the following: suspending agents, suspending aids, thickeners, colorants, and antioxidants.

[0061] The excipients also include one or more of the following: pH adjuster, osmotic pressure adjuster, thickener, wetting agent, and coating material.

[0062] The pharmaceutical composition is an oral or injectable formulation.

[0063] The oral preparations are granules, tablets, capsules, powders, syrups, oral liquids, or tinctures.

[0064] The injectable preparation is an intravenous injection preparation, an intramuscular injection preparation, or an injectable powder.

[0065] Example 4: This invention discloses the use of a MYEOV expression inhibitor in the preparation of a gemcitabine-resistant drug for pancreatic cancer. The MYEOV expression inhibitor is shRNA, and the nucleotide sequence of the shRNA is shown in SEQ ID NO. 1.

[0066] B. Western blot analysis to verify MYEOV protein expression levels at different drug resistance stages: Western blot experiments were performed on the constructed gemcitabine-resistant pancreatic cancer cells to verify the expression level of MYEOV protein at different drug resistance stages.

[0067] The specific steps are as follows: 1) Total protein extraction: Discard the culture medium from the cells prepared one day in advance and wash them three times with PBS; add RIPA protein lysis buffer containing 1% protease inhibitor and lyse the cells on ice for 30 minutes; scrape off the cells and transfer the lysis buffer to EP tubes; sonicate the cells using an ultrasonic cell disruptor under ice conditions to more thoroughly lyse the cells and break down DNA. Centrifuge at low temperature and high speed: 4°C, 12000g, 15 min; transfer the supernatant to a new EP tube; determine protein concentration using the BCA method. 2) Protein sample preparation: Based on the actual measured protein concentration, the proteins in each group were diluted to a uniform concentration using RIPA lysis buffer. An appropriate volume of 5× loading buffer containing 10% β-mercaptoethanol was added to each tube, and the mixture was vortexed. The tubes were then boiled in a 95°C metal bath for 5 minutes to denature the proteins. 3) Western blot analysis: ① Protein electrophoresis: Denatured proteins are separated according to molecular weight using SDS-PAGE electrophoresis; ② Protein transfer: The separated protein was transferred from the gel to a PVDF membrane. Wet transfer was used, with the membrane transferred at 350 mA for 1 hour under ice bath conditions. ③ Seal: Incubate with 5% skim milk powder at room temperature for 1 hour; ④ Primary antibody incubation: Add the primary antibody that specifically binds to MYEOV and ACTB, and incubate overnight at 4 °C; ⑤ Washing the membrane: Wash the PVDF membrane with TBST 3 times, 10 minutes each time; ⑥ Secondary antibody incubation: Add HRP-labeled secondary antibody and incubate at room temperature for 1 hour; ⑦ Washing the membrane: Wash the PVDF membrane with TBST 3 times, 10 min each time; ⑧ Imaging: Expose the imager, record and analyze the imaging results.

[0068] The Western blot results of Examples 1, 2, Comparative Examples 1 and 2 are as follows: Figure 1 As shown.

[0069] Figure 1 The PANC-1 cell line included a control group (PANC-1 / WT) and GR drug treatment groups (0.25 μM, 0.5 μM, 1 μM).

[0070] Figure 1 The CFPAC-1 cell line included a control group (CFPAC-1 / WT) and GR drug treatment groups (0.25 μM, 0.5 μM, 1 μM).

[0071] MYEOV protein (-33KDA): In PANC-1 cells, the MYEOV band in the control group (PANC-1 / WT) was faint; as the concentration of GR treatment increased (0.25μM→0.5μM→1μM), the brightness of the MYEOV band gradually increased (expression level increased). In CFPAC-1 cells, the control group (CFPAC-1 / WT) had almost no MYEOV band; as the concentration of GR treatment increased, the brightness of the MYEOV band also gradually increased, and was most obvious at 1μM.

[0072] Internal control ACTB (-42 KDA): The ACTB band brightness was uniform across all groups in both cell lines, indicating consistent loading amounts and validating the reliability of the experimental loading.

[0073] The results showed that in PANC-1 and CFPAC-1 cells, the protein expression level of MYEOV gradually increased as gemcitabine resistance gradually increased.

[0074] C. Constructing pancreatic cancer cell lines that knock down and overexpress MYEOV: a. Lentiviral infection: Example 1 and Example 2: Knockdown and overexpression of MYEOV in PANC-1 / GR and CFPAC-1 / GR cells: 5×10 4 PANC-1 / GR and CFPAC-1 / GR cells were seeded in 24-well culture plates and cultured for 24 hours to allow the cells to adhere and recover. At an MOI (multiple of infection) of 10, 5 μL of virus solution was added to each well, the culture plate was gently shaken, and the cells were cultured for another 24 hours before being replaced with fresh culture medium. 72 hours after virus infection, the cell culture medium was replaced with medium containing 2 μg / mL puromycin to obtain stable MYEOV knockdown and overexpression cell lines.

[0075] b. Verification of knockdown and overexpression efficiency: 1) qRT-PCR verification of transcriptional knockdown and overexpression effects: Total RNA was extracted from cells using Trizol reagent. Using a reverse transcription kit, cDNA was synthesized by reverse transcription using 1 μg of RNA as a template and then used for qRT-PCR analysis. Primers are shown in Table 1 below.

[0076] Table 1. Gene primer sequences: ; 2) Western blot experiments to verify the effects of protein knockdown and overexpression: As shown in the Western blot experiment steps in the above implementation case, sh-MYEOV, OE-MYEOV and total protein of control cells were extracted from PANC-1 / GR and CFPAC-1 / GR cells, and the changes in MYEOV protein expression level were detected.

[0077] The results showed that the knockdown efficiency of MYEOV mRNA levels in PANC-1 / GR and CFPAC-1 / GR cells in Examples 1 and 2 was 99%, and the expression level increased 12-fold after overexpression.

[0078] like Figure 2As shown, Figure 2 These are two bar charts showing the MYEOV mRNA levels in pancreatic cancer cells after lentiviral knockdown and overexpression of MYEOV in Examples 1, 2, 1, and 2, respectively. They illustrate the changes in MYEOV gene mRNA expression levels in different cell lines (PANC-1 / GR, CFPAC-1 / GR) after different treatments. The left figure shows a comparison of the expression of the "control group" and the "sh-MYEOV group (MYEOV gene silence)".

[0079] The MYEOV mRNA expression level in the control group was high (close to 1), while the expression in the sh-MYEOV group was significantly reduced (close to 0); indicating that the difference between the two groups was statistically significant.

[0080] The right-hand figure compares the expression of the "control group" and the "OE-MYEOV group (MYEOV gene overexpression)".

[0081] The expression level of MYEOV mRNA in the control group was very low (close to 1), while the expression in the OE-MYEOV group was significantly increased (more than 10); this also indicates that the difference was statistically significant.

[0082] The two figures verified that sh-MYEOV effectively silences the MYEOV gene, while OE-MYEOV effectively overexpresses the MYEOV gene, with consistent results in both cell lines. The results showed a 90% knockdown efficiency and a 3-fold overexpression efficiency.

[0083] D. Regarding the increased sensitivity of pancreatic cancer cells to gemcitabine after MYEOV knockdown: a. Clonogenic assay: Pancreatic cancer cells in logarithmic growth phase, including sh-MYEOV, OE-MYEOV, and control cells, were digested with trypsin, collected, resuspended in complete culture medium to form a single-cell suspension, and counted. 1000 cells were seeded into each well of a 6-well plate. The cells were cultured for approximately 10-14 days, with the medium changed every 3 days and cell status observed. After culture, the culture medium was discarded, and 1 mL of 4% paraformaldehyde was added to each well to fix the cells for 15 min. After washing three times with PBS, 1 mL of 0.1% crystal violet solution was added to each well for staining for 15 min. The stain was washed away with running water, and the cells were air-dried. The entire 6-well plate and each well were photographed, and the number of clones with more than 10 cells was counted. The clonogenic rate of MYEOV knockdown, overexpression, and control cells was calculated: Clonogenic rate = (number of clones / number of seeded cells) × 100%.

[0084] like Figure 3As shown, the target protein MYEOV (molecular weight approximately 33 KDA) was used to verify the gene expression regulation effect. The internal control protein ACTB (β-actin, molecular weight approximately 42 KDA) was used to control the consistency of sample loading. In both cell types, the MYEOV band brightness in the sh-MYEOV group was weaker than that in the sh-control group, proving that the MYEOV gene silencing was successful.

[0085] In both cell types, the MYEOV band brightness in the OE-MYEOV group was significantly stronger than that in the OE-control group, demonstrating successful MYEOV gene overexpression. Furthermore, the uniformity of ACTB band brightness across all groups indicates consistent loading amounts and reliable experimental results.

[0086] The results showed that knockdown of MYEOV in PANC-1 / GR and CFPAC-1 / GR cells in Examples 1 and 2 significantly inhibited the colony formation of pancreatic cancer cells, while overexpression of MYEOV significantly promoted colony formation. In other words, knockdown of MYEOV significantly inhibited gemcitabine resistance in pancreatic cancer cells.

[0087] b. EdU-based cell proliferation assay: Pancreatic cancer cells in logarithmic growth phase, including sh-MYEOV from Example 1, OE-MYEOV from Example 2, the control group from Comparative Example 1, and the control group from Comparative Example 2, were analyzed at a concentration of 2 × 10⁻⁶ cells / mL. 4 Cells were seeded per well in a 24-well plate and cultured overnight until they recovered to normal condition. EdU reagent was added, and the cells were incubated for another 4 hours. The culture medium was discarded, and 1 mL of 4% paraformaldehyde was added to fix the cells at room temperature for 15 minutes. The fixative was discarded, and each well was washed three times with 1 mL of PBS for 5 minutes each time. The PBS was discarded, and 1 mL of permeabilization buffer was added to each well, and the cells were incubated at room temperature for 15 minutes. The permeabilization buffer was discarded, and each well was washed twice with 1 mL of PBS for 5 minutes each time. The PBS was discarded, and 0.21 mL of Click reaction solution was added to each well. The mixture was gently shaken to ensure that the reaction mixture could evenly cover the sample, and the cells were incubated at room temperature in the dark for 30 minutes. The Click reaction solution was discarded, and each well was washed three times with 11 mL of PBS for 5 minutes each time. Hoechst 33342 was diluted 1:1000 with PBS. After removing the PBS, 1 mL of 1X Hoechst 33342 solution was added to each well, and the cells were incubated at room temperature in the dark for 10 minutes. The 1X Hoechst 33342 solution was aspirated, and 1 mL of PBS was added to each well. Wash three times with mLPBS for 5 minutes each time; then observe and count EdU-positive cells using a fluorescence microscope.

[0088] like Figure 4a As shown, in which, 1) the top row consists of PCNAGR cells.

[0089] sh-control group: The culture dish contains a large number of dense purple clonal spots, representing the clonal formation capacity of the cells.

[0090] In the sh-MYEOV group, the number of purple clonal spots was significantly reduced and their distribution was sparse, indicating that the clonogenic ability of PCNAGR cells was suppressed after the MYEOV gene was silenced.

[0091] OE-control group: The number of clonal spots is similar to that of the sh-control group, serving as the baseline control for overexpression experiments.

[0092] The OE-MYEOV group showed a greater number and density of purple clonal spots, indicating that overexpression of the MYEOV gene enhanced the clonogenic ability of PCNAGR cells.

[0093] 2) The bottom row consists of CFPAC-1 / GR cells.

[0094] sh-control group: A certain number of purple clonal spots are present in the culture dish, which is the background clonal formation ability of the cells.

[0095] In the sh-MYEOV group, the number of purple clonal spots was significantly reduced, with only a few sparse spots remaining, indicating that silencing MYEOV also inhibited the clonogenic ability of CFPAC-1 / GR cells.

[0096] OE-control group: The number of clonal spots is similar to that of the sh-control group, serving as the baseline control for the overexpression experiment.

[0097] The OE-MYEOV group had significantly more purple clonal spots than the OE-control group, indicating that overexpression of MYEOV also enhanced the clonogenic ability of CFPAC-1 / GR cells.

[0098] like Figure 4b As shown, the sh-control group (dark blue column) has approximately 150 colonies, which represents the baseline clonogenic capacity of the cells.

[0099] sh-MYEOV group (yellow column): Colony formation count decreased to approximately 50, marked as "" compared to the sh-control group. (This usually represents P<0.01, indicating a highly significant difference), demonstrating that silencing the MYEOV gene significantly inhibits the clonogenic ability of PANC-1 / GR cells.

[0100] OE - Control group (green column): The number of colonies formed was approximately 110, which is the baseline level of the overexpression experiment.

[0101] OE-MYEOV group (orange column): Colony formation count increased to approximately 180, marked with "" compared to the OE-control group. "(The difference was extremely significant), demonstrating that the clonogenic ability of PANC-1 / GR cells was significantly enhanced after overexpression of the MYEOV gene."

[0102] like Figure 4c As shown, the sh-control group (dark blue column) has approximately 60 colonies, which represents the baseline clonogenic capacity of the cells.

[0103] sh-MYEOV group (yellow column): Colony formation count decreased to approximately 15, marked as "" compared to the sh-control group. (P<0.01, indicating extremely significant difference), demonstrating that silencing the MYEOV gene significantly inhibited the clonogenic ability of CFPAC-1 / GR cells.

[0104] OE - Control group (green column): The number of colonies formed was approximately 45, which is the baseline level of the overexpression experiment.

[0105] OE-MYEOV group (orange column): Colony formation increased to approximately 80, compared to the OE-control group (marked ""). (representing P) <0.05 (significant difference), demonstrating that overexpression of the MYEOV gene significantly enhanced the clonogenic ability of CFPAC-1 / GR cells.

[0106] like Figure 5a As shown, the effect of knocking down MYEOV (sh-control group vs sh-MYEOV): the sh-control group had a higher number of EdU-positive cells (red); while the sh-MYEOV group had a significantly reduced number of EdU-positive cells, indicating that knocking down MYEOV inhibits the proliferation of PANC-1 / GR cells.

[0107] Effects of MYEOV overexpression (OE-control group vs OE-MYEOV): The OE-control group had fewer EdU-positive cells, while the OE-MYEOV group had a significantly increased number of EdU-positive cells, indicating that MYEOV overexpression promotes the proliferation of PANC-1 / GR cells.

[0108] Clearly, the MYEOV gene is a proliferation promoter of PANC-1 / GR cells: knocking down the gene inhibits cell proliferation, while overexpression enhances it.

[0109] like Figure 5b As shown, the effect of knocking down MYEOV was that the EdU-positive cell rate in the sh-MYEOV group (approximately 0.15%) was much lower than that in the sh-control group (approximately 0.45%), and the difference was extremely significant, verifying that "knockdown of MYEOV inhibits cell proliferation".

[0110] Effects of MYEOV overexpression: The EdU-positive cell rate in the OE-MYEOV group (approximately 0.65%) was significantly higher than that in the OE-control group (approximately 0.35%), confirming that "MYEOV overexpression promotes cell proliferation".

[0111] like Figure 6a As shown, knockdown of MYEOV (sh-MYEOV group): Compared with the sh-control group, EdU-positive cells (red) were significantly reduced, indicating that MYEOV knockdown inhibits cell proliferation.

[0112] Overexpression of MYEOV (OE-MYEOV group): Compared with the OE-control group, the number of EdU-positive cells increased significantly, indicating that MYEOV overexpression promotes cell proliferation.

[0113] like Figure 6b As shown, knockdown of MYEOV (sh-MYEOV group): compared with the sh-control group, the EdU positive cell rate decreased significantly (from about 0.25 to 0.1), verifying that "MYEOV knockdown inhibits cell proliferation".

[0114] Overexpression of MYEOV (OE-MYEOV group): Compared with the OE-control group, the EdU-positive cell rate increased significantly (from about 0.19 to 0.37), verifying that "MYEOV overexpression promotes cell proliferation".

[0115] In summary, fluorescence microscopy revealed that MYEOV knockdown significantly reduced cell proliferation, while MYEOV overexpression promoted cell proliferation.

[0116] c. Animal experiments: A nude mouse model of pancreatic cancer was established. Pancreatic cancer cells from Example 1 (sh-MYEOV), Example 2 (OE-MYEOV), the control group of Comparative Example 1, and the control group of Comparative Example 2 were subcutaneously inoculated to form tumors. All mice were treated with gemcitabine for 21 days.

[0117] like Figure 7 As shown, specifically, 2.5 × 10⁻⁶ mg / L was injected into nude mice. 6 One CFPAC-1 cell. -14d: Nude mice were injected with CFPAC-1 cells for tumor xenograft; 0d: When the tumor volume in the nude mice grew to approximately 100 mm. 3 At this time, subsequent experiments were initiated; 0d, 4d, 8d, 12d, 16d, and 20d: gemcitabine injections and tumor size measurements were performed at these time points. Nude mice were sacrificed after 20 days (or at the end of the experimental cycle). The gemcitabine dose during this period was 50 mg / kg.

[0118] During the treatment, the weight and subcutaneous tumor volume of nude mice were measured every two days. After the treatment, the nude mice were sacrificed, and the final weight and volume of the subcutaneous tumor were recorded. The effects of MYEOV knockdown and overexpression on gemcitabine resistance in pancreatic cancer were analyzed.

[0119] like Figure 8 As shown in the figures on the left and right, the subcutaneous tumor volume in nude mice of the sh-control group was significantly larger than that in nude mice of the sh-MYEOV group. Conversely, the subcutaneous tumor volume in nude mice of the OE-control group was significantly smaller than that in nude mice of the OE-MYEOV group.

[0120] like Figure 9 As shown, the tumor volume growth rate in the sh-MYEOV group was significantly slower than that in the sh-control group; the tumor volume growth rate in the OE-MYEOV group was significantly faster than that in the OE-control group. The figure shows that MYEOV promotes the growth of CFPAC-1 transplanted tumors in vivo.

[0121] like Figure 10 As shown, MYEOV knockdown (sh-MYEOV group): compared with the sh-control group, tumor weight was significantly reduced, indicating that MYEOV knockdown inhibited tumor growth in vivo.

[0122] Overexpression of MYEOV (OE-MYEOV group): Compared with the OE-control group, the tumor weight was significantly increased, indicating that MYEOV overexpression promoted tumor growth in vivo.

[0123] like Figure 11 As shown, the weight of nude mice in the four groups showed a slight fluctuation and a slow upward trend throughout the experimental period. The weight of each group remained in a similar range of about 22g, and there was no significant difference between the groups.

[0124] In summary, the subcutaneous tumors in the MYEOV knockdown group of Example 1 were significantly smaller in volume than those in the control group after gemcitabine treatment, while the subcutaneous tumors in the MYEOV overexpression group of Example 2 were significantly larger in volume than those in the control group after gemcitabine treatment. Furthermore, the growth curves of the subcutaneous tumors during treatment also showed the same trend. That is, in a nude mouse model of pancreatic cancer, MYEOV knockdown in Example 1 significantly reduced gemcitabine resistance, while MYEOV overexpression in Example 2 significantly increased gemcitabine resistance, indicating that MYEOV is a key target for gemcitabine resistance in pancreatic cancer in vivo.

[0125] The above description is merely a preferred embodiment of the present invention and does not limit the scope of patent protection of the present invention. Any equivalent structural transformations made based on the description and drawings of the present invention, whether directly or indirectly applied to other related technical fields, are similarly included within the scope of protection of the present invention.

Claims

1. A MYEOV expression inhibitor, characterized in that, Includes shRNA, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The MYEOV expression inhibitor as described in claim 1, characterized in that, MYEOV expression inhibitors were knocked down and overexpressed in PANC-1 / GR and CFPAC-1 / GR cells.

3. A pharmaceutical composition, characterized in that, The invention includes a MYEOV expression inhibitor and gemcitabine, wherein the MYEOV expression inhibitor comprises shRNA, the nucleotide sequence of which is shown in SEQ ID NO.

1.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition includes pharmaceutically acceptable excipients or carriers.

5. The pharmaceutical composition according to claim 4, characterized in that, The excipients include one or more of the following: suspending agents, suspending aids, thickeners, colorants, and antioxidants.

6. The pharmaceutical composition according to claim 4, characterized in that, The excipients also include one or more of the following: pH adjuster, osmotic pressure adjuster, thickener, wetting agent, and coating material.

7. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is an oral or injectable formulation.

8. The pharmaceutical composition according to claim 7, characterized in that, The oral preparations are granules, tablets, capsules, powders, syrups, oral liquids, or tinctures.

9. The pharmaceutical composition according to claim 7, characterized in that, The injectable preparation is an intravenous injection preparation, an intramuscular injection preparation, or an injectable powder.

10. The use of a MYEOV expression inhibitor in the preparation of a gemcitabine-resistant drug for pancreatic cancer, characterized in that, The MYEOV expression inhibitor includes shRNA, the nucleotide sequence of which is shown in SEQ ID NO. 1.

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