Markers for diagnosis and treatment of tumors

Abstract

The application discloses a marker for diagnosing and treating tumors. It is found 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; and inhibition of circTMEM45A can inhibit the proliferation, migration and invasion of esophageal cancer cells, and further verified through a mouse tumor transplantation experiment, so that a new thought and direction are provided for the diagnosis and treatment of gastric cancer and esophageal cancer, and have a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a marker for diagnosing and treating tumors. BACKGROUND

[0002] Esophageal cancer, cardia cancer and gastric cancer are the most common malignant tumors in the digestive tract. The incidence of esophageal cancer and gastric cancer in the world ranks the seventh and fifth respectively, and the tumor-related mortality rates rank the sixth and third respectively.

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

[0004] Circular RNA (circular RNA / circRNA) is a large class of non-coding RNA covalently combined to form a ring structure. Circular RNA was first discovered in plant viruses in 1976, and it was initially considered as a kind of wrong splicing and no research significance RNA. Subsequent studies have found that circRNA has an important regulatory effect on gene expression in vivo, and its structural characteristics are a closed ring structure without 5' cap and 3' tail. This ring structure is formed by connecting the upstream 3' end acceptor and the downstream 5' end donor through reverse splicing. This covalently bonded closed ring structure makes circRNA more stable than linear RNA and not affected by exonuclease. In recent years, studies have found that circRNA has the following basic functions: as a competitive endogenous RNA (ceRNA) to absorb microRNA; regulating transcription and alternative 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 study of the role of circRNA in digestive tract tumors has potential great significance for the diagnosis and treatment of digestive tract tumors. SUMMARY

[0005] In order to make up for the shortcomings of the prior art, the present application provides a marker for diagnosing and treating tumors.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions.

[0007] The first aspect of the present application provides use of a promoter of circRANBP9 in the preparation of a pharmaceutical composition for treating gastric cancer.

[0008] Further, the promoter is selected from a plasmid for overexpression of circRANBP9.

[0009] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

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

[0011] Further, the inhibitor is selected from a nucleic acid inhibitor.

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

[0013] Further, the sequence of the siRNA is shown in any one of SEQ ID NO: 4-6.

[0014] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0015] The third aspect of the present application provides use of circRANBP9 as a target in screening of a candidate drug for treating gastric cancer.

[0016] Further, the method for screening of a candidate drug for treating gastric cancer comprises testing the effect of the candidate drug on the level of circRANBP9 in a sample obtained from a subject, wherein the level of circRANBP9 is increased after using the candidate drug, indicating that the candidate drug has an effect of treating gastric cancer.

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

[0018] Further, the method for screening of a candidate drug for treating esophageal cancer comprises testing the effect of the candidate drug on the level of circTMEM45A in a sample obtained from a subject, wherein the level of circTMEM45A is decreased after using the candidate drug, indicating that the candidate drug has an effect of treating esophageal cancer.

[0019] The fifth aspect of the present application provides use of a promoter of circRANBP9 in the preparation of a product for inhibiting proliferation / migration of gastric cancer cells, inhibiting lymphangiogenesis / migration, inhibiting anaplasia, or promoting senescence of gastric cancer cells.

[0020] Further, the promoter is selected from a plasmid for overexpression of circRANBP9.

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

[0022] Further, the inhibitor is selected from a nucleic acid inhibitor.

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

[0024] Further, the sequence of the siRNA is shown in any one of SEQ ID NO: 4-6.

[0025] The seventh aspect of the present application provides a pharmaceutical composition comprising a promoter of circRANBP9 or an inhibitor of circTMEM45A.

[0026] The eighth aspect of the present application provides any one of the following methods:

[0027] (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 circRANBP9 in a sample obtained from a subject, wherein the level of circRANBP9 is increased after using the candidate drug, indicating that the candidate drug has an effect of treating gastric cancer;

[0028] (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 the level of circTMEM45A is decreased after using the candidate drug, indicating that the candidate drug has an effect of treating esophageal cancer;

[0029] (3) A method for in vitro regulation of the level of a marker, the method comprising administering a promoter or inhibitor of circRANBP9 / circTMEM45A, the marker comprising a muscle atrophy marker, a lymphangiogenesis marker, a mesenchymal cell marker, an epithelial cell marker, a proliferation marker, a metastasis marker;

[0030] (4) A method for regulating apoptosis / migration / senescence of gastric cancer cells, the method comprising administering a promoter of circRANBP9;

[0031] (5) A method for regulating apoptosis / migration of esophageal cancer cells, the method comprising administering an inhibitor of circTMEM45A.

[0032] Further, the method is a method for non-therapeutic purposes.

[0033] Further, the promoter is selected from a plasmid overexpressing circRANBP9.

[0034] Further, the inhibitor is selected from a nucleic acid inhibitor.

[0035] Further, the nucleic acid inhibitor is selected from siRNA.

[0036] Further, the sequence of the siRNA is shown in any one of SEQ ID NO: 4-6.

[0037] Further, the muscle atrophy marker in (3) comprises Atrogen-1 and / or MURF1.

[0038] Further, the lymphangiogenesis marker comprises LYVE1.

[0039] Further, the mesenchymal cell marker comprises N-cadherin and / or Vimentin.

[0040] Further, the epithelial cell marker comprises E-cadherin.

[0041] Further, the proliferation marker comprises Ki67 and / or PCNA.

[0042] Further, the metastasis marker comprises MMP2 and / or MMP9.

[0043] The ninth aspect of the present application provides the use of a reagent for detecting the expression level of circRANBP9 / circTMEM45A in the preparation of a product for diagnosing gastric cancer / esophageal cancer.

[0044] Further, the reagent is selected from a probe specifically recognizing circRANBP9 / circTMEM45A, a primer specifically amplifying circRANBP9 / circTMEM45A, or a binding agent specifically binding to a protein encoded by the circRANBP9 / circTMEM45A gene.

[0045] Further, the product comprises a chip, a kit, a test paper, or a nucleic acid film strip.

[0046] Further, the kit further comprises a detectable label.

[0047] Advantages and beneficial effects of the present application:

[0048] The present application first found that overexpression of circRANBP9 can inhibit gastric cancer cell proliferation, migration and invasion, and promote gastric cancer cell aging; inhibition of circTMEM45A can inhibit esophageal cancer cell proliferation, migration and invasion, and further verified by mouse xenograft experiment, which provides a new idea and direction for the diagnosis and treatment of gastric cancer and esophageal cancer, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 is a biological function diagram of circRANBP9, wherein 1A is a diagram of the expression level of circRANBP9 in a gastric cancer cell line, 1B is a diagram of siRNA knockdown efficiency verification, 1C is a diagram of the influence of knockdown of circRANBP9 on the mRNA levels of circRANBP9 and RANBP9, and 1D is a diagram of the influence of overexpression of circRANBP9 on the mRNA levels of circRANBP9 and RANBP9;

[0050] Figure 2 is a diagram of the influence of knockdown of circRANBP9 and overexpression of circRANBP9 on the proliferation and migration of gastric cancer cells, wherein 2A is a diagram of cell EdU determination and data statistics, 2B is a diagram of cell colony formation and data statistics, 2C is a diagram of CCK8 of cells in which circRANBP9 is knocked down, 2D is a diagram of CCK8 of cells in which circRANBP9 is overexpressed, 2E is a diagram of cell invasion and data statistics, and 2F is a diagram of cell migration and data statistics;

[0051] Figure 3 is a diagram of the influence of knockdown of circRANBP9 and overexpression of circRANBP9 on the aging of gastric cancer cells, wherein 3A is a diagram of tube formation, migration and data statistics of HLEC, 3B is a diagram of β-galactosidase staining and data statistics, 3C is a diagram of immunoblotting of aging-related proteins and data statistics, 3D is a diagram of muscle atrophy marker proteins, 3E is a diagram of a popliteal lymph node metastasis model and data statistics, 3F is a diagram of a mouse xenograft tumor, 3G is a diagram of weight statistics of a transplanted tumor, 3H is a diagram of volume statistics of a transplanted tumor, 3I is a diagram of leg TA muscle in a dysplasia model and data statistics, and 3J is a diagram of HE staining of TA muscle and data statistics;

[0052] Figure 4 is a diagram of IHC staining of overexpression of circRANBP9, wherein 4A is a diagram of IHC staining of Ki-67, N-cadherin and Vimentin of a transplanted tumor, 4B is a diagram of IHC staining data statistics of Ki-67, N-cadherin and Vimentin of a transplanted tumor, 4C is a diagram of IHC staining of LYVE1, N-cadherin and Vimentin of a lymph node, and 4D is a diagram of IHC staining data statistics of LYVE1, N-cadherin and Vimentin of a lymph node;

[0053] Figure 5is a biological function diagram of circTMEM45A, wherein 5A is a circTMEM45A expression level diagram in an esophageal cancer cell line, 1B is a siRNA knockdown efficiency verification diagram, 1C is a diagram of the influence of knockdown of circTMEM45A on circTMEM45A and TMEM45A mRNA levels, and 1D is a diagram of the influence of overexpression of circTMEM45A on circTMEM45A and TMEM45A mRNA levels;

[0054] Figure 6are the effects of knocking down circTMEM45A and overexpressing circTMEM45A on gastric cancer cells and transplanted tumor mice, wherein 6A is a CCK8 data statistical diagram of KYSE30 cells overexpressing circTMEM45A, 6B is a colony formation and data statistical diagram of KYSE30 cells overexpressing circTMEM45A, 6C is an EdU assay and data statistical diagram of KYSE30 cells overexpressing circTMEM45A, 6D is migration and data statistical diagram of KYSE30 cells overexpressing circTMEM45A, 6E is a CCK8 data statistical diagram of TE1 cells overexpressing circTMEM45A, 6F is a colony formation and data statistical diagram of TE1 cells overexpressing circTMEM45A, 6G is an EdU assay and data statistical diagram of TE1 cells overexpressing circTMEM45A, 6H is migration and data statistical diagram of TE1 cells overexpressing circTMEM45A, 61 is invasion and data statistical diagram of KYSE30 cells overexpressing circTMEM45A, 6J is invasion and data statistical diagram of TE1 cells overexpressing circTMEM45A, 6K is a CCK8 data statistical diagram of KYSE30 cells knocking down circTMEM45A, 6L is a colony formation and data statistical diagram of KYSE30 cells knocking down circTMEM45A, 6M is an EdU assay and data statistical diagram of KYSE30 cells knocking down circTMEM45A, 6N is migration and data statistical diagram of KYSE30 cells knocking down circTMEM45A, 60 is a CCK8 data statistical diagram of TE1 cells knocking down circTMEM45A, 6P is a colony formation and data statistical diagram of TE1 cells knocking down circTMEM45A, 6Q is an EdU assay and data statistical diagram of TE1 cells knocking down circTMEM45A, 6R is migration and data statistical diagram of TE1 cells knocking down circTMEM45A, 6S is invasion and data statistical diagram of KYSE30 cells knocking down circTMEM45A, 6T is invasion and data statistical diagram of TE1 cells knocking down circTMEM45A, 6U is a mouse construction diagram knocking down circTMEM45A, 6V is a transplanted tumor mouse and growth curve, transplanted tumor weight statistical diagram, 6W is a transplanted tumor mouse volume statistical diagram, 6X is a transplanted tumor mouse volume diagram, 6Y is a transplanted tumor mouse HE staining diagram, and 6Z is a transplanted tumor mouse IHC staining diagram. DETAILED DESCRIPTION

[0055] The following provides definitions for some of the terms used in this specification. 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 application belongs and the same meaning as those described in the specification.

[0056] The application provides an application of a promoter of circRANBP9 in preparation of a pharmaceutical composition for treating gastric cancer.

[0057] In some embodiments, the promoter of circRANBP9 refers to a substance capable of promoting expression of circRANBP9, including but not limited to a circRANBP9 overexpression plasmid and a lentivirus, a naturally purified substance capable of promoting expression of circRANBP9, a modified naturally purified substance, a semi-synthetic substance, a chemically synthesized substance and / or any combination thereof, and any substance capable of promoting expression of circRANBP9 is within the protection scope of the present application.

[0058] In specific embodiments, the promoter of circRANBP9 is selected from a plasmid for overexpression of circRANBP9.

[0059] The application provides an application of an inhibitor of circTMEM45A in preparation of a pharmaceutical composition for treating esophageal cancer.

[0060] In some embodiments, the inhibitor refers to a substance capable of inhibiting activity and / or expression of a circTMEM45A protein or polynucleotide, including a nucleic acid inhibitor, a protein inhibitor, a compound.

[0061] In preferred embodiments, the inhibitor of circTMEM45A is selected from a nucleic acid inhibitor, including siRNA, shRNA, ribozyme.

[0062] In specific embodiments, the nucleic acid inhibitor of circTMEM45A is selected from siRNA.

[0063] The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0064] In some embodiments, the pharmaceutically acceptable excipient includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. These media and agents are those used in the art of pharmaceutical preparations of active agents. Unless otherwise specified, the use of any and all agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents is contemplated to be within the scope of the present application. In addition, various adjuvants such as those commonly used in the art can be included. Considerations for inclusion of various components in pharmaceutical compositions are described, for example, in Gilman et al. (Eds.) (1990), Goodman and Gilman’s: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press, which is incorporated herein by reference in its entirety.

[0065] Examples of pharmaceutically acceptable excipients include sugars, such as lactose, dextrose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powdered tragacanth; 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 oil of theobroma; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the TWEENS; wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; and phosphate buffer solutions.

[0066] The choice of a pharmaceutically acceptable excipient depends primarily on the form of the active substance being administered.

[0067] The pharmaceutical compositions described herein are preferably provided in unit dosage form. As used herein, a unit dosage form is a composition containing an amount of a drug that is suitable for administration to an animal, preferably a mammalian subject, in a single dose according to good pharmaceutical practice. The preparation of a single dose or unit dosage form, however, does not imply that the dosage form is to be administered once per day or once per course of treatment. Some dosage forms contemplate administration once, twice, three times, or more per day, and can be administered as an infusion over a period of time (e.g., from about 30 minutes to about 2-6 hours), or as a continuous infusion, and can be given more than once in a course of treatment, although a single administration is not specifically excluded. The skilled artisan will recognize that the formulation does not specifically contemplate the entire course of treatment, and that these decisions are left to those skilled in the art of treatment rather than formulation.

[0068] The useful drugs described above can be in any of a variety of suitable forms for a variety of routes of administration, e.g., for oral, nasal, rectal, topical (including transdermal), ocular, intracerebral, intracranial, intrathecal, intraarterial, intravenous, intramuscular, or other parenteral routes of administration. The skilled artisan will appreciate that oral and nasal compositions include compositions for administration by inhalation and are prepared using available methods. A variety of pharmaceutically acceptable excipients well known in the art can be used depending on the particular mode of administration desired. Pharmaceutically acceptable excipients include, for example, solid or liquid fillers, diluents, solubilizers, surface-active agents, and encapsulating substances. Optional pharmaceutically active materials can be included which do not substantially interfere with the inhibitory activity of the drug. The amount of excipient employed in conjunction with the drug is sufficient to provide the physical stability needed to administer the actual quantity of material per unit dose. Techniques and compositions for preparing the dosage forms useful in the methods described herein are described in the following references, all incorporated herein by reference: Modern Pharmaceutics, 4th Ed., Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms 8th Edition (2004).

[0069] The present application provides a method of in vitro regulating a marker level, the method comprising administering a promoter or an inhibitor of circRANBP9 / circTMEM45A, the marker comprising a muscle atrophy marker, a lymphangiogenesis marker, a mesenchymal cell marker, an epithelial cell marker, a proliferation marker, a metastasis marker.

[0070] In some embodiments, administration of a promoter of circRANBP9 is capable of significantly increasing the expression of muscle atrophy markers Atrogen-1 and MURF1 protein, significantly reducing the expression of lymphangiogenesis marker LYVE1, proliferation marker Ki-67, and mesenchymal cell markers N-cadherin and Vimentin, and significantly increasing the expression of epithelial cell marker E-cadherin, and administration of an inhibitor of circRANBP9 has the opposite effect.

[0071] Administration of an inhibitor of circTMEM45A is capable of significantly reducing the expression of proliferation markers Ki67 and PCNA, and metastasis markers MMP2 and MMP9, and administration of a promoter of circTMEM45A has the opposite effect.

[0072] The application provides use of a reagent for detecting expression levels of circRANBP9 / circTMEM45A in preparation of a product for diagnosing gastric cancer / oesophageal cancer.

[0073] The product includes a chip, a kit, a test paper or a nucleic acid film strip.

[0074] The kit further comprises a detectable label.

[0075] In some embodiments, if the expression level of circRANBP9 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, oesophageal cancer is diagnosed.

[0076] In some embodiments, the detectable label refers to a composition capable of generating a detectable signal indicating the presence of a target polynucleotide in a test sample. Suitable labels include but are not limited to radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties. Therefore, the label is any composition capable of being detected by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical detection devices or any other suitable device. In some embodiments, the label can be detected visually without the aid of a device.

[0077] Among them, the radioisotope includes but is not limited to 3 H, 14 C, 35 S, 125 I, 131 I.

[0078] The enzyme includes but is not limited to horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase.

[0079] The fluorescent molecule includes but is not limited to FITC, rhodamine, lanthanide phosphors.

[0080] In some embodiments, the 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.

[0081] The kit includes a gene detection kit comprising a reagent or chip for detecting the transcription level of circRANBP9 / circTMEM45A gene, and a protein detection kit comprising a reagent or chip for detecting the expression level of circRANBP9 / circTMEM45A protein.

[0082] The kit of this application also includes one or more substances from the following group: container, positive control, negative control, buffer, preservative, and protein stabilizer.

[0083] The kit may also include an instruction manual, which explains how to use the kit for testing and how to use the test results to assess tumor development and select treatment options.

[0084] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0085] Example 1: Biomarkers for the Diagnosis and Treatment of Gastric Cancer

[0086] 1. Experimental Methods

[0087] Cell culture

[0088] The four gastric cancer cell lines confirmed to be mycoplasma-free were all obtained from Suzhou Haixing Biotechnology (HGC-27, AGS, MKKN45 and MKN74) and cultured in RPMI 1640 or F-12 medium. The medium was supplemented with 10% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin (GIBCO, USA) at a temperature of 37℃ and a humidity of 5% CO2.

[0089] Plasmid construction and cell transfection

[0090] An overexpression plasmid of circRANBP9 was constructed by amplifying and cloning the hsa_circ_0001577 (circRANBP9) sequence into the pLC5-ciR vector (Geneseed Biotech, Guangzhou). Gastric cancer cells were introduced into the pLC5-ciR vector at a density of 1 × 10⁻⁶ cells per well, according to the kit instructions. 6 Cells were seeded at a density in 6-well plates. Plasmids or siRNA (RiboBio, Guangzhou; siRNA sequences are shown in Table 1) were then transfected into the cells using PolyFect or HiperFect transfection reagent (Promega, USA). After 6 hours, the culture medium was changed, and the cells were cultured in complete growth medium for another 48 hours before harvesting for analysis.

[0091] Table 1. siRNA sequence of hsa_circ_0001577

[0092]

[0093]

[0094] RNA extraction, reverse transcription, quantitative real-time PCR (qRT-PCR)

[0095] Total RNA was isolated from cell lines using TRIzol reagent (Invitrogen, USA), and GoTaq qPCR and GoTaq Green Master Mixes were used for reverse transcription and amplification, respectively. The thermal cycling conditions included an 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, and gene expression levels were calculated by 2 -ΔΔCT Method calculation.

[0096] CCK-8 assay

[0097] 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 hours using a cell counting kit-8 (CCK-8) reagent (MCE, MedChemExpress, Shanghai, China) for 5 days. At each time point, 10 μL of CCK-8 solution was added to each well and incubated at 37 °C for 1 hour. The absorbance at 450 nm was then measured using a microplate reader (Tecan).

[0098] Colony formation assay Gastric cancer cells from different treatment groups were seeded in 6-well plates at a density of 1 x 10 3 cells per well and incubated at 37 °C for about 10 days to allow colony formation and become visible to the naked eye. The colonies were then fixed with 4% paraformaldehyde and stained with crystal violet. Colony images were taken, and the number of colonies was quantified by microscopic examination.

[0099] 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay The EdU assay was used to assess the proliferation of gastric cancer cells. Cells from different treatment groups were cultured to the desired confluency, and then an EdU solution was added to the culture medium and incubated for the specified time to allow EdU incorporation 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 with a confocal microscope to determine the cell proliferation rate.

[0100] After transfection with the designated plasmids for wound healing assay, gastric cancer cells were seeded in 6-well plates and incubated for 24 hours until reaching approximately 80% confluency. A scratch was made on the cell monolayer using a 10 pL pipette tip, followed by washing the cells three times and placing them in fresh serum-free medium. An initial scratch image was captured (0 h), 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] x 100%.

[0101] Transwell migration and invasion assay

[0102] Cell migration and invasion were assessed using Transwell chambers. For migration assay, cells from different treatment groups (4 x 10 4 ) were suspended in serum-free medium and incubated at 37 °C for 12 to 24 hours, with the lower chamber containing medium supplemented with 10% FBS to stimulate migration. For invasion assay, Matrigel (Corning, USA) was coated on the bottom of the cavity and incubated at 37 °C for 12 hours to form a barrier. The remaining steps followed the steps of the migration assay: cells were fixed with 4% paraformaldehyde, stained with crystal violet, and counted in at least five random microscopic fields.

[0103] Tube formation assay

[0104] Matrigel (Corning, USA) was added to the wells of a 96-well plate and incubated at 37 °C for 30 minutes. After incubation, an equal volume of cell supernatant from different treatment groups was mixed with extracellular matrix (ECM) medium to prepare conditioned medium. Human lymphatic endothelial cells (HLECs) were suspended in 100 pL of conditioned medium at 12,000 cells per 100 pL and seeded into the Matrigel-coated wells. The culture dishes were incubated for another 4 to 6 hours, after which the formation of tubes was assessed by microscopy and quantified using ImageJ software.

[0105] Immunohistochemistry (IHC)

[0106] Tissue sections were baked at 60 °C for 2 hours and deparaffinated with xylene. Antigen retrieval was performed using an EDTA solution, and endogenous peroxidase activity was blocked with a 3% hydrogen peroxide solution. The sections were then blocked with a 5% bovine serum albumin (BSA) solution to prevent non-specific binding and incubated with primary antibodies overnight at 4 °C. The next day, secondary antibodies were added, followed by staining of antigens and nuclei using diaminobenzidine (DAB) and hematoxylin, respectively. Finally, the slides were mounted and examined under a microscope to assess the staining pattern.

[0107] Western blotting Western blotting 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, USA). Membranes were blocked with blocking buffer to prevent non-specific binding, then incubated with primary antibodies against target proteins overnight at 4°C, and then incubated with secondary antibodies for 1 hour at room temperature. Finally, PVDF membranes were imaged using enhanced chemiluminescence reagents (Solarbio) according to the manufacturer's instructions.

[0108] In vivo xenograft tumor model and lymph node metastasis model

[0109] For the xenograft tumor model, 10 BALB / c nude mice (Sibeifu (Beijing) Biotechnology Co., Ltd.) were randomly divided into two groups and subcutaneously inoculated with 1 x 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. Meanwhile, the lymph node metastasis model was injected with 1 x 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.

[0110] 2. Experimental results

[0111] The biological function of circRANBP9 was studied in vitro and in vivo models. First, qRT-PCR results showed that the expression of endogenous circRANBP9 was higher in AGS cells and lower in HGC-27 among the four gastric cancer cell lines (A). Next, three siRNAs were designed targeting the reverse splicing sequence of circRANBP9, and qRT-PCR verified the knockdown efficiency of the three siRNA sequences, and 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 (B), and siRNA-1 was used to construct AGS cell lines stably knocking down circRANBP9. Further qRT-PCR experiments proved that AGS cells stably knocking down circRANBP9 only reduced the expression of circRANBP9 without affecting the expression of linear RANBP9 mRNA (C). Figure 1 A). Next, three siRNAs were designed targeting the reverse splicing sequence of circRANBP9, and qRT-PCR verified the knockdown efficiency of the three siRNA sequences, and 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 (B), and siRNA-1 was used to construct AGS cell lines stably knocking down circRANBP9. Further qRT-PCR experiments proved that AGS cells stably knocking down circRANBP9 only reduced the expression of circRANBP9 without affecting the expression of linear RANBP9 mRNA (C). Figure 1 A). Next, three siRNAs were designed targeting the reverse splicing sequence of circRANBP9, and qRT-PCR verified the knockdown efficiency of the three siRNA sequences, and 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 (B), and siRNA-1 was used to construct AGS cell lines stably knocking down circRANBP9. Further qRT-PCR experiments proved that AGS cells stably knocking down circRANBP9 only reduced the expression of circRANBP9 without affecting the expression of linear RANBP9 mRNA (C). Figure 1C). Subsequently, HGC-27 cells were forced to overexpress exogenous circRANBP9 by transfecting artificially synthesized circular plasmids, qRT-PCR verified the overexpression efficiency of circRANBP9, and at the same time verified that the exogenous plasmid only affected the expression of circRANBP9, but the expression of linear transcript of RANBP9 did not increase Figure 1 D). The above experiments show that interfering with the endogenous expression of circRANBP9 does not affect the biological function changes of gastric cancer cells, and linear transcript RANBP9 does not affect the biological function changes of gastric cancer cells.

[0112] EdU assay and CCK-8 and colony formation test showed that the DNA replication activity, proliferation and colony formation ability of AGS cells with knockdown of circRANBP9 were significantly increased, while the opposite results were observed in HGC-27 cells with overexpression of circRANBP9 Figure 2 A-D). Wound healing and transwell test showed that the migration and invasion ability of AGS cells with knockdown of circRANBP9 were significantly increased compared with si-NC group, while overexpression of circRANBP9 had the opposite effect in HGC-27 cells Figure 2 E-F).

[0113] Given that senescence is one of the 14 hallmarks of cancer, promoting the senescence of cancer cells has always been a hot spot in the research of inhibiting cancer progression. Next, the effect of circRANBP9 on the aging process of AGS and HGC-27 cells was studied. β-galactosidase staining and Western blot analysis showed that knockdown of circRANBP9 promoted the aging process of AGS cells and the expression of senescence-related proteins, while overexpression of circRANBP9 promoted the aging process of HGC-27 cells Figure 3 B-C).

[0114] To verify whether circRANBP9 affects lymph node metastasis and anaplasia in vivo and in vitro, HLEC cells were used for lymphangiogenesis experiments, and C2C12 cells were used for detection of anaplasia-related proteins. 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), opposite results were observed when co-cultured with HGC-27 cells overexpressing circRANBP9. In addition, Atrogen-1 and MURF1 proteins are the hallmark proteins of muscle atrophy, which significantly increase when cachexia occurs. When co-cultured with AGS cells with knockdown of circRANBP9, the Atrogen-1 and MURF1 proteins in C2C12 cells decreased compared with the si-NC group, while significantly increased when co-cultured with HGC-27 cells overexpressing circRANBP9 Figure 3 D).

[0115] In vivo experiments of circRANBP9 overexpression were also performed using BALB / C mice. Popliteal lymph node metastasis model and cachexia model were constructed. Figure 3 E shows representative images of the popliteal lymph node metastasis model. Compared with the control group (pLC5-ciR), the volume of metastatic lymph nodes in the circRANBP9 overexpression group was significantly reduced. Meanwhile, 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 C-D). Figure 3 F shows images of mouse xenograft tumors. Compared with the control group, the tumor volume in the circRANBP9 overexpression group was significantly reduced, the weight of the tumor was significantly reduced, and the growth of the tumor was significantly slowed down Figure 3 F-H). Meanwhile, 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 A-B). Figure 3 I shows images of TA muscle in the cachexia model. Compared with the control group, the TA muscle in the circRANBP9 overexpression group atrophied less, the grip strength of BALB / C mice and the overall body weight of BALB / C mice were significantly higher Figure 3 F-H). Meanwhile, HE staining also proved that the TA muscle of the circRANBP9 overexpression group had a larger cross-sectional area Figure 3 J).

[0116] Example 2 Marker for diagnosing and treating esophageal cancer

[0117] 1. Experimental method

[0118] Cell culture

[0119] Five human esophageal cancer cell lines without mycoplasma were all from Suzhou Haixing Biological (TE1, TE10, KYSE30, KYSE150 and KYSE410) and were cultured in RPMI1640 or F-12 medium with 10% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin (GIBCO, USA) at 37℃ and 5% CO2.

[0120] Plasmid construction and cell transfection

[0121] The overexpression plasmid of circTMEM45A was constructed by amplifying and cloning the hsa_circ_0066658 (circTMEM45A) sequence into the pLC5-ciR vector (Geneseed Biotech, Guangzhou). According to the kit instructions, esophageal cancer cells were transfected with 1 x 10 6 Cells were seeded in 6-well plates at a density. Then the plasmid or siRNA (Guangzhou RiboBio, the sequence of siRNA is shown in Table 2) was transfected into cells using PolyFect or HiperFect transfection reagent (Promega, USA). After 6 hours, the medium was replaced and the cells were cultured in complete growth medium for another 48 hours, then harvested for analysis.

[0122] Table 2 siRNA sequence of hsa_circ_0066658

[0123]

[0124] RNA extraction, reverse transcription, quantitative real-time PCR (qRT-PCR)

[0125] Total RNA was isolated from cell lines using TRIzol reagent (Invitrogen, USA), and GoTaq qPCR and GoTaq Green Master Mixes were used for reverse transcription and amplification, respectively. The thermal cycling conditions included initial denaturation at 95℃ for 5 minutes, followed by 40 cycles of 95℃ for 15 seconds, 58℃ for 30 seconds and 72℃ for 30 seconds, and finally extension at 72℃ for 10 minutes. U6 small nuclear RNA and GAPDH were used as internal controls for circRNA and mRNA quantification, respectively, and gene expression levels were calculated by 2 -ΔΔCT Method calculation.

[0126] CCK-8 detection

[0127] 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 hours for 5 days using Cell Counting Kit-8 (CCK-8) reagent (MCE, MedChemExpress, Shanghai, China). At each time point, 10 pL of CCK-8 solution was added to each well and incubated at 37 °C for 1 hour. Absorbance at 450 nm was then measured using a microplate reader (Tecan).

[0128] Colony formation assay Esophageal cancer cells from different treatment groups were seeded in 6-well plates at a density of 1 x 10 3 cells per well and incubated at 37 °C for about 10 days to allow colony formation and become macroscopically visible. Colonies were then fixed with 4% paraformaldehyde and stained with crystal violet. Colony images were taken and the number of colonies was quantified by microscopic examination.

[0129] 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay EdU assay was used to assess the proliferation of esophageal cancer cells. Cells from different treatment groups were cultured to the desired confluency, then EdU solution was added to the culture medium and incubated for the specified time to allow EdU incorporation into newly synthesized DNA. After incubation, cells were fixed and permeabilized, and incorporated EdU was labeled with a fluorescent marker. Fluorescence intensity was then measured with a confocal microscope to determine the cell proliferation rate.

[0130] Wound healing assay After transfection with the indicated plasmids, esophageal cancer cells were seeded in 6-well plates and incubated for 24 hours until reaching about 80% confluency. A scratch was made on the cell monolayer using a 10 pL pipette tip, then the cells were washed three times and placed in fresh serum-free medium. An initial scratch image was captured (0h), then a second image was captured at 24h. The migration distance of cells into the scratch area was measured and normalized to the 0h width. Scratch healing rate (representing cell migration) was calculated as: [(initial scratch width - 24h scratch width) / initial scratch width] x 100%.

[0131] Transwell migration and invasion assay

[0132] Cell migration and invasion were assessed using Transwell chambers. For migration assay, 4 x 10 4Cell suspensions of the cells of the application were incubated in serum-free medium at 37°C for 12 to 24 hours, and the lower chamber contained medium supplemented with 10% FBS to stimulate migration. For invasion assay, Matrigel (Coming, USA) was coated at the bottom of the chamber and incubated at 37°C for 12 hours to form a barrier. The remaining steps followed the steps of migration assay: cells were fixed with 4% paraformaldehyde, stained with crystal violet, and counted in at least five random microscopic fields.

[0133] Tube formation assay

[0134] Matrigel (Coming, USA) was added to the wells of a 96-well plate and incubated at 37°C for 30 minutes. After incubation, an equal volume of cell supernatant from different treatment groups was mixed with extracellular matrix (ECM) medium to prepare conditioned medium. Human lymphatic endothelial cells (HLECs) were suspended in 100 μΐ of conditioned medium at 12,000 cells per 100 μΐ and seeded into Matrigel-coated wells. The culture dishes were incubated for another 4 to 6 hours, and then the formation of tubes was evaluated by microscopy and quantified using ImageJ software.

[0135] Immunohistochemistry (IHC)

[0136] Tissue sections were baked at 60°C for 2 hours and deparaffinated with xylene. Antigen retrieval was performed using an EDTA solution, and endogenous peroxidase activity was blocked with a 3% hydrogen peroxide solution. The sections were then blocked with a 5% bovine serum albumin (BSA) solution to prevent non-specific binding and incubated with primary antibodies overnight at 4°C. The next day, secondary antibodies were added, and the antigen and nucleus were stained using diaminobenzidine (DAB) and hematoxylin, respectively. Finally, the slides were mounted and examined under a microscope to evaluate the staining pattern.

[0137] Western blotting Western blotting was performed according to standard protocols for Western blot analysis. Total cellular proteins were extracted from esophageal cancer cells using RIPA lysis buffer containing 1% PMSF. Protein lysates were quantified to ensure equal loading amounts, separated by SDS-PAGE, and transferred to PVDF membranes (Millipore, USA, Massachusetts). The membranes were blocked with blocking buffer to prevent non-specific binding and then incubated with primary antibodies against target proteins overnight at 4°C, followed by incubation with secondary antibodies for 1 hour at room temperature. Finally, the PVDF membranes were imaged using enhanced chemiluminescence reagents (Solarbio) according to the manufacturer's instructions.

[0138] In vivo xenograft tumor model and lymph node metastasis model

[0139] For the xenograft tumor model, 10 BALB / c nude mice (Sibeifu (Beijing) Biotechnology Co., Ltd.) were randomly divided into two groups and subcutaneously inoculated with 1 x 107 KYSE150 cells. Ten days later, cholesterol-modified si-circTMEM45A or si-NC (5 nmol / 20 g, RioBio, Guangzhou, China) was injected into the tumor every three days according to the protocol. Four weeks later, the mice were euthanized, and the tumors were harvested for HE staining and IHC analysis.

[0140] 2. Experimental Results

[0141] The biological functions of circTMEM45A were investigated in in vitro and in vivo models. First, qRT-PCR results showed that endogenous circTMEM45A expression was higher in KYSE150 and KYSE410 cells, while expression was lower in KYSE30 and TE1. Figure 5 A). Next, three siRNAs were designed targeting the reverse splicing sequence of circTMEM45A. qRT-PCR was used to verify the knockdown efficiency of the three siRNA sequences. siRNA-1 (si-hsa_circ_0066658_001) was selected for subsequent experimental verification. Figure 5 B), further qRT-PCR experiments demonstrated that KYSE150 cells with stable knockdown of circTMEM45A only reduced the expression of circTMEM45A without affecting the expression of linear TMEM45Am RNA. Figure 5 C). Subsequently, KYSE30 cells were forced to overexpress exogenous circTMEM45A by transfection with a synthetically produced circular plasmid. qRT-PCR verified the overexpression efficiency of circTMEM45A and confirmed that the exogenous plasmid only affected the expression of circTMEM45A, while the expression of the linear transcript of TMEM45A was not increased. Figure 5 D). The above experiments demonstrate that while interfering with the endogenous expression of circTMEM45A, the non-linear transcript TMEM45A affects the biological functional changes of esophageal cancer cells.

[0142] EdU assays, CCK-8 assays, and colony formation assays showed that KYSE30 and TE1 cells overexpressing circTMEM45A exhibited significantly increased DNA replication activity, proliferation, and colony formation capacity. Figure 6 AC, EG), while the opposite results were observed in KYSE150 and KYSE410 cells with knockdown of circTMEM45A (AC, EG). Figure 6 KM, OQ). Wound healing and transwell assays showed that KYSE30 and TE1 cells overexpressing the circTMEM45A plasmid had significantly increased migration and invasion abilities compared to those overexpressing the empty vector plasmid. Figure 6D, 6H-J), while knockdown of circTMEM45A had the opposite effect in KYSE150 and KYSE410 cells Figure 6 N, 6R-T).

[0143] By randomly dividing 10 BALB / C mice into two groups, 5 in each group, injecting KYSE150-luc cells at the beginning stage, and according to the flow chart, injecting si-NC and si-circTMEM45A into the control group and the experimental group mice respectively, and recording the size change of the tumor at the same time Figure 6 U), and on the 28th day, the mice were sacrificed, and it was found that compared with the si-NC group, the tumor growth curve of the si-circTMEM45A group was slower, and the tumor volume was smaller Figure 6 V-X). At the same time, HE and IHC staining of the tumor showed that the proliferation markers ki67 and PCNA and the metastasis markers MMP2 and MMP9 of the si-circTMEM45A group mice were significantly reduced compared with the si-NC group Figure 6 Y-Z).

[0144] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. Use of a promoter of circRANBP9 in the preparation of a pharmaceutical composition for treating gastric cancer; the promoter is selected from a plasmid overexpressing circRANBP9, and the circRANBP9 is hsa_circ_0001577.

2. Use according to claim 1, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

3. Use of an inhibitor of circTMEM45A in the preparation of a pharmaceutical composition for treating esophageal cancer; the inhibitor is selected from an siRNA; the sequence of the siRNA is shown as SEQ ID NO: 4; and the circTMEM45A is hsa_circ_0066658.

4. Use according to claim 3, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

5. Use of a promoter of circRANBP9 in the preparation of a product for inhibiting the proliferation / migration of gastric cancer cells or promoting the senescence of gastric cancer cells; the promoter is selected from a plasmid overexpressing circRANBP9, and the circRANBP9 is hsa_circ_0001577.

6. Use of an inhibitor of circTMEM45A in the preparation of a product for inhibiting the proliferation / migration of esophageal cancer cells; the inhibitor is selected from an siRNA; the sequence of the siRNA is shown as SEQ ID NO: 4; and the circTMEM45A is hsa_circ_0066658.

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