New use of marchf6 in diagnosis or treatment of thoracic tumor

Abstract

The application discloses a new use of MARCHF6 in diagnosis or treatment of chest tumors, and first uses MARCHF6 in diagnosis and treatment of esophageal cancer, and finds, through verification in real clinical samples collected in the application, that the MARCHF6 has good diagnostic efficiency for diagnosis of esophageal cancer, and knocking down expression of the MARCHF6 can significantly inhibit proliferation of esophageal cancer cells, in addition, the application further provides an esophageal cancer diagnosis kit which is simple and convenient to operate, rapid, high in sensitivity and specificity, and can be used in early diagnosis and screening of esophageal cancer, improves survival rate of patients, and is easy to popularize and apply.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to a thoracic tumor marker MARCHF6, and more specifically, it relates to a novel use of MARCHF6 in the diagnosis or treatment of thoracic tumors. Background Technology

[0002] With the rapid growth of the global population and the aggravation of environmental pollution, human health problems, including various diseases, are becoming increasingly serious. Cancer is one of the leading causes of death worldwide, and among all cancers, thoracic tumors have the highest incidence and mortality rates. Thoracic tumors are diseases that occur in the thoracic cavity, mainly including esophageal cancer (EC), which is the seventh leading cause of cancer death globally. The main symptoms of thoracic tumors are cough, sputum production, hemoptysis, chest pain, chest tightness, shortness of breath, back pain, and shoulder pain. Imaging examinations can provide a preliminary assessment of whether the tumor is benign or malignant. In addition to preliminary assessment based on imaging, the research and identification of specific biomarkers for thoracic tumors, and the preparation of sensitive and effective reagent kits, can further improve the accuracy of thoracic tumor diagnosis and enable qualitative and quantitative detection.

[0003] Worldwide, there are over 470,000 new cases of esophageal cancer each year. Based on biological differences, esophageal cancer can be mainly divided into two subtypes: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). Although significant breakthroughs have been made in the diagnosis and treatment of esophageal cancer, the prognosis remains poor due to the late diagnosis in most patients. Currently, early diagnosis of esophageal adenocarcinoma and squamous cell carcinoma, including the identification of Barrett's esophagus and squamous cell dysplasia, has greatly helped improve the prognosis of esophageal cancer. However, current mainstream methods such as confocal laser endoscopy and high-resolution microendoscopy, and the detection of cell-free cfDNA, still face limitations in sensitivity. Therefore, it is more important to combine cytological assessment with the detection of biomarkers (such as DNA methylation status, miRNA detection, or cell surface proteins) to develop next-generation precise predictive models.

[0004] The lack of ideal diagnostic and treatment technologies is a major reason for the current poor outcomes in the diagnosis and treatment of thoracic tumors. Traditional preoperative diagnostic methods, such as endoscopy, computed tomography (CT), and positron emission tomography-computed tomography (PET-CT), often limit their use for large-scale screening due to the trauma or radiation exposure they cause. Because early-stage thoracic tumors lack characteristic clinical manifestations, early diagnosis is difficult, and many patients are already in the middle or late stages at their first visit, thus missing the optimal treatment opportunity. Furthermore, the accuracy of tumor margin assessment during cancer surgery is crucial; relying solely on pathological examination increases the likelihood of postoperative recurrence because pathological examination involves many subjective factors, and its reliability largely depends on the pathologist's experience and skills. Therefore, providing a specific biomarker related to thoracic tumors is of great significance for the early diagnosis and treatment of these tumors.

[0005] Membrane-associated ring finger 6 (MARCHF6) is a member of the MARCH family (which primarily functions as an E3 ubiquitin ligase, broadly regulating various cellular activities such as signal transduction, metabolism, proliferation, and differentiation through protein ubiquitination). It mainly regulates the ubiquitination of key enzymes and modulates metabolic pathways such as cholesterol synthesis. The role of MARCHF6 in different types of tumors is controversial, possibly due to differences in the microenvironment and molecular regulatory characteristics of different tumors, leading to variations in MARCHF6 function. Currently, there are no studies or reports on MARCHF6 in esophageal cancer. This study explored the expression characteristics, clinical significance, and biological functions of MARCHF6 in esophageal cancer, providing insights for research on the carcinogenic mechanisms of esophageal cancer, early screening, and even the exploration of new targets for precision prevention and treatment. Summary of the Invention

[0006] In order to overcome the above-mentioned technical problems existing in the field, the purpose of this invention is to provide a new use of MARCHF6 in the diagnosis or treatment of thoracic tumors.

[0007] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0008] A first aspect of the present invention provides the use of a reagent for detecting the expression level of MARCHF6 in a sample in the preparation of products for diagnosing or identifying esophageal cancer.

[0009] Furthermore, the reagent includes:

[0010] Reagents for detecting the mRNA expression level of MARCHF6 in samples;

[0011] Reagents for detecting the protein expression level of MARCHF6 in a sample; or

[0012] A reagent for detecting the number of MARCHF6-positive cells in a sample;

[0013] Optionally, the reagents for detecting the mRNA expression level of MARCHF6 in the sample include:

[0014] Primers specifically amplifying MARCHF6; or

[0015] Probes that specifically recognize MARCHF6;

[0016] Optionally, the reagents for detecting the protein expression level of MARCHF6 in the sample include:

[0017] Antibodies that specifically bind to the protein encoded by MARCHF6; or

[0018] An affinity protein that specifically binds to the protein encoded by MARCHF6;

[0019] Optionally, the reagent for detecting the number of MARCHF6-positive cells in the sample includes a reagent for detecting the number of MARCHF6-positive cells by immunohistochemistry.

[0020] Furthermore, the identification of esophageal cancer includes differentiating between esophageal squamous cell carcinoma and esophageal adenocarcinoma.

[0021] Furthermore, the MARCHF6 can also be used to differentiate between Barrett's esophagus, esophageal adenocarcinoma, esophageal squamous cell carcinoma, and / or normal esophagus.

[0022] In this invention, the information of the gene MARCHF6 is as follows: The gene ID of gene MARCHF6 (membrane associatedring-CH-type finger 6 [Homo sapiens (human)]) is 10299, and detailed information about the gene can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0023] In this invention, the diagnosis refers to the identification or classification of a molecular or pathological state, disease, or symptom. For example, through molecular characteristics (e.g., specific genes, proteins encoded by specific genes, specific microbial communities, specific metabolites), an early differential diagnosis can be made as to whether a subject has esophageal cancer or the risk of having esophageal cancer, or an early differential diagnosis as to whether a subject has esophageal squamous cell carcinoma or esophageal adenocarcinoma.

[0024] In some embodiments, the primers are the same as amplification primers, which are oligonucleotides that can be used in amplification methods (such as polymerase chain reaction (PCR)) to amplify nucleotide sequences based on a polynucleotide sequence corresponding to a target gene (e.g., a sequence of MARCHF6 or a portion thereof). The primers typically refer to nucleic acid fragments containing 5-100 nucleotides. In preferred embodiments, the primers or amplification primers contain 15-30 nucleotides capable of initiating an enzymatic reaction (e.g., an enzymatic amplification reaction). In specific embodiments of the invention, the primers are primers specifically amplifying the gene MARCHF6.

[0025] In some embodiments, the probe refers to a molecule capable of binding to a specific sequence, subsequence, or other portion of another molecule. In a specific embodiment of the invention, the probe refers to a probe that specifically recognizes MARCHF6. In other embodiments, the probe refers to any molecule or molecule that can bind indirectly or directly, covalently or nonvalently, to or associated with any substrate and / or reaction product and / or protease disclosed herein, and whose association or binding can be detected using the methods disclosed herein. In other embodiments, the probe is a fluorescent probe, an antibody, or an absorbance-based probe. If it is an absorbance-based probe, the chromophore pNA (p-nitroaniline) can be used as a probe for detecting and / or quantifying the target nucleic acid sequences disclosed herein. In some embodiments, the probe may be a nucleic acid sequence comprising a fluorescent molecule or substrate that becomes fluorescent upon exposure to an enzyme, and the nucleic acid sequence is complementary to a fragment of a nucleic acid sequence.

[0026] Unless otherwise specified, in this invention, a probe generally refers to a polynucleotide probe capable of binding to another polynucleotide (often called a target polynucleotide) through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. Hybridization methods include, but are not limited to, solution-phase, solid-phase, mixed-phase, or in situ hybridization assays. Exemplary probes in this invention include gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection assay probes, probes linked to molecular barcodes, and probes immobilized on beads.

[0027] The stringency of hybridization reactions can be readily determined by those skilled in the art and is typically calculated empirically based on probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization usually relies on the ability of denatured DNA to re-anneal when the complementary strand is present in an environment below its denaturation temperature. The higher the expected degree of homology between the probe and the hybridizable sequence, the higher the usable relative temperature. As a result, it is inferred that higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures are less stringent.

[0028] In some embodiments, the reagents that specifically bind to the protein encoded by MARCHF6 include, but are not limited to, antibodies, affinity proteins, and also include peptides, aptamers, and / or compounds that specifically bind to the protein encoded by MARCHF6.

[0029] In some embodiments, the antibody is well known in the art and refers to a specific immunoglobulin targeting an antigenic site. The antibody described in this invention refers to an antibody that specifically binds to the protein encoded by MARCHF6 described in this invention, and can be manufactured according to conventional methods in the art.

[0030] Antibodies can take the form of polyclonal or monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2 and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antigen-binding sites, and any other modified immunoglobulin molecules containing antigen-binding sites, provided that the antibody exhibits the desired biological binding activity.

[0031] In some embodiments, the peptide has a high binding capacity to the target substance (the protein encoded by MARCHF6 as described in this invention) and does not denature during heat or chemical treatment. Furthermore, due to its small size, it can be used as a fusion protein by attaching it to other proteins. Specifically, because it can specifically attach to high molecular weight protein chains, it can be used as a diagnostic kit and drug delivery substance.

[0032] In some embodiments, the aptamer refers to a polynucleotide composed of a specific type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and the ability to bind to a target molecule (the protein encoded by MARCHF6 as described in this invention) with high affinity and specificity. As mentioned above, because aptamers can specifically bind to antigenic substances like antibodies, but are more stable than proteins, have a simpler structure, and are composed of easily synthesized polynucleotides, they can be used as an alternative to antibodies.

[0033] In some embodiments, the reagents for detecting the number of MARCHF6-positive cells by immunohistochemistry include, but are not limited to, any reagents required for detecting the number of MARCHF6-positive cells by immunohistochemistry.

[0034] In some implementations, the reagents required for detecting the number of MARCHF6-positive cells by immunohistochemistry include, but are not limited to: fixatives, buffers, chromogenic solutions, adhesives, sealing agents, enzyme digestion solutions, and sucrose solutions.

[0035] In some embodiments, the fixative includes, but is not limited to, formaldehyde, glutaraldehyde, paraformaldehyde, ethanol, HneFIX, and acetone.

[0036] In some implementations, the buffer includes, but is not limited to, PBS buffer, citrate buffer, EDTA buffer, and TBS buffer.

[0037] In some embodiments, the colorimetric solution includes, but is not limited to: DAB colorimetric solution, 4-chloro-1-naphthol (4-Cl-1-Naphthol) colorimetric solution, 3-amino-9-ethylcarbozole (3-amino-9-ethylcarbozole, AEC) colorimetric solution, TMB colorimetric solution, and NBT colorimetric solution.

[0038] In some embodiments, the adhesive includes, but is not limited to, gelatin, resin glue, polylysine, and commercially available adhesives.

[0039] In some embodiments, the sealing agent includes, but is not limited to, skim milk powder, BSA, serum, and Fab fragment single-chain secondary antibody.

[0040] In some embodiments, the enzyme digestion solution includes, but is not limited to, trypsin digestion solution and pepsin digestion solution.

[0041] In some embodiments, the expression level of the present invention refers to the absolute or relative amount of MARCHF6. The expression level of MARCHF6 of the present invention can be determined by various techniques known to those skilled in the art. In particular, the absolute or relative amount of MARCHF6 of the present invention can be detected by immunohistochemical detection methods known to those skilled in the art.

[0042] In some embodiments, the sample refers to a composition obtained from or derived from the target subject, which contains cellular entities and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical and / or physiological characteristics.

[0043] Furthermore, the sample may be obtained from the subject's tissue sample, blood sample, or other fluid sample of biological origin, such as a biopsy tissue sample or tissue culture or cells derived therefrom. The source of the tissue sample may be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy tissue or aspirates; blood or any blood component; body fluids; cells from any stage of an individual's pregnancy or development; or plasma. The term "sample" includes biological samples that have been processed in any way after their acquisition, such as being treated with reagents, stabilized, or enriched for certain components (such as proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes.

[0044] In some embodiments, the sample includes, but is not limited to: subject-derived tissue, blood, serum, plasma, blood-derived cells, lymph, synovial fluid, cerebrospinal fluid, pleural fluid, peritoneal fluid, bladder irrigation fluid, secretions (e.g., breast secretions), oral irrigation fluid, swabs (e.g., oral swabs), touch preparations, fine needle aspiration materials, cell extracts, and combinations thereof. In a specific embodiment of the invention, the sample is preferably a subject-derived tissue sample.

[0045] In a specific embodiment of the present invention, the receiver operating characteristic (ROC) analysis method is used to evaluate the performance of the MARCHF6 in the differential diagnosis of esophageal cancer, or to test its accuracy in separating patients into different groups.

[0046] The ROC curve is a graphical representation of sensitivity and specificity spectra generated using sensitivity as the y-axis and 1-specificity as the x-axis, with various cutoff values. In the ROC curve, the true positive rate (sensitivity) is plotted as a function of the FP rate (100-specificity) for different cutoff points. Each point on the ROC curve represents a sensitivity / specificity pair corresponding to a specific decision threshold. The ROC curve for a test with perfect discriminative power (no overlap between the two distributions) passes through the upper left corner (sensitivity 100%, specificity 100%). Therefore, qualitatively, the closer the curve is to the upper left corner, the higher the overall accuracy of the test. The area under the ROC curve (AUC) reflects the accuracy of the test and is displayed in the lower left corner of the curve. For an AUC greater than 0.5, the closer it is to 1, the better the diagnostic or predictive performance of the indicator.

[0047] In a specific embodiment of the present invention, it has been verified that the AUC value of MARCHF6 in the real clinical sample set collected in the present invention (40 esophageal cancer tissue samples and corresponding 40 adjacent normal tissue samples) is 0.9113, the specificity is 85%, and the sensitivity is 100%, which proves that MARCHF6 has extremely high diagnostic efficacy for esophageal cancer, with high accuracy, specificity and sensitivity.

[0048] A second aspect of the present invention provides a diagnostic kit or detection chip for diagnosing or identifying esophageal cancer.

[0049] Furthermore, the diagnostic kit or detection chip contains the reagent described in the first aspect of the present invention for detecting the expression level of MARCHF6 in the sample.

[0050] Furthermore, the diagnostic kit contains primers or probes that specifically bind to the MARCHF6;

[0051] Optionally, the detection chip includes a solid support and a probe attached to the solid support that specifically identifies the MARCHF6.

[0052] In some embodiments, the diagnostic kit further includes instructions for use or a label, a positive control, a negative control, a buffer, an adjuvant, or a solvent, and one or more containers for containing the compositions contained in the diagnostic kit. The compositions may be in liquid form or lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials, including glass or plastic. The instructions for use or label details how to use the diagnostic kit to test a sample and how the diagnostic kit is used to diagnose or assist in the diagnosis of esophageal cancer.

[0053] In some embodiments, the diagnostic kit may also include a variety of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed in this invention. Any reagent that is based on the detection of MARCHF6 to diagnose or assist in the diagnosis of esophageal cancer falls within the protection scope of this invention.

[0054] In some implementations, the diagnostic kits include, but are not limited to: qPCR kits, Western blot assay kits, immunochromatographic assay kits, flow cytometry kits, immunohistochemical assay kits, ELISA kits, and electrochemiluminescence assay kits.

[0055] In some embodiments, the detection chip can be prepared using conventional biochip preparation methods known to those skilled in the art, including but not limited to: using a solid-phase carrier of a modified glass slide or silicon wafer, having an amino-modified polydT string at the 5' end of the probe, preparing an oligonucleotide probe into a solution, and then spotting it onto the modified glass slide or silicon wafer using a spotting instrument to arrange it into a predetermined sequence or array, and then fixing it by leaving it overnight to obtain the detection chip of the present invention.

[0056] In some embodiments, the primers that specifically bind to the MARCHF6 can be prepared by chemical synthesis, appropriately designed using methods well known to those skilled in the art and with reference to known information, and prepared by chemical synthesis.

[0057] A third aspect of the invention provides the use of a reagent for detecting the expression level of MARCHF6 in a sample in the preparation of systems and / or devices for diagnosing or identifying esophageal cancer.

[0058] Furthermore, the system and / or device includes the following units:

[0059] Input unit: The input unit is used to input the data on the MARCHF6 expression level obtained from the subject sample into the processing unit;

[0060] Processing unit: The processing unit is used to analyze and process the expression level data of MARCHF6 input by the input unit to obtain the diagnostic or differential diagnosis results of the subject;

[0061] Output unit: The output unit is used to output the diagnostic or identification results of the subject obtained by the processing unit.

[0062] A fourth aspect of the invention provides the use of an agent that inhibits MARCHF6 expression in the preparation of a medicament for the treatment and / or prevention of esophageal cancer.

[0063] Furthermore, the reagents for inhibiting MARCHF6 expression include siRNA targeting the MARCHF6 gene, shRNA targeting the MARCHF6 gene, reagents for inhibiting the transcriptional activity of the MARCHF6 gene, reagents for inhibiting the transcriptional level of MARCHF6 mRNA, reagents for promoting the degradation of MARCHF6 mRNA, or reagents for inhibiting the translation of MARCHF6 mRNA.

[0064] Optionally, the reagent for inhibiting MARCHF6 expression is siRNA targeting the MARCHF6 gene;

[0065] Optionally, the sequence of the siRNA is as shown in SEQ ID NO:1-2 or as shown in SEQ ID NO:3-4;

[0066] Optionally, the dosage form of the drug is selected from gastrointestinal dosage forms and non-gastrointestinal dosage forms;

[0067] Optionally, the gastrointestinal dosage form is selected from solutions, granules, tablets, capsules, suspensions, powders, sustained-release preparations, effervescent tablets, emulsions, syrups, drops, and chewable tablets;

[0068] Optionally, the non-gastrointestinal dosage form is selected from injection dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

[0069] In some embodiments, the reagents for inhibiting MARCHF6 expression according to the present invention include, but are not limited to: reagents for inhibiting the expression of the gene encoding the MARCHF6 protein or reducing the expression level of the gene, and reagents for reducing the activity of the MARCHF6 protein. For example, reagents for inhibiting the expression of the MARCHF6 gene or reducing its expression level include, but are not limited to: reagents for inhibiting the transcriptional activity of the MARCHF6 gene, reagents for inhibiting the transcriptional level of MARCHF6 mRNA, reagents for promoting the degradation of MARCHF6 mRNA, siRNA targeting the MARCHF6 gene, shRNA targeting the MARCHF6 gene, reagents for inhibiting the translation of MARCHF6 mRNA, reagents for specifically recognizing and cleaving guide nucleic acids of the MARCHF6 gene to reduce its expression level, dsRNA targeting the MARCHF6 gene, microRNA targeting the MARCHF6 gene, and antisense nucleic acids targeting the MARCHF6 gene.

[0070] In other embodiments, the entire MARCHF6 gene can be knocked out by administering a targeting vector, thereby inhibiting or reducing MARCHF6 gene expression. In other embodiments, the agent for reducing MARCHF6 protein activity can be, for example, a specific antibody targeting MARCHF6 or a small molecule compound having the ability to inhibit MARCHF6 protein activity. In a specific embodiment of the invention, the agent for inhibiting MARCHF6 expression is an siRNA encoding a sequence as shown in SEQ ID NO:1-2 or as shown in SEQ ID NO:3-4.

[0071] In some embodiments, the activity of the MARCHF6 protein can also be reduced by introducing mutations into it. In other embodiments, mutations that weaken or eliminate the corresponding activity are introduced into the functional domains of the MARCHF6 protein. Mutations can be the insertion, deletion, or substitution of one or more (e.g., more than 10, 20, or 30) amino acids. Mutations that weaken or eliminate the associated biological activity can be introduced into the functional domains of the encoded MARCHF6 protein by administering a reagent that acts on the MARCHF6 gene. Such reagents can alter the sequence of the MARCHF6 gene, resulting in a corresponding mutation in the encoded MARCHF6 protein, thereby exhibiting weakened or lost activity. For example, the wild-type MARCHF6 gene can be replaced with a mutated MARCHF6 gene using homologous recombination technology, resulting in the expression of a weakly active or inactive MARCHF6 protein.

[0072] In this invention, siRNA refers to small interfering ribonucleic acid, i.e., a relatively short double-stranded nucleic acid or optionally its longer precursor. In some embodiments, the length of the siRNA usable in this invention is preferably about 20-50 bp. However, there is no particular limitation on the length of the usable siRNA in this invention. For example, siRNA may initially exist in the cell in a precursor form, which is substantially different from the final or processed form of siRNA that exhibits and exerts gene silencing activity when or after delivery to the target cell. For example, the precursor form of siRNA may include precursor sequence elements that are processed, degraded, altered, or cleaved during or after delivery to produce siRNA with gene silencing activity mediating within the cell.

[0073] In this invention, the shRNA refers to short hairpin RNA, which comprises two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector consists of two short inverted repeat sequences separated by a stem-loop sequence, forming a hairpin structure controlled by the polIII promoter. Subsequently, 5-6 T molecules are added as a transcription terminator for RNA polymerase III. The shRNA can stably integrate into the cell's genome, allowing for long-term gene knockout.

[0074] In this invention, dsRNA refers to double-stranded ribonucleic acid, an RNA molecule formed by the renaturation of two complementary strands, which can be cleaved by the Dicer enzyme to form siRNA. dsRNA inhibits gene expression through RNA interference (RNAi). dsRNA does not need to have 100% homology with the target gene sequence, as long as it can inhibit the expression of the target gene.

[0075] In this invention, the microRNA refers to microRNA, which is a non-coding RNA approximately 22 nt in length and is widely found in various organisms, from viruses to humans. Mature miRNAs primarily function to negatively regulate gene transcription at the post-transcriptional level, participating in numerous life processes such as cell proliferation, apoptosis, immunity, neuroendocrine processes, and stem cell differentiation by causing the degradation of their target mRNAs or interrupting translation.

[0076] In this invention, the antisense nucleic acid refers to a nucleic acid containing a sequence complementary to the encoding MARCHF6. The antisense nucleic acid can be composed of DNA, RNA, or both. It may contain non-complementary bases, provided it can specifically hybridize under stringent conditions. When introduced into a cell, the antisense nucleic acid binds to the target polynucleotide and inhibits transcription, RNA processing, or stability. In addition to the antisense polynucleotide, the antisense nucleic acid also includes a polynucleotide mimic containing a modified backbone and 3' and 5' end portions. Such antisense nucleic acids can be appropriately designed based on MARCHF6 sequence information and prepared using methods known to those skilled in the art.

[0077] The fifth aspect of the invention provides any of the following products:

[0078] (1) A pharmaceutical composition comprising the reagent for inhibiting MARCHF6 expression as described in the fourth aspect of the present invention;

[0079] (2) A pharmaceutical preparation comprising the reagent for inhibiting MARCHF6 expression as described in the fourth aspect of the present invention.

[0080] In some embodiments, the pharmaceutical composition or pharmaceutical formulation may further comprise pharmaceutically acceptable carriers and / or excipients, which are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used as needed to aid in the stability of the formulation or to help improve its activity or bioavailability. The pharmaceutically acceptable carriers and / or excipients include, but are not limited to: buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); preservatives; and any other pharmaceutically or physiologically acceptable carriers or excipients reported in the prior art that can be used in the pharmaceutical composition.

[0081] In some embodiments, the dosage form of the pharmaceutical composition or pharmaceutical preparation includes, but is not limited to, parenteral dosage forms or oral preparations. Exemplary parenteral dosage forms include injections, aerosols, suppositories, or subcutaneous dosage forms. Exemplary oral preparations include tablets, capsules, pills, granules, microcapsule tablets, suspensions, pellets, and oral liquid preparations.

[0082] A sixth aspect of the present invention provides a method for screening candidate drugs for the treatment and / or prevention of esophageal cancer.

[0083] Furthermore, the method includes the following steps:

[0084] (1) Treat the system expressing or containing MARCHF6 with the analyte;

[0085] (2) Detect the expression of MARCHF6 in the system;

[0086] (3) Select the test substance that can inhibit the expression of MARCHF6 as a candidate drug.

[0087] In some implementations, the system is selected from cellular systems, subcellular systems, solution systems, tissue systems, organ systems, or animal systems.

[0088] In some implementations, the analyte includes, but is not limited to, interfering molecules designed for MARCHF6, nucleic acid inhibitors, small molecule compounds, etc.

[0089] In some implementations, the methods for detecting MARCHF6 expression in the detection system include, but are not limited to: reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, ribonuclease protection assay (RPA), RNA blotting, and DNA microarray.

[0090] In some implementations, reagents for detecting MARCHF6 expression include, but are not limited to, primers, probes, or antisense nucleotides. Those skilled in the art can design primers, probes, or antisense nucleotides that specifically bind to MARCHF6 based on its sequence information.

[0091] In some implementations, the selected candidate drug is an analyte that, in the presence of the candidate drug, can inhibit the expression level of MARCHF6 in the system compared to the expression level of MARCHF6 detected in the absence of the candidate drug.

[0092] In addition, the present invention provides a method for diagnosing or identifying esophageal cancer, the method comprising detecting the expression level of MARCHF6 in a sample from a subject in need, and determining, based on the expression level of MARCHF6, whether the subject is an esophageal cancer patient, the risk of having esophageal cancer, or the specific type of esophageal cancer.

[0093] Furthermore, the present invention also provides a method for treating and / or preventing esophageal cancer, the method comprising administering to a subject in need a therapeutically and / or preventively effective amount of the reagent for inhibiting MARCHF6 expression as described in the fourth aspect of the present invention, the pharmaceutical composition as described in the fifth aspect of the present invention, or the pharmaceutical preparation as described in the fifth aspect of the present invention.

[0094] In some embodiments, the subject of this invention refers to any animal, including both human and non-human animals. Non-human animals include all vertebrates, such as mammals like non-human primates (especially higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; as well as non-mammals such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human.

[0095] In some embodiments, the routes of administration of the reagents, pharmaceutical compositions, or pharmaceutical preparations described in this invention are not limited, as long as they achieve the desired therapeutic or preventative effect. These routes of administration include, but are not limited to: local, via skin, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesical, intramuscular, intratracheal, subcutaneous, inhalation, via pleura, via mucous membranes, skin, gastrointestinal tract, intra-articular, intraventricular, rectal, vaginal, intraskull, intraurethral, ​​and intrahepatic administration. In some cases, systemic administration may be used; in others, local administration may be used.

[0096] In some embodiments, the dosage of the reagents, pharmaceutical compositions, or pharmaceutical preparations described in this invention is not limited, as long as the desired therapeutic or preventive effect can be obtained, and can be appropriately determined based on the subject's symptoms, gender, age, etc. The dosage of the reagents, pharmaceutical compositions, or pharmaceutical preparations described in this invention can be determined in detail using, for example, the therapeutic or preventive effect on the disease as an indicator.

[0097] In some implementations, the treatment and / or prevention refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of existing symptoms, conditions, ailments, or diseases (e.g., esophageal cancer). Desired therapeutic effects include, but are not limited to: preventing disease onset or recurrence, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

[0098] In some embodiments, the effective amount refers to a therapeutically effective amount, which, as used herein, means an amount of pharmaceutical composition sufficient to treat a specified disorder, condition, or disease (e.g., improve, alleviate, reduce, and / or delay one or more of its symptoms). In some embodiments, the effective amount is an amount sufficient to delay the development of a disease (e.g., esophageal cancer). In some embodiments, the effective amount is an amount sufficient to prevent or delay disease recurrence. An effective dose can be administered in a single or multiple-dose manner. A pharmaceutically effective therapeutic amount depends on a variety of factors, including but not limited to: characteristics of the treatment subject (e.g., height, weight, sex, age, and medication history), the severity of the disease, etc.

[0099] Compared with the prior art, the present invention has the following beneficial effects:

[0100] This invention is the first to use MARCHF6 in the diagnosis and treatment of esophageal cancer. Validation in real clinical samples collected during this invention revealed that MARCHF6 has good diagnostic efficacy for esophageal cancer, with high accuracy, sensitivity, and specificity, making it suitable for effective diagnosis. Knockdown of MARCHF6 expression significantly inhibits the proliferation of esophageal cancer cells. Furthermore, this invention provides a simple, rapid, and highly sensitive and specific esophageal cancer diagnostic kit that can be used for early diagnosis and screening of esophageal cancer, improving patient survival rates and facilitating widespread application. Attached Figure Description

[0101] Figure 1 Gene expression matrices and clinical information from different esophageal cancer datasets;

[0102] Figure 2The gene expression characteristics of MARCHF6 in esophageal cancer-normal tissues are shown in Figure A: Unlabeled UMAP plots of 8 esophageal cancer datasets; Figure B: Integrated UMAP plots of the esophageal cancer dataset; Figures C and D: Gene expression characteristics of MARCHF6 in esophageal cancer tissues and normal tissues in the esophageal cancer dataset (training and validation sets); Figure EF: ROC curves showing the distinguishing effect between esophageal cancer tissues and normal tissues (training and validation sets).

[0103] Figure 3 Gene expression characteristics of MARCHF6 in esophageal squamous cell carcinoma and normal tissues. Figure AB: Gene expression characteristics of MARCHF6 in esophageal squamous cell carcinoma and normal tissues in esophageal cancer datasets (training and validation sets); Figure CD: ROC curves showing the distinguishing effect between esophageal squamous cell carcinoma and normal tissues (training and validation sets).

[0104] Figure 4 The gene expression characteristics of MARCHF6 in esophageal adenocarcinoma-normal tissues are shown in Figures A, C, and E: Esophageal cancer datasets (training and validation sets), illustrating the gene expression characteristics of MARCHF6 in esophageal adenocarcinoma-normal tissues, Barrett's esophagus and esophageal adenocarcinoma, and Barrett's esophagus and normal tissues. Figures B, D, and F: ROC curves showing the discriminative effect in esophageal adenocarcinoma-normal tissues, Barrett's esophagus and esophageal adenocarcinoma, and Barrett's esophagus and normal tissues (training and validation sets).

[0105] Figure 5 The gene expression characteristics of MARCHF6 in esophageal adenocarcinoma and esophageal squamous cell carcinoma. Figure A: Gene expression characteristics of MARCHF6 in esophageal adenocarcinoma and esophageal squamous cell carcinoma tissues in esophageal cancer datasets (training and validation sets); Figure B: ROC curves showing the discriminative effect in esophageal adenocarcinoma and esophageal squamous cell carcinoma tissues (training and validation sets).

[0106] Figure 6 Biological function analysis of the MARCHF6 gene in esophageal adenocarcinoma and esophageal squamous cell carcinoma. Figure A shows that in the Hallmark database, MARCHF6 activation in esophageal adenocarcinoma is associated with important cancer features, such as proliferation and KRAS / AKT activation. Figure B shows that in the Hallmark database, MARCHF6 in esophageal squamous cell carcinoma is also associated with various cancer features, such as proliferation and KRAS / AKT activation. Figure C shows the biological function analysis of the MARCHF6 gene in esophageal adenocarcinoma in the Reactome database. Figure D shows the biological function analysis of the MARCHF6 gene in esophageal squamous cell carcinoma in the Reactome database.

[0107] Figure 7 : Tissue microarray analysis results of MARCHF6 in real clinical esophageal cancer tissue samples and adjacent normal tissue samples collected in this invention;

[0108] Figure 8 : Protein quantity analysis of MARCHF6 in esophageal cancer, wherein, Figure A: Differential expression analysis results of MARCHF6 in real clinical samples collected in this invention (immunohistochemical quantitative analysis of MARCHF6 in esophageal cancer tissue-normal tissue); Figure B: Diagnostic efficacy analysis results of MARCHF6 in real clinical samples collected in this invention (ROC curve shows the distinguishing effect in esophageal cancer tissue-normal tissue).

[0109] Figure 9 MARCH1 has no diagnostic efficacy for esophageal cancer. Figure A shows no difference in MARCH1 expression between esophageal cancer and normal tissues; Figure B shows no difference in diagnostic ROC curves; Figure C shows no difference in MARCH1 expression between esophageal squamous cell carcinoma and normal tissues; Figure D shows no difference in diagnostic ROC curves; Figure E shows no difference in MARCH1 expression between esophageal adenocarcinoma and normal tissues; Figure F shows no difference in diagnostic ROC curves.

[0110] Figure 10 The effect of knocking down MARCHF6 expression on esophageal cancer cells: Figure A: Establishment of an esophageal cancer cell line model with low MARCHF6 expression using siRNA technology; Figure B: Plate cloning assay to detect the effect of knocking down MARCHF6 expression on the cloning ability of esophageal cancer cells; Figure C: CCK8 cell proliferation assay to detect the effect of knocking down MARCHF6 expression on the proliferation ability of esophageal cancer cells. Detailed Implementation

[0111] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents.

[0112] The reagents and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0113] Example 1: Study on the Diagnosis and Biological Function of MARCHF6 in Esophageal Cancer

[0114] 1. Materials and Methods

[0115] 1.1 Public Dataset Information

[0116] Gene expression matrices and clinical information for different esophageal cancer datasets were obtained from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ) (see details). Figure 1 ).

[0117] Eight esophageal cancer datasets were integrated for gene expression characterization analysis, including: datasets (number of tumor tissue / normal tissue samples), GSE1420 (16 / 8), GSE20347 (17 / 17), GSE23400 (53 / 53), GSE26886 (50 / 19), GSE38129 (30 / 30), GSE5364 (16 / 13), GSE77861 (7 / 7), and GSE92396 (12 / 9).

[0118] 1.2 Patients and Organizations

[0119] Forty esophageal cancer tissue samples (corresponding to 40 adjacent normal tissue samples) were obtained from the Department of Thoracic Surgery, Second Affiliated Hospital of Air Force Medical University. Detailed clinical information was collected, organized, and analyzed simultaneously. The tissues were divided into two parts: one part was used for paraffin embedding and immunohistochemical experiments, and the other part was frozen tissue used for protein and RNA extraction and subsequent experiments. The use of human pathological specimens in this study complied with the ethical requirements of the Ethics Committee of the Second Affiliated Hospital of Air Force Medical University, as well as the 1964 Declaration of Helsinki and its subsequent amendments and supplements. All study subjects provided informed consent and signed informed consent forms.

[0120] 1.3 Immunohistochemistry

[0121] After collecting the aforementioned esophageal cancer and adjacent normal tissues, they were fixed with 4% paraformaldehyde, dehydrated with graded ethanol, cleared with xylene, impregnated with paraffin, and embedded in paraffin. Paraffin sections were dried, dewaxed with xylene, hydrated with graded ethanol, washed with phosphate buffer, and microwaved for antigen retrieval. After treatment with 3% hydrogen peroxide to remove endogenous peroxidase, the sections were blocked, incubated overnight with primary antibody, washed the next day, incubated with enzyme-labeled secondary antibody, washed, and then subjected to DAB staining. The protein content in immunohistochemistry was measured using a semi-quantitative method: H-score = Σ(pi*i), where "pi" represents the percentage of positive cells and "i" represents the signal intensity.

[0122] 1.4 Western blot

[0123] Frozen tissues were lysed using RIPA lysis buffer containing phosphatase and protease inhibitors. The lysates were added to loading buffer containing sodium dodecyl sulfate (SDS) and boiled in water, followed by separation by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were then transferred to nitrocellulose membranes. After blocking with 5% skim milk, the membranes were incubated overnight with specific primary antibodies, then incubated with species-specific enzyme-labeled secondary antibodies, washed, and detected using an ECL kit.

[0124] 1.5 Methods and Statistical Analysis

[0125] Gene expression data were standardized using Z-scaling; MARCHF6 gene expression levels in esophageal cancer tissue-normal tissue and MARCHF6 protein expression levels in esophageal cancer tissue-adjacent tissue were assessed using the Wilcoxon test; the correlation between MARCHF6 and genes was evaluated using Pearson correlation analysis, and the bioaccumulation of MARCHF6 in the Hallmark and Reactome databases was assessed using gene set enrichment analysis (GSEA); graphical plotting and statistical analysis were performed using R language (statistically significant: two-sided). p <0.05).

[0126] 2. Experimental Results

[0127] 2.1 MARCHF6 shows high expression in esophageal cancer.

[0128] Because the sample size of both normal tissue and esophageal cancer tissue in the above individual datasets is small, statistically significant comparisons cannot be made. Therefore, this embodiment integrates the above gene sets. It can be seen that there is a significant batch effect before annotation. Figure 2 A) After integration, the batch effect is significantly reduced (through U-map). Figure 2 B). We stratified the dataset into training and validation sets in a 1:1 ratio based on the ratio of esophageal cancer to normal tissue (Normal tissue / Tumor tissue, 156 / 173). We can see that, in both the validation and training sets, the MARCHF6 gene is significantly more expressed in esophageal cancer tissue compared to normal tissue. Figure 2 C, training set; Figure 2 D, the validation set). Simultaneously, the ROC curve reveals a significant discrimination effect (…). Figure 2 E, the training set; Figure 2 F, the validation set).

[0129] 2.2 MARCHF6 is highly expressed in esophageal squamous cell carcinoma, and it can be used to differentiate between esophageal squamous cell carcinoma and normal esophageal tissue.

[0130] Esophageal squamous cell carcinoma is an important pathological subtype of esophageal cancer. We also stratified the dataset into training and validation sets in a 1:1 ratio based on the ratio of esophageal squamous cell carcinoma to normal tissue (Normal tissue / squamous cell carcinoma, 156 / 116). We can see that, in both the validation and training sets, the MARCHF6 gene shows significantly higher expression in esophageal squamous cell carcinoma tissue compared to normal tissue. Figure 3 A, the training set; Figure 3 B, the validation set). Simultaneously, the ROC curve reveals a significant discrimination effect (…). Figure 3 C, training set; Figure 3 D, the validation set).

[0131] 2.3 Analysis of MARCHF6 gene expression levels in esophageal adenocarcinoma, Barrett's esophagus, and normal tissues

[0132] Due to the limited number of esophageal adenocarcinoma cases, cross-validation was not feasible. Therefore, we uniformly analyzed expression levels within the overall dataset, as esophageal adenocarcinoma follows a transitional pattern from normal tissue to Barrett's esophagus and then to esophageal adenocarcinoma. We compared the relationships among these three groups (Normal tissue / Barrett's esophagus / adenocarcinoma, 156 / 28 / 41). We found that the MARCHF6 gene exhibited significantly higher expression in esophageal adenocarcinoma tissue compared to normal tissue. Figure 4 A). Simultaneously, the ROC curve reveals a significant distinguishing effect ( Figure 4 B). No significant difference was observed in MARCHF6 between Barrett's esophagus and adenocarcinoma. Figure 4 C) and the differentiation effect ( Figure 4 D). Between Barrett's esophagus and normal tissue, Barrett's esophagus showed high expression of MARCHF6 (D). Figure 4 E), while the diagnostic ROC curve shows critical diagnostic characteristics ( Figure 4 F). This reflects the potential for cancerous transformation in Barrett's esophagus.

[0133] 2.4 Analysis of MARCHF6 gene expression in esophageal adenocarcinoma and esophageal squamous cell carcinoma

[0134] Due to the limited number of esophageal adenocarcinoma cases, cross-validation was not feasible. Therefore, we unified the expression analysis within the overall dataset, examining both esophageal adenocarcinoma (AD) and esophageal squamous cell carcinoma (SQC) (adenocarcinoma / squamous cell carcinoma, 41 / 116). We found that the expression level of the MARCHF6 gene was lower in esophageal adenocarcinoma tissues than in esophageal squamous cell carcinoma tissues (AD). Figure 5 A). Simultaneously, the ROC curve reveals a significant distinguishing effect ( Figure 5 B). This suggests that MARCHF6 has the function of differentiating between esophageal squamous cell carcinoma and adenocarcinoma.

[0135] 2.5 Biological function analysis of the MARCHF6 gene in esophageal adenocarcinoma and esophageal squamous cell carcinoma

[0136] We analyzed the biological significance of MARCHF6 in esophageal adenocarcinoma and esophageal squamous cell carcinoma, respectively. We used Pearson correlation analysis to identify genes associated with MARCHF6 and performed GSEA analysis based on correlation coefficient ranking. We found that in Hallmark data, MARCHF6 activation in esophageal adenocarcinoma was associated with important cancer features, such as proliferation and KRAS / AKT activation. Figure 6 A), while MARCHF6 in esophageal squamous cell carcinoma is also associated with various cancer characteristics, such as proliferation and KRASAKT activation ( Figure 6 B). The results of biological function analysis of the MARCHF6 gene in esophageal adenocarcinoma and esophageal squamous cell carcinoma in the Reactome database are shown below. Figure 6 C and Figure 6 D.

[0137] 2.6 Analysis of MARCHF6 expression levels in esophageal cancer and normal tissues

[0138] This embodiment uses tissue microarray method to further verify the expression of MARCHF6 in real clinical sample set, and calculates the area under the receiver operating characteristic curve (AUC) by ROC analysis to analyze the accuracy (AUC value), sensitivity and specificity of MARCHF6 in diagnosing esophageal cancer, so as to further determine the diagnostic efficacy of MARCHF6 in the above real clinical sample set.

[0139] In 40 esophageal cancer tissue samples and 40 corresponding adjacent normal tissue samples collected by our department (Department of Thoracic Surgery, Second Affiliated Hospital of Air Force Medical University), immunohistochemistry revealed that MARCHF6 showed high expression of a cancer-associated protein compared to adjacent normal tissue. Figure 8 A) The tissue microarray analysis results are as follows: Figure 7 As shown. Simultaneously, ROC curve analysis revealed a significant distinguishing effect (…). Figure 8B) The above results indicate that MARCHF6 has an accurate role in differentiating and diagnosing esophageal cancer, with an AUC value of 0.9113, a specificity of 85%, and a sensitivity of 100%.

[0140] Example 2: Application of MARCHF6 in the treatment of esophageal cancer

[0141] 1. qPCR experiment

[0142] After lysing the extracted cells with Trizol solution, chloroform was added to extract RNA. Following centrifugation, anhydrous ethanol was added to the supernatant to precipitate the RNA. After centrifugation, the damaged cells were discarded, and the cells were washed with anhydrous ethanol, dried, and the RNA was dissolved. The concentration of the extracted RNA was determined using a Nanodrop instrument. Using the extracted RNA as a template, cDNA was synthesized under the action of reverse transcriptase. A qPCR reaction solution containing primers and template was prepared, spotted onto a PCR plate, and the reaction conditions of the PCR instrument were set for amplification. Finally, the target DNA content was determined through data analysis.

[0143] 2. CCK8 cell proliferation experiment

[0144] Cells in good logarithmic growth phase were thoroughly diluted into single-cell suspensions, counted, and then arranged at equal cell densities (10T) per group. 4 Cells were seeded in 96-well plates (8 wells per group, 100 μL per well). Cells were cultured normally, and assays were performed daily at fixed times (100 μL of 10% CCK8 reagent was added to each well, incubated for 2 h, and the absorbance was measured at 450 nm using a microplate reader). The relative proliferation of cells was plotted based on absorbance versus time.

[0145] 3. Plate cloning experiment

[0146] Cells in good logarithmic growth phase were thoroughly diluted into single-cell suspensions and then seeded into 6-well plates (keeping the cell count consistent across groups, 500-2000 cells per well). After 2-3 weeks of normal culture (until visible clones formed), the cells were fixed with methanol, washed, stained with crystal violet, photographed, and the clone formation was statistically analyzed.

[0147] 4. Small interfering RNA transfection

[0148] The mixture of liposomes (Lipofectamine 2000) and MARCHF6 small interfering RNA (siRNA) was transfected into the esophageal cancer cell line TE1. The transfection effect was detected by Western blotting after 48-74 hours. Once the conditions were met, it was used for subsequent functional experiments.

[0149] The sequences of si-MARCHF6-1 and si-MARCHF6-2 are shown below:

[0150] si-MARCHF6-1 Justice Chain: 5'-GCUCUACAGUGAUGCUCCA-3' (SEQ ID NO:1);

[0151] si-MARCHF6-1 antisense chain: 5'-UGGAGCAUCACUGUAGAGC-3' (SEQ ID NO:2);

[0152] si-MARCHF6-2 Justice Chain: 5'-CCCUUACCAUAUUGGUCAU-3' (SEQ ID NO:3);

[0153] si-MARCHF6-2 antisense chain: 5'-AUGACCAAUAUGGUAAGGG-3' (SEQ ID NO:4).

[0154] 5. Experimental Results

[0155] Given the positive proliferative potential of MARCHF6 in esophageal cancer tissues, this invention further analyzed the proliferative function of MARCHF6 in esophageal cancer cell lines. Firstly, a low-expression model of MARCHF6 was established in the esophageal cancer cell line (TE1) using siRNA technology. The results are as follows: Figure 10 As shown in Figure A, the results indicated that both si-MARCHF6-1 and si-MARCHF6-2 significantly knocked down the expression of MARCHF6 in the esophageal cancer cell line TE1. Further plate colony assays revealed that knocking down MARCHF6 expression significantly reduced the cloning ability of the esophageal cancer cell line, and si-MARCHF6-2 showed a more significant inhibitory effect on the proliferation of esophageal cancer cells. Figure 10 B). Finally, CCK8 cell proliferation experiments revealed that knocking down MARCHF6 expression significantly inhibited the proliferation of esophageal cancer cell lines. Figure 10 C).

[0156] Contrast ratios show that not all members of the MARCH family have the ability to diagnose esophageal cancer.

[0157] MARCH1 is also a member of the MARCH family. Verification using the methods described in the previous examples revealed that MARCH1 cannot effectively diagnose esophageal cancer, and its expression is not significantly different between esophageal cancer and normal tissues. Figure 9 A), ROC curve diagnosis showed no difference ( Figure 9 B). MARCH1 expression showed no difference between esophageal squamous cell carcinoma and normal tissue. Figure 9 C), ROC curve diagnosis showed no difference ( Figure 9D). MARCH1 expression showed no difference between esophageal adenocarcinoma and normal tissue. Figure 9 E), ROC curve diagnosis showed no difference ( Figure 9 F).

[0158] The above results indicate that not all members of the MARCH family have the function of diagnosing esophageal cancer. The fact that MARCHF6, which was discovered for the first time in this invention, has the function of accurately identifying and diagnosing esophageal cancer is a technical effect that was not expected by those skilled in the art.

Claims

1. Application of reagents for detecting the protein expression level of MARCHF6 in samples in the preparation of products for esophageal cancer diagnosis.

2. The application according to claim 1, characterized in that, The reagents used to detect the protein expression level of MARCHF6 in the sample include: Antibodies that specifically bind to the protein encoded by MARCHF6; or An affinity protein that specifically binds to the protein encoded by MARCHF6.

3. Application of reagents for detecting the protein expression level of MARCHF6 in samples in the preparation of systems and / or devices for diagnosing esophageal cancer.

4. The use of reagents that inhibit MARCHF6 expression in the preparation of drugs for the treatment and / or prevention of esophageal cancer; The reagent used to inhibit MARCHF6 expression is siRNA targeting the MARCHF6 gene; The sequence of the siRNA is shown in SEQ ID NO:1-2 or SEQ ID NO:3-4.

5. The application according to claim 4, characterized in that, The dosage form of the drug is selected from gastrointestinal dosage forms and non-gastrointestinal dosage forms.

6. The application according to claim 5, characterized in that, The enteral dosage form is selected from solutions, granules, tablets, capsules, suspensions, powders, emulsions, and syrups.

7. The application according to claim 6, characterized in that, The solution is a drop.

8. The application according to claim 6, characterized in that, The tablets are effervescent tablets or chewable tablets.

9. The application according to claim 5, characterized in that, The gastrointestinal dosage form is a sustained-release formulation.

10. The application according to claim 5, characterized in that, The non-gastrointestinal dosage form is selected from injection dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

Images