Markers for diagnosis and treatment of tumors

Abstract

The invention discloses markers for diagnosing and treating tumors. It is found for the first time that overexpressed circRNABP9 can inhibit proliferation, migration and invasion of gastric cancer cells and promote aging of the gastric cancer cells; the circTMEM45A can inhibit proliferation, migration and invasion of esophageal cancer cells, and further through mouse transplantation tumor experiment verification, a new thought and direction are provided for diagnosis and treatment of gastric cancer and esophageal cancer, and the circTMEM45A has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to markers for diagnosing and treating tumors. Background Art

[0002] Digestive tract tumors are not only the most common tumors in the world, but also in my country. Esophageal cancer, cardia cancer, and gastric cancer are the most common malignant tumors of the digestive tract. The global incidence of esophageal cancer and gastric cancer are ranked seventh and fifth, respectively, and the tumor-related mortality rates are ranked sixth and third, respectively. In my country, the incidence and mortality rates of esophageal cancer and gastric cancer are at the forefront of malignant tumors.

[0003] Digestive tract tumors have an insidious onset, with no obvious early symptoms and a low early detection rate. When diagnosed, the disease is already in a late stage, with a wide range of invasion and high malignancy, which has seriously affected the possibility of patients receiving radical treatment and the treatment effect. The prognosis of digestive tract tumors is closely related to the course of the disease at the time of diagnosis. Early detection, early diagnosis, and early treatment are crucial to improving the survival of patients with digestive tract tumors. Therefore, it is urgent to find a digestive tract-related molecular marker with high sensitivity and specificity, and use these indicators as the basis for clinical diagnosis, prognosis judgment, and individualized treatment.

[0004] Circular RNA (circRNA) is a large class of non-coding RNAs that covalently bind to form a circular structure. Circular RNA was first discovered in plant viruses in 1976. Initially, it was considered to be a type of RNA that was mis-spliced ​​and had no research significance. Subsequent studies have found that circRNA plays an important regulatory role in gene expression in vivo. Its structural characteristics are a closed circular structure without a 5' cap end and a 3' tail end. This circular structure is formed by connecting the upstream 3' end splice acceptor with the downstream 5' end splice donor through reverse splicing. This covalently bound circular closed structure causes circRNA to be unaffected by nuclease exonucleases and is more stable than linear RNA. In recent years, studies have found that circRNA has the following basic functions: adsorbing microRNA as a competitive endogenous RNA (ceRNA); regulating transcription and selective splicing; acting on RNA binding proteins; and translating into proteins. Based on the above functions of circRNA, related studies have confirmed that circRNA is closely related to human diseases, including tumors. Therefore, in-depth research on the role of circRNA in gastrointestinal tumors has potential great significance for the diagnosis and treatment of gastrointestinal tumors. Summary of the invention

[0005] To make up for the deficiencies of the prior art, the present invention provides markers for diagnosing and treating tumors.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] The first aspect of the present invention provides the use of a promoter of circRNABP9 in the preparation of a pharmaceutical composition for treating gastric cancer.

[0008] Furthermore, the promoter is selected from a plasmid for overexpression of circRNABP9.

[0009] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0010] A second aspect of the present invention provides use of an inhibitor of circTMEM45A in the preparation of a pharmaceutical composition for treating esophageal cancer.

[0011] Furthermore, the inhibitor is selected from nucleic acid inhibitors.

[0012] Furthermore, the nucleic acid inhibitor is selected from siRNA.

[0013] Furthermore, the sequence of the siRNA is shown in any one of SEQ ID NOs: 4-6.

[0014] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0015] The third aspect of the present invention provides the use of circRNABP9 as a target in screening candidate drugs for treating gastric cancer.

[0016] Furthermore, the method for screening candidate drugs for treating gastric cancer includes: testing the effect of the candidate drug on the level of circRNABP9 in a sample obtained from a subject, wherein, after using the candidate drug, the level of circRNABP9 increases, indicating that the candidate drug has an effect in treating gastric cancer.

[0017] The fourth aspect of the present invention provides the use of circTMEM45A as a target in screening candidate drugs for treating esophageal cancer.

[0018] Furthermore, the method for screening candidate drugs for treating esophageal cancer includes: testing the effect of the candidate drug on the level of circTMEM45A in a sample obtained from a subject, wherein, after using the candidate drug, the level of circTMEM45A decreases, indicating that the candidate drug has an effect in treating esophageal cancer.

[0019] The fifth aspect of the present invention provides the use of a promoter of circRNABP9 in the preparation of a product for inhibiting gastric cancer cell proliferation / migration, inhibiting lymphangiogenesis / migration, inhibiting cachexia or promoting gastric cancer cell senescence.

[0020] Furthermore, the promoter is selected from a plasmid for overexpression of circRNABP9.

[0021] A sixth aspect of the present invention provides the use of an inhibitor of circTMEM45A in the preparation of a product for inhibiting the proliferation / migration of esophageal cancer cells.

[0022] Furthermore, the inhibitor is selected from nucleic acid inhibitors.

[0023] Furthermore, the nucleic acid inhibitor is selected from siRNA.

[0024] Furthermore, the sequence of the siRNA is shown in any one of SEQ ID NOs: 4-6.

[0025] The seventh aspect of the present invention provides a pharmaceutical composition, which comprises a promoter of circRNABP9 or an inhibitor of circTMEM45A.

[0026] An eighth aspect of the present invention provides any of the following methods: (1) A method for screening a candidate drug for treating gastric cancer, the method comprising: testing the effect of the candidate drug on the level of circRNABP9 in a sample obtained from a subject, wherein after using the candidate drug, the level of circRNABP9 increases, indicating that the candidate drug has an effect of treating gastric cancer; (2) A method for screening a candidate drug for treating esophageal cancer, the method comprising: testing the effect of the candidate drug on the level of circTMEM45A in a sample obtained from a subject, wherein after using the candidate drug, the level of circTMEM45A decreases, indicating that the candidate drug has an effect of treating esophageal cancer; (3) A method for regulating marker levels in vitro, the method comprising administering a promoter or inhibitor of circRNABP9 / circTMEM45A, the marker comprising a muscle atrophy marker, a lymphangiogenesis marker, a mesenchymal cell marker, an epithelial cell marker, a proliferation marker, and a metastasis marker; (4) A method for regulating apoptosis / migration / senescence of gastric cancer cells, the method comprising administering a promoter of circRNABP9; (5) A method for regulating apoptosis / migration of esophageal cancer cells, comprising administering an inhibitor of circTMEM45A.

[0027] Furthermore, the method is a non-therapeutic method.

[0028] Furthermore, the promoter is selected from a plasmid for overexpression of circRNABP9.

[0029] Furthermore, the inhibitor is selected from nucleic acid inhibitors.

[0030] Furthermore, the nucleic acid inhibitor is selected from siRNA.

[0031] Furthermore, the sequence of the siRNA is shown in any one of SEQ ID NOs: 4-6.

[0032] Furthermore, the muscle atrophy markers described in (3) include Atrogen-1 and / or MURF1.

[0033] Furthermore, the lymphangiogenesis marker includes LYVE1.

[0034] Furthermore, the mesenchymal cell markers include N-cadherin and / or Vimentin.

[0035] Furthermore, the epithelial cell marker includes E-cadherin.

[0036] Furthermore, the proliferation markers include Ki67 and / or PCNA.

[0037] Furthermore, the metastasis markers are MMP2 and / or MMP9.

[0038] A ninth aspect of the present invention provides the use of a reagent for detecting the expression level of circRNABP9 / circTMEM45A in the preparation of a product for diagnosing gastric cancer / esophageal cancer.

[0039] Furthermore, the reagent is selected from a probe that specifically recognizes circRNABP9 / circTMEM45A, a primer that specifically amplifies ccircRNABP9 / circTMEM45A, or a binder that specifically binds to a protein encoded by the circRNABP9 / circTMEM45A gene.

[0040] Furthermore, the product includes a chip, a test kit, a test paper or a nucleic acid membrane strip.

[0041] Furthermore, the kit also includes a detectable label.

[0042] Advantages and beneficial effects of the present invention: This application discovered for the first time that overexpression of circRNABP9 can inhibit the proliferation, migration and invasion of gastric cancer cells and promote the aging of gastric cancer cells; inhibition of circTMEM45A can inhibit the proliferation, migration and invasion of esophageal cancer cells, and further verified it through mouse transplant tumor experiments, providing new ideas and directions for the diagnosis and treatment of gastric cancer and esophageal cancer, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1It is a biological function diagram of circRNABP9, among which 1A is the expression level diagram of circRNABP9 in gastric cancer cell lines, 1B is the siRNA knockdown efficiency verification diagram, 1C is the effect diagram of knocking down circRANBP9 on circRANBP9 and RANBP9 mRNA levels, and 1D is the effect diagram of overexpressing circRANBP9 on circRANBP9 and RANBP9 mRNA levels; Figure 2 Figure 2 is the effect of knocking down circRANBP9 and overexpressing circRANBP9 on the proliferation and migration of gastric cancer cells, wherein 2A is the cell EdU assay and data statistics, 2B is the cell colony formation and data statistics, 2C is the CCK8 graph of cells with knocking down circRANBP9, 2D is the CCK8 graph of cells with overexpressing circRANBP9, 2E is the cell invasion and data statistics, and 2F is the cell migration and data statistics; Figure 3 Figure 3 is the effect of knocking down circRANBP9 and overexpressing circRANBP9 on gastric cancer cell senescence, among which, 3A is the tube formation, migration and statistical data of HLEC, 3B is β-galactosidase staining and statistical data, 3C is immunoblotting of aging-related proteins and statistical data, 3D is a muscle atrophy marker protein, 3E is a popliteal lymph node metastasis model and statistical data, 3F is a mouse xenograft tumor, 3G is a statistical graph of transplanted tumor weight, 3H is a statistical graph of transplanted tumor volume, 3I is a leg TA muscle in the cachexia model and statistical data, 3J is HE staining of TA muscle and statistical data; Figure 4 are IHC staining images of overexpressed circRANBP9, wherein 4A is an IHC staining image of Ki-67, N-cadherin and Vimentin in transplanted tumors, 4B is an IHC staining data statistical image of Ki-67, N-cadherin and Vimentin in transplanted tumors, 4C is an IHC staining image of LYVE1, N-cadherin and Vimentin in lymph nodes, and 4D is an IHC staining data statistical image of LYVE1, N-cadherin and Vimentin in lymph nodes; Figure 5 is a diagram of the biological function of circTMEM45A, wherein 5A is a diagram of the expression level of circTMEM45A in esophageal cancer cell lines, 1B is a diagram of the siRNA knockdown efficiency verification, 1C is a diagram of the effect of knocking down circTMEM45A on the mRNA levels of circTMEM45A and TMEM45A, and 1D is a diagram of the effect of overexpressing circTMEM45A on the mRNA levels of circTMEM45A and TMEM45A; Figure 6Figure 6 is the effect of knocking down circTMEM45A and overexpressing circTMEM45A on gastric cancer cells and transplanted tumor mice. Among them, 6A is the CCK8 data statistics of KYSE30 cells overexpressing circTMEM45A, 6B is the colony formation and data statistics of KYSE30 cells overexpressing circTMEM45A, 6C is the EdU determination and data statistics of KYSE30 cells overexpressing circTMEM45A, 6D is the migration and data statistics of KYSE30 cells overexpressing circTMEM45A, 6E is the 6A is the CCK8 statistical graph of TE1 cells, 6F is the colony formation and statistical graph of TE1 cells overexpressing circTMEM45A, 6G is the EdU assay and statistical graph of TE1 cells overexpressing circTMEM45A, 6H is the migration and statistical graph of TE1 cells overexpressing circTMEM45A, 6I is the invasion and statistical graph of KYSE30 cells overexpressing circTMEM45A, 6J is the invasion and statistical graph of TE1 cells overexpressing circTMEM45A, 6K is the KYSE knockdown of circTMEM45A 6L is the colony formation and statistical graph of KYSE30 cells with circTMEM45A knocked down, 6M is the EdU assay and statistical graph of KYSE30 cells with circTMEM45A knocked down, 6N is the migration and statistical graph of KYSE30 cells with circTMEM45A knocked down, 6O is the CCK8 statistical graph of TE1 cells with circTMEM45A knocked down, 6P is the colony formation and statistical graph of TE1 cells with circTMEM45A knocked down, 6Q is the TE1 cells with circTMEM45A knocked down EdU determination and statistical graphs of cells, 6R is the migration and statistical graphs of TE1 cells with circTMEM45A knockdown, 6S is the invasion and statistical graphs of KYSE30 cells with circTMEM45A knockdown, 6T is the invasion and statistical graphs of TE1 cells with circTMEM45A knockdown, 6U is the construction diagram of mice with circTMEM45A knockdown, 6V is the statistical graphs of tumor-transplanted mice and growth curves, and tumor weight, 6W is the statistical graph of tumor-transplanted mice volume, 6X is the volume graph of tumor-transplanted mice, 6Y is the HE staining graph of tumor-transplanted mice, and 6Z is the IHC staining graph of tumor-transplanted mice. DETAILED DESCRIPTION

[0044] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0045] The present invention provides the use of a promoter of circRNABP9 in preparing a pharmaceutical composition for treating gastric cancer.

[0046] In some embodiments, a promoter of circRNABP9 refers to a substance that can promote the expression of circRNABP9, including but not limited to circRNABP9 overexpression plasmids and lentiviruses, natural purified substances that can promote the expression of circRNABP9, modified natural purified substances, semi-synthetic substances, chemically synthesized substances and / or any combination thereof. Any substance that can promote the expression of circRNABP9 is within the scope of protection of this application.

[0047] In a specific embodiment, the promoter of circRNABP9 is selected from a plasmid for overexpression of circRNABP9.

[0048] The present invention provides use of a circTMEM45A inhibitor in preparing a pharmaceutical composition for treating esophageal cancer.

[0049] In some embodiments, the inhibitor refers to a substance that can inhibit the activity and / or expression of circTMEM45A protein or polynucleotide, including nucleic acid inhibitors, protein inhibitors, and compounds.

[0050] In a preferred embodiment, the inhibitor of circTMEM45A is selected from nucleic acid inhibitors, including siRNA, shRNA, and ribozyme.

[0051] In a specific embodiment, the nucleic acid inhibitor of circTMEM45A is selected from siRNA.

[0052] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0053] In some embodiments, pharmaceutically acceptable excipients include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. These media and solvents are well known in the art for pharmaceutically active substances. Unless any conventional media or medicaments are incompatible with the active ingredient so far, its use in therapeutic compositions is considered. In addition, various adjuvants such as those commonly used in the art may be included. Describe the consideration of the inclusion of various components in pharmaceutical compositions, for example, in Gilman et al. (Eds.) (1990), Goodman and Gilman's: The PHarmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which are incorporated herein by reference in their entirety.

[0054] Examples of pharmaceutically acceptable excipients include sugars, e.g., lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and methylcellulose; powdered yarrow; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa oil; polyols, such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as TWEENS; wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffered solution.

[0055] The choice of pharmaceutically acceptable excipients depends essentially on the way in which the active substance is to be administered.

[0056] The pharmaceutical composition described in the present application is preferably provided in unit dosage form. As used in the present application, a unit dosage form is a composition containing a certain amount of medicine, suitable for administering to animals with a single dose according to good pharmaceutical practice, preferably mammalian subjects. However, the preparation of a single dose or unit dosage form does not mean that the dosage form is administered once a day or once per course of treatment. Some dosage forms are considered to be administered once, twice, three times or more per day, and can be administered with infusion over a period of time (e.g., from about 30 minutes to about 2-6 hours), or administered with continuous infusion, and can be given more than once in a course of treatment, although single administration is not particularly excluded. The technician will recognize that the preparation does not particularly consider the entire course of treatment, and these decisions are left to those technicians in the field of treatment rather than in the field of formulations.

[0057] The useful medicine described above can be any one of a variety of suitable forms, for a variety of routes of administration, for example, for oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intraarterial, intravenous, intramuscular or other parenteral routes of administration. It will be appreciated by the technician that oral and nasal compositions comprise compositions administered by inhalation, and are prepared using available methods. According to the desired specific mode of administration, a variety of pharmaceutically acceptable adjuvants well known in the art can be used. Pharmaceutically acceptable adjuvants include, for example, solid or liquid fillers, diluents, cosolvents, surfactants and encapsulated materials. Optional pharmaceutically active materials can be included, which do not substantially affect the inhibitory activity of the medicine. The amount of the adjuvant used together with the medicine is enough to provide the actual number of per unit dose materials for administering the medicine. Techniques and compositions for preparing dosage forms useful in the methods described herein are described in the following references, all of which are incorporated herein by reference: Modern PHarmaceuticals, 4th edition, Chapters 9 and 10 (Banker & Rhodes, eds., 2002); Lieberman et al., PHarmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to PHarmaceutical Dosage Forms 8th edition (2004).

[0058] The present invention provides a method for regulating marker levels in vitro, the method comprising administering a promoter or inhibitor of circRNABP9 / circTMEM45A, wherein the markers include muscle atrophy markers, lymphangiogenesis markers, mesenchymal cell markers, epithelial cell markers, proliferation markers, and metastasis markers.

[0059] In some embodiments, administration of a promoter of circRNABP9 can significantly increase the expression of muscle atrophy markers Atrogen-1 and MURF1 proteins, significantly reduce the expression of lymphangiogenesis marker LYVE1, proliferation marker Ki-67, and mesenchymal cell markers N-cadherin and Vimentin, and significantly increase the expression of epithelial cell marker E-cadherin, while administration of an inhibitor of circRNABP9 has the opposite effect.

[0060] Administration of circTMEM45A inhibitors significantly reduced the expression of proliferation markers Ki67 and PCNA and metastasis markers MMP2 and MMP9, whereas administration of circTMEM45A promoters had the opposite effect.

[0061] The present invention provides use of a reagent for detecting the expression level of circRNABP9 / circTMEM45A in preparing a product for diagnosing gastric cancer / esophageal cancer.

[0062] The products include chips, test kits, test strips or nucleic acid membrane strips.

[0063] The kit also includes a detectable label.

[0064] In some embodiments, if the expression level of circRNABP9 is significantly lower than that of a healthy control, gastric cancer is diagnosed; if the expression level of circTMEM45A is significantly higher than that of a healthy control, esophageal cancer is diagnosed.

[0065] In some embodiments, detectable label refers to a composition that can produce a detectable signal indicating the presence of a target polynucleotide in a determination sample. Suitable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties. Therefore, labeling is any composition that can be detected by a device or method, including but not limited to spectroscopy, photochemistry, biochemistry, immunochemistry, electricity, optics, chemical detection devices or any other suitable device. In some embodiments, labeling can be detected visually without the aid of a device.

[0066] Among them, radioactive isotopes include but are not limited to 3 H. 14 C. 35 S. 125 I. 131 I.

[0067] Enzymes include, but are not limited to, horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase.

[0068] Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors.

[0069] In some embodiments, a kit refers to a set of components provided in the context of a system for sequencing nucleotides and / or isolating nucleotide sequences and / or diagnosing a subject with a disease or infection based on the presence, absence, and / or amount of expressed nucleotide sequences from a sample or cell.

[0070] The kit includes a gene detection kit and a protein detection kit. The gene detection kit includes a reagent or a chip for detecting the transcription level of the circRNABP9 / circTMEM45A gene, and the protein detection kit includes a reagent or a chip for detecting the expression level of the circRNABP9 / circTMEM45A protein.

[0071] The kit of the present application further comprises one or more substances of the following group: a container, a positive control substance, a negative control substance, a buffer, a preservative, and a protein stabilizer.

[0072] The kit may also be accompanied by an instruction manual for use of the kit, which describes how to use the kit for testing, and how to use the test results to judge tumor development and select a treatment plan.

[0073] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0074] Example 1 Markers for diagnosis and treatment of gastric cancer 1. Experimental methods Cell culture Four gastric cancer cell lines that were confirmed to be mycoplasma-free were all obtained from Suzhou Haixing Biotechnology (HGC-27, AGS, MKKN45, and MKN74) and cultured in RPMI1640 or F-12 medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin (GIBCO, USA) at 37°C and 5% CO 2 .

[0075] Plasmid construction and cell transfection The overexpression plasmid of circRNABP9 was constructed by amplifying the hsa_circ_0001577 (circRNABP9) sequence and cloning it into the pLC5-ciR vector (Geneseed Biotech, Guangzhou). According to the instructions of the kit, gastric cancer cells were plated at 1 × 10 6 The cells were seeded at a density of 100 μg / mL in 6-well plates. The cells were then transfected with plasmids or siRNA (RiboBio, Guangzhou, the sequences of siRNA are shown in Table 1) using PolyFect or HiperFect transfection reagents (Promega, USA). After 6 hours, the medium was replaced and the cells were cultured in complete growth medium for another 48 hours before being harvested for analysis.

[0076] Table 1 siRNA sequences of hsa_circ_0001577

[0077] RNA extraction, reverse transcription, quantitative real-time PCR (qRT-PCR) Total RNA was isolated from the cell lines using TRIzol reagent (Invitrogen, USA), and reverse transcription and amplification were performed using GoTaq qPCR and GoTaq Green Master Mixes, respectively. The thermal cycling conditions included initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 10 min. U6 small nuclear RNA and GAPDH were used as internal controls for circRNA and mRNA quantification, respectively. Gene expression levels were measured by 2 -ΔΔCT Method calculation.

[0078] CCK-8 assay Gastric cancer cells from different treatment groups were seeded in 96-well plates at a density of 2000 cells per well. After cell attachment, cell proliferation was assessed every 24 h for 5 days using the Cell Counting Kit-8 (CCK-8) reagent (MCE, MedChemExpress, Shanghai, China). At each time point, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 1 h. The absorbance at 450 nm was then measured using a microplate reader (Tecan).

[0079] Colony formation assay Gastric cancer cells from different treatment groups were plated at 1 × 10 3 The cells were seeded in 6-well plates at a density of 10 cells and incubated at 37°C for about 10 days to allow colonies to form and become visible to the naked eye. The colonies were then fixed with 4% paraformaldehyde and stained with crystal violet. Images of the colonies were taken and the number of colonies was quantified by microscopic examination.

[0080] 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay The EdU assay is used to evaluate the proliferation of gastric cancer cells. Cells from different treatment groups were cultured to the desired confluence, and then EdU solution was added to the culture medium and incubated for the specified time to allow EdU to be incorporated into newly synthesized DNA. After incubation, the cells were fixed and permeabilized, and the incorporated EdU was labeled with a fluorescent marker. The fluorescence intensity was then measured using a confocal microscope to determine the cell proliferation rate.

[0081] Wound healing assay After transfection with the indicated plasmids, gastric cancer cells were seeded in 6-well plates and incubated for 24 h until approximately 80% confluence was reached. A scratch was made on the cell monolayer using a 10 μL pipette tip, and the cells were then washed three times and placed in fresh serum-free medium. An initial scratch image (0 h) was captured, followed by a second image at 24 h. The migration distance of cells into the scratch area was measured and normalized to the 0 h width. The scratch healing rate (representing cell migration) was calculated as: [(initial scratch width - 24 h scratch width) / initial scratch width] × 100%.

[0082] Transwell migration and invasion assays Transwell chambers were used to assess cell migration and invasion. For the migration assay, cells from different treatment groups (4 × 10 4 ) were suspended in serum-free medium and incubated at 37°C for 12 to 24 hours, and the lower chamber contained medium supplemented with 10% FBS to stimulate migration. For the invasion assay, Matrigel (Corning, USA) was coated on the bottom of the chamber and incubated at 37°C for 12 hours to form a barrier. The remaining steps followed those of the migration assay: cells were fixed with 4% paraformaldehyde, stained with crystal violet, and counted in at least five random microscopic fields.

[0083] Tube formation assay Matrigel (Corning, USA) was added to the wells of a 96-well plate and incubated at 37°C for 30 minutes. After incubation, equal volumes of cell supernatants from different treatment groups were mixed with extracellular matrix (ECM) medium to prepare conditioned medium. Human lymphatic endothelial cells (HLECs) were suspended at 12,000 cells per 100 μL of conditioned medium and seeded into Matrigel-coated wells. The culture dishes were incubated for an additional 4 to 6 hours, and tube formation was assessed by microscopy and quantified using ImageJ software.

[0084] Immunohistochemistry (IHC) Tissue sections were baked at 60°C for 2 hours and dewaxed with xylene. Antigen retrieval was performed using EDTA solution, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution. Sections were then blocked with 5% bovine serum albumin (BSA) solution to prevent nonspecific binding and incubated with primary antibodies overnight at 4°C. The next day, secondary antibodies were added, followed by staining of antigens and cell nuclei using diaminobenzidine (DAB) and hematoxylin, respectively. Finally, slides were mounted and examined under a microscope to evaluate the staining pattern.

[0085] Western blotting Western blot analysis was performed according to standard protocols. Total cellular proteins were extracted from gastric cancer cells using RIPA lysis buffer containing 1% PMSF. Protein lysates were quantified to ensure equal loading, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked with blocking buffer to prevent nonspecific binding and then incubated with primary antibodies against the target proteins overnight at 4°C, followed by incubation with secondary antibodies for 1 h at room temperature. Finally, the PVDF membranes were imaged using enhanced chemiluminescence reagents (Solarbio) according to the manufacturer's instructions.

[0086] In vivo xenograft tumor models and lymph node metastasis models For the xenograft tumor model, 10 BALB / c nude mice (Beijing Sibeifu Biotechnology Co., Ltd.) were randomly divided into two groups and inoculated subcutaneously with 1×10 7 AGS cells and AGS cells stably overexpressing circRANBP9. After 4 weeks, the mice were euthanized and the tumors were harvested for HE staining and IHC analysis. At the same time, the lymph node metastasis model was injected with 1×10 7 AGS cells and AGS cells stably overexpressing circRANBP9. After 4 weeks, the mice were euthanized and the tumors were harvested for HE staining and IHC analysis.

[0087] 2. Experimental results The biological functions of circRNABP9 were investigated in in vitro and in vivo models. First, qRT-PCR results showed that among the four gastric cancer cell lines, AGS cells had higher expression of endogenous circRANBP9, while HGC-27 cells had lower expression ( Figure 1 A). Next, three siRNAs were designed for the reverse cleavage sequence of circRANBP9. The knockdown efficiency of the three siRNA sequences was verified by qRT-PCR. siRNA-1 (si-hsa_circ_0001577_001) and siRNA-2 (si-hsa_circ_0001577_002) with better knockdown efficiency were selected for subsequent experimental verification ( Figure 1 B) At the same time, siRNA-1 was used to construct an AGS cell line with stable knockdown of circRANBP9. Further qRT-PCR experiments demonstrated that AGS cells with stable knockdown of circRANBP9 only reduced the expression of circRANBP9 without affecting the expression of linear RANBP9 mRNA ( Figure 1C). Subsequently, HGC-27 cells were forced to overexpress exogenous circRANBP9 by transfection with artificially synthesized circular plasmids. qRT-PCR verified the overexpression efficiency of circRANBP9 and also verified that the exogenous plasmid only affected the expression of circRANBP9 while the expression of the linear transcript of RANBP9 did not increase ( Figure 1 D). The above experiments show that when interfering with the endogenous expression of circRANBP9, it is not the linear transcript RANBP9 that affects the biological function changes of gastric cancer cells.

[0088] EdU assay as well as CCK-8 and colony formation assays showed that the DNA replication activity, proliferation and colony formation ability of AGS cells with circRNABP9 knockdown were significantly increased, while the opposite results were observed in HGC-27 cells overexpressing circRANBP9 ( Figure 2 AD). Wound healing and transwell assays showed that the migration and invasion abilities of AGS cells with circRNABP9 knockdown were significantly increased compared with those in the si-NC group, whereas overexpression of circRANBP9 had the opposite effect in HGC-27 cells ( Figure 2 EF).

[0089] Given that senescence is one of the 14 major characteristics of cancer, promoting the senescence of cancer cells has always been a hot topic in inhibiting cancer progression. Next, the effect of circRANBP9 on the senescence process of AGS and HGC-27 cells was studied. β-Galactosidase staining and Western blot analysis showed that knocking down circRANBP9 promoted and inhibited the senescence process of AGS cells and the expression of senescence-related proteins, while overexpression of circRANBP9 promoted the senescence process of HGC-27 cells ( Figure 3 BC).

[0090] In order to verify whether circRANBP9 affects lymph node metastasis and cachexia in vivo and in vitro, lymphangiogenesis experiments were performed using HLEC cells, and cachexia-related proteins were detected using C2C12 cells. The experimental results showed that when co-cultured with AGS cells with knockdown of circRANBP9, the tube formation and migration ability of HLEC were significantly enhanced ( Figure 3A), the opposite results were observed when co-cultured with HGC-27 cells overexpressing circRANBP9. In addition, Atrogen-1 and MURF1 proteins are markers of muscle atrophy, which increase significantly when cachexia occurs. When co-cultured with AGS cells with knockdown of circRANBP9, Atrogen-1 and MURF1 proteins in C2C12 cells decreased compared with the si-NC group, but increased significantly when co-cultured with HGC-27 cells overexpressing circRANBP9 ( Figure 3 D).

[0091] We also performed in vivo experiments on circRANBP9 overexpression using BALB / C mice, and constructed popliteal lymph node metastasis models and cachexia models. Figure 3 E shows a representative image of the popliteal lymph node metastasis model. Compared with the control group (pLC5-ciR), the metastatic lymph node volume of the circRANBP9 overexpression group was significantly reduced. At the same time, IHC staining showed that circRANBP9 significantly reduced the expression of lymphangiogenesis marker LYVE1 and mesenchymal cell markers N-cadherin and Vimentin, but increased the expression of epithelial cell marker E-cadherin ( Figure 4 CD). Figure 3 F shows images of mouse xenograft tumors. Compared with the control group, the tumor volume, tumor weight, and tumor growth of the circRANBP9 overexpression group were significantly reduced ( Figure 3 FH). At the same time, IHC staining showed that circRANBP9 significantly reduced the expression of proliferation marker Ki-67 and mesenchymal cell markers N-cadherin and Vimentin, but increased the expression of epithelial cell marker E-cadherin ( Figure 4 AB). Figure 3 I shows images of the leg TA muscle in the cachexia model. Compared with the control group, the TA muscle atrophy was less in the circRANBP9 overexpression group, and the grip strength of BALB / C mice and the body weight of BALB / C mice were significantly higher ( Figure 3 FH). HE staining also demonstrated that the TA muscle in the circRANBP9 overexpression group had a larger cross-sectional area ( Figure 3 J).

[0092] Example 2 Markers for diagnosis and treatment of esophageal cancer 1. Experimental methods Cell culture Five human esophageal cancer cell lines that were confirmed to be mycoplasma-free were all obtained from Suzhou Haixing Biotechnology (TE1, TE10, KYSE30, KYSE150, and KYSE410) and cultured in RPMI1640 or F-12 medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin (GIBCO, USA). The culture temperature was 37°C and the humidity was 5% CO 2 .

[0093] Plasmid construction and cell transfection The overexpression plasmid of circTMEM45A was constructed by amplifying the hsa_circ_0066658 (circTMEM45A) sequence and cloning it into the pLC5-ciR vector (Geneseed Biotech, Guangzhou). Esophageal cancer cells were plated at 1 × 10 6 The cells were seeded at a density of 100 μg / mL in 6-well plates. The cells were then transfected with plasmids or siRNA (RiboBio, Guangzhou, the sequences of siRNA are shown in Table 2) using PolyFect or HiperFect transfection reagents (Promega, USA). After 6 hours, the medium was replaced and the cells were cultured in complete growth medium for another 48 hours before being harvested for analysis.

[0094] Table 2 siRNA sequences of hsa_circ_0066658

[0095] RNA extraction, reverse transcription, quantitative real-time PCR (qRT-PCR) Total RNA was isolated from the cell lines using TRIzol reagent (Invitrogen, USA), and reverse transcription and amplification were performed using GoTaq qPCR and GoTaq Green Master Mixes, respectively. The thermal cycling conditions included initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 10 min. U6 small nuclear RNA and GAPDH were used as internal controls for circRNA and mRNA quantification, respectively. Gene expression levels were measured by 2 -ΔΔCT Method calculation.

[0096] CCK-8 assay Esophageal cancer cells from different treatment groups were seeded in 96-well plates at a density of 2000 cells per well. After cell attachment, cell proliferation was assessed every 24 h for 5 days using the Cell Counting Kit-8 (CCK-8) reagent (MCE, MedChemExpress, Shanghai, China). At each time point, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 1 h. The absorbance at 450 nm was then measured using a microplate reader (Tecan).

[0097] Colony formation assay Esophageal cancer cells from different treatment groups were plated at 1 × 10 3 The cells were seeded in 6-well plates at a density of 10 cells and incubated at 37°C for about 10 days to allow colonies to form and become visible to the naked eye. The colonies were then fixed with 4% paraformaldehyde and stained with crystal violet. Images of the colonies were taken and the number of colonies was quantified by microscopic examination.

[0098] 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay The EdU assay is used to evaluate the proliferation of esophageal cancer cells. Cells from different treatment groups are cultured to the desired confluence, and then EdU solution is added to the culture medium and incubated for the specified time to allow EdU to be incorporated into newly synthesized DNA. After incubation, the cells are fixed and permeabilized, and the incorporated EdU is labeled with a fluorescent marker. The fluorescence intensity is then measured using a confocal microscope to determine the cell proliferation rate.

[0099] Wound healing assay After transfection with the indicated plasmids, esophageal cancer cells were seeded in 6-well plates and incubated for 24 h until approximately 80% confluence was reached. A scratch was made on the cell monolayer using a 10 μL pipette tip, and the cells were then washed three times and placed in fresh serum-free medium. An initial scratch image (0 h) was captured, followed by a second image at 24 h. The migration distance of cells into the scratch area was measured and normalized to the 0 h width. The scratch healing rate (representing cell migration) was calculated as: [(initial scratch width - 24 h scratch width) / initial scratch width] × 100%.

[0100] Transwell migration and invasion assays Transwell chambers were used to assess cell migration and invasion. For the migration assay, cells from different treatment groups (4 × 10 4) were suspended in serum-free medium and incubated at 37°C for 12 to 24 hours, and the lower chamber contained medium supplemented with 10% FBS to stimulate migration. For the invasion assay, Matrigel (Corning, USA) was coated on the bottom of the chamber and incubated at 37°C for 12 hours to form a barrier. The remaining steps followed those of the migration assay: cells were fixed with 4% paraformaldehyde, stained with crystal violet, and counted in at least five random microscopic fields.

[0101] Tube formation assay Matrigel (Corning, USA) was added to the wells of a 96-well plate and incubated at 37°C for 30 minutes. After incubation, equal volumes of cell supernatants from different treatment groups were mixed with extracellular matrix (ECM) medium to prepare conditioned medium. Human lymphatic endothelial cells (HLECs) were suspended at 12,000 cells per 100 μL of conditioned medium and seeded into Matrigel-coated wells. The culture dishes were incubated for an additional 4 to 6 hours, and tube formation was assessed by microscopy and quantified using ImageJ software.

[0102] Immunohistochemistry (IHC) Tissue sections were baked at 60°C for 2 hours and dewaxed with xylene. Antigen retrieval was performed using EDTA solution, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution. Sections were then blocked with 5% bovine serum albumin (BSA) solution to prevent nonspecific binding and incubated with primary antibodies overnight at 4°C. The next day, secondary antibodies were added, followed by staining of antigens and cell nuclei using diaminobenzidine (DAB) and hematoxylin, respectively. Finally, slides were mounted and examined under a microscope to evaluate the staining pattern.

[0103] Western blotting Western blot analysis was performed according to standard protocols. Total cellular proteins were extracted from esophageal cancer cells using RIPA lysis buffer containing 1% PMSF. Protein lysates were quantified to ensure equal loading, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore, MA, USA). The membranes were blocked with blocking buffer to prevent nonspecific binding and then incubated with primary antibodies against the target proteins overnight at 4°C, followed by incubation with secondary antibodies for 1 h at room temperature. Finally, the PVDF membranes were imaged using enhanced chemiluminescence reagents (Solarbio) according to the manufacturer's instructions.

[0104] In vivo xenograft tumor models and lymph node metastasis models For the xenograft tumor model, 10 BALB / c nude mice (Beijing Sibeifu Biotechnology Co., Ltd.) were randomly divided into two groups and inoculated subcutaneously with 1×10 7KYSE150 cells. After 10 days, cholesterol-modified si-circTMEM45A or si-NC (5 nmol / 20 g, RioBio, Guangzhou, China) were injected intratumorally every three days according to the protocol. After 4 weeks, the mice were euthanized and the tumors were harvested for HE staining and IHC analysis.

[0105] 2. Experimental results The biological functions of circTMEM45A were investigated in in vitro and in vivo models. First, qRT-PCR results showed that among the four esophageal cancer cell lines, KYSE150 cells and KYSE410 cells had higher expression of endogenous circTMEM45A, while KYSE30 and TE1 cells had lower expression ( Figure 5 A). Next, three siRNAs were designed for the reverse cleavage sequence of circTMEM45A. The knockdown efficiency of the three siRNA sequences was verified by qRT-PCR. siRNA-1 (si-hsa_circ_0066658_001) was selected for subsequent experimental verification ( Figure 5 B) Further qRT-PCR experiments demonstrated that stable knockdown of circTMEM45A in KYSE150 cells only reduced the expression of circTMEM45A without affecting the expression of linear TMEM45A mRNA ( Figure 5 C). Subsequently, KYSE30 cells were forced to overexpress exogenous circTMEM45A by transfection with artificially synthesized circular plasmids. qRT-PCR verified the overexpression efficiency of circTMEM45A and also verified that the exogenous plasmid only affected the expression of circTMEM45A while the expression of the linear transcript of TMEM45A did not increase ( Figure 5 D). The above experiments show that when interfering with the endogenous expression of circTMEM45A, it is not the linear transcript TMEM45A that affects the biological function changes of esophageal cancer cells.

[0106] EdU assay, CCK-8 and colony formation assays showed that the DNA replication activity, proliferation and colony formation ability of KYSE30 and TE1 cells overexpressing circTMEM45A were significantly increased ( Figure 6 AC, EG), whereas the opposite results were observed in KYSE150 and KYSE410 cells with circTMEM45A knockdown ( Figure 6 KM, OQ). Wound healing and transwell assays showed that the migration and invasion abilities of KYSE30 and TE1 cells overexpressing circTMEM45A plasmid were significantly increased compared with those overexpressing empty plasmid ( Figure 6D, 6H-J), while knockdown of circTMEM45A had the opposite effect in KYSE150 and KYSE410 cells ( Figure 6 N, 6R-T).

[0107] Ten BALB / C mice were randomly divided into two groups, with 5 mice in each group. All mice were injected with KYSE150-luc cells at the beginning. According to the flowchart, si-NC and si-circTMEM45A were injected into the tumor of the control group and the experimental group, respectively, and the changes in tumor size were recorded. Figure 6 U), mice were killed on the 28th day and it was found that compared with the si-NC group, the tumor growth curve of mice in the si-circTMEM45A group was slower and the tumor volume was smaller ( Figure 6 VX). HE and IHC staining of the tumors revealed that the proliferation markers ki67 and PCNA as well as the metastasis markers MMP2 and MMP9 in the si-circTMEM45A group were significantly reduced compared with those in the si-NC group ( Figure 6 YZ).

[0108] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of a promoter of circRNABP9 in the preparation of a pharmaceutical composition for treating gastric cancer; Preferably, the promoter is selected from a plasmid overexpressing circRNABP9; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

2. Use of circTMEM45A inhibitors in the preparation of pharmaceutical compositions for the treatment of esophageal cancer; Preferably, the inhibitor is selected from nucleic acid inhibitors; Preferably, the nucleic acid inhibitor is selected from siRNA; Preferably, the sequence of the siRNA is as shown in any one of SEQ ID NOs: 4-6; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

3. Application of circRNABP9 as a target in screening candidate drugs for the treatment of gastric cancer; Preferably, the method for screening candidate drugs for treating gastric cancer comprises: The effect of the candidate drug on the level of circRNABP9 in a sample obtained from a subject is tested, wherein after using the candidate drug, the level of circRNABP9 increases, indicating that the candidate drug has an effect of treating gastric cancer.

4. Application of circTMEM45A as a target in screening candidate drugs for the treatment of esophageal cancer; Preferably, the method for screening candidate drugs for treating esophageal cancer comprises: The effect of the candidate drug on the level of circTMEM45A in a sample obtained from the subject is tested, wherein after administration of the candidate drug, the level of circTMEM45A is reduced, indicating that the candidate drug has an effect in treating esophageal cancer.

5. Use of promoters of circRNABP9 in the preparation of products that inhibit gastric cancer cell proliferation / migration, inhibit lymphangiogenesis / migration, inhibit cachexia, or promote gastric cancer cell senescence; Preferably, the promoter is selected from a plasmid for overexpression of circRNABP9.

6. Application of circTMEM45A inhibitors in the preparation of products for inhibiting the proliferation / migration of esophageal cancer cells; Preferably, the inhibitor is selected from nucleic acid inhibitors; Preferably, the nucleic acid inhibitor is selected from siRNA; Preferably, the sequence of the siRNA is shown in any one of SEQ ID NOs: 4-6.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a promoter of circRNABP9 or an inhibitor of circTMEM45A.

8. Any of the following methods: (1) A method for screening candidate drugs for treating gastric cancer, characterized in that: The method comprises: testing the effect of the candidate drug on the level of circRNABP9 in a sample obtained from a subject, wherein, after using the candidate drug, the level of circRNABP9 increases, indicating that the candidate drug has an effect of treating gastric cancer; (2) A method for screening a candidate drug for treating esophageal cancer, characterized in that the method comprises: testing the effect of the candidate drug on the level of circTMEM45A in a sample obtained from a subject, wherein after using the candidate drug, the level of circTMEM45A decreases, indicating that the candidate drug has an effect of treating esophageal cancer; (3) A method for regulating marker levels in vitro, characterized in that the method comprises administering a promoter or inhibitor of circRNABP9 / circTMEM45A, wherein the marker comprises a muscle atrophy marker, a lymphangiogenesis marker, a mesenchymal cell marker, an epithelial cell marker, a proliferation marker, and a metastasis marker; (4) A method for regulating apoptosis / migration / senescence of gastric cancer cells, characterized in that the method comprises administering a promoter of circRNABP9; (5) A method for regulating apoptosis / migration of esophageal cancer cells, characterized in that the method comprises administering an inhibitor of circTMEM45A; Preferably, the promoter is selected from a plasmid overexpressing circRNABP9; Preferably, the inhibitor is selected from nucleic acid inhibitors; Preferably, the nucleic acid inhibitor is selected from siRNA; Preferably, the sequence of the siRNA is shown in any one of SEQ ID NOs: 4-6.

9. The method according to claim 8, characterized in that (3) The muscle atrophy markers described in the invention include Atrogen-1 and / or MURF1; Preferably, the lymphangiogenesis marker includes LYVE1; Preferably, the mesenchymal cell markers include N-cadherin and / or Vimentin; Preferably, the epithelial cell marker includes E-cadherin; Preferably, the proliferation markers include Ki67 and / or PCNA; Preferably, the metastasis markers are MMP2 and / or MMP9.

10. Application of reagents for detecting the expression level of circRNABP9 / circTMEM45A in the preparation of products for diagnosing gastric cancer / esophageal cancer; Preferably, the reagent is selected from a probe that specifically recognizes circRNABP9 / circTMEM45A, a primer that specifically amplifies ccircRNABP9 / circTMEM45A, or a binding agent that specifically binds to a protein encoded by the circRNABP9 / circTMEM45A gene; Preferably, the product comprises a chip, a test kit, a test paper or a nucleic acid membrane strip; Preferably, the kit further comprises a detectable label.

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