New application of MARCHF6 in breast tumor diagnosis or treatment

Abstract

The invention discloses a new application of MARCHF6 in diagnosis or treatment of breast tumors, MARCHF6 is applied to diagnosis and treatment of esophageal cancer for the first time, verification in a real clinical sample collected by the application shows that the MARCHF6 has better diagnosis efficiency in diagnosis of esophageal cancer, proliferation of esophageal cancer cells can be remarkably inhibited by knocking down expression of the MARCHF6, and in addition, the MARCHF6 can be applied to diagnosis or treatment of breast tumors. The invention further provides an esophageal cancer diagnostic kit which is simple, convenient and rapid to operate and relatively high in sensitivity and specificity, can be applied to early diagnosis and screening of esophageal cancer, improves the survival rate of patients, and is easy to popularize and apply.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular, relates to a chest tumor marker MARCHF6, and more particularly, to a new use of MARCHF6 in the diagnosis or treatment of chest tumors. Background Art

[0002] With the rapid growth of the global population and the intensification of environmental pollution, human health problems, including various diseases, are becoming increasingly severe. Cancer is one of the main diseases that cause human death. Among them, the incidence and mortality of chest tumors rank first among all tumors. Chest tumors are diseases that occur in the chest cavity, mainly including esophageal cancer (EC), etc. Esophageal cancer is the seventh leading cause of cancer death worldwide. The main symptoms of chest tumors are cough, sputum, hemoptysis, chest pain, chest tightness, shortness of breath, back pain, shoulder pain, etc. The benign and malignant nature of the tumor can be preliminarily determined through imaging examinations. In addition to making preliminary judgments based on imaging, the research and determination of chest tumor-specific markers, as well as the preparation of sensitive and effective test kits using them, can further improve the accuracy of chest tumor diagnosis and complete qualitative and quantitative detection.

[0003] Worldwide, there are more than 470,000 new cases of esophageal cancer each year. Based on biological differences, esophageal cancer can be divided into two subtypes: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). Although there have been major breakthroughs in the diagnosis and treatment of esophageal cancer, the prognosis of esophageal cancer is still poor because most patients are diagnosed late. At present, early diagnosis of esophageal adenocarcinoma and squamous cell carcinoma, including the identification of Barrett's esophagus and squamous cell dysplasia, has provided great help in improving the prognosis of esophageal cancer. However, the current mainstream methods such as confocal laser endoscopy and high-resolution microendoscopy, detection of free cfDNA and other methods still face shortcomings such as limited sensitivity. Therefore, it is more important to combine cytological evaluation with the detection of biomarkers (such as DNA methylation status, miRNA detection or cell surface proteins) to develop a new generation of accurate prediction models.

[0004] The lack of ideal diagnostic and treatment technology is the main reason for the poor diagnosis and treatment of chest tumors. Traditional preoperative diagnostic methods such as endoscopy, computed tomography (CT) and positron emission tomography (PET-CT) are often limited to large-scale screening due to trauma or radiation exposure. Due to the lack of characteristic clinical manifestations in the early stage of chest tumors, it is difficult to achieve early diagnosis. Most patients with chest tumors have progressed to the middle and late stages when they first visit the doctor, thus missing the best treatment opportunity. In addition, the accuracy of tumor margin assessment in cancer surgery is very high. Relying solely on pathological examinations for judgment will increase the possibility of postoperative recurrence. This is because pathological examinations have many subjective factors in diagnosis, and the reliability of diagnosis depends on the relevant experience and skills of the pathologist. Therefore, providing a specific biomarker related to chest tumors is of great significance for the early diagnosis and treatment of chest tumors.

[0005] Membrane-associated ring finger 6 (MARCHF6) is a member of the MARCH family (this family mainly plays the role of E3 ubiquitin ligase, and widely regulates various cellular life activities such as signal transduction, metabolism, proliferation and differentiation through protein ubiquitination modification). It mainly regulates the ubiquitination modification of key enzymes and regulates metabolic pathways such as cholesterol synthesis. The role of MARCHF6 in different types of tumors is controversial. Different tumors may have different microenvironmental characteristics and molecular regulatory characteristics, and the function of MARCHF6 is different. At present, there is no relevant research or report on MARCHF6 in esophageal cancer. This study explored the expression characteristics, clinical significance and biological functions of MARCHF6 in esophageal cancer, which provided some inspiration for the study of the carcinogenic mechanism, early screening and even the exploration of new targets for precise prevention and treatment of esophageal cancer. Summary of the invention

[0006] In order to overcome the above technical problems existing in the art, the present invention aims to provide a new use of MARCHF6 in the diagnosis or treatment of chest tumors.

[0007] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives: The 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 a product for diagnosing or identifying esophageal cancer.

[0008] Further, the reagent includes: A reagent for detecting the mRNA expression level of MARCHF6 in a sample; A reagent for detecting the protein expression level of MARCHF6 in a sample; or Reagent for detecting the number of MARCHF6-positive cells in a sample; Optionally, the reagent for detecting the mRNA expression level of MARCHF6 in the sample comprises: Primers that specifically amplify MARCHF6; or A probe that specifically recognizes MARCHF6; Optionally, the reagent for detecting the protein expression level of MARCHF6 in the sample comprises: An antibody that specifically binds to a protein encoded by MARCHF6; or Affinity protein that specifically binds to the protein encoded by MARCHF6; Optionally, the reagent for detecting the number of MARCHF6-positive expressing cells in a sample includes a reagent for detecting the number of MARCHF6-positive expressing cells by immunohistochemistry.

[0009] Furthermore, the identification of esophageal cancer includes identifying and distinguishing esophageal squamous cell carcinoma and esophageal adenocarcinoma.

[0010] Furthermore, the MARCHF6 can also be used to differentiate Barrett's esophagus, esophageal adenocarcinoma, esophageal squamous cell carcinoma and / or normal esophagus.

[0011] In the present invention, the information of the gene MARCHF6 is as follows: the Gene ID of the gene MARCHF6 (membrane associatedring-CH-type finger 6 [Homo sapiens (human)]) is 10299, and the detailed information of the gene can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0012] In the present invention, the diagnosis refers to the identification or classification of a molecular or pathological state, disease or condition. For example, by using molecular features (e.g., specific genes, proteins encoded by specific genes, specific microbial communities, specific metabolites), early differential diagnosis of whether a subject has esophageal cancer or the risk of esophageal cancer, or early differential diagnosis of whether a subject has esophageal squamous cell carcinoma or esophageal adenocarcinoma.

[0013] In some embodiments, the primer is the same as an amplification primer, which refers to an oligonucleotide that can be used in an amplification method (such as polymerase chain reaction (PCR)) to amplify a nucleotide sequence based on a polynucleotide sequence corresponding to a target gene (e.g., the sequence of MARCHF6 or a portion thereof). The primer generally refers to a nucleic acid fragment containing 5-100 nucleotides. In a preferred embodiment, the primer or amplification primer contains 15-30 nucleotides that can initiate an enzymatic reaction (e.g., an enzymatic amplification reaction). In a specific embodiment of the present invention, the primer refers to a primer that specifically amplifies the gene MARCHF6.

[0014] In some embodiments, the probe refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule. In a specific embodiment of the present invention, the probe refers to a probe that specifically recognizes MARCHF6. In other embodiments, the probe refers to any molecule that can be indirectly or directly, covalently or non-covalently bound to any substrate and / or reaction product and / or protease disclosed herein or associated with it, and its 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 sequence disclosed herein. In some embodiments, the probe can be a nucleic acid sequence comprising a fluorescent molecule or substrate that becomes fluorescent when exposed to an enzyme, and the nucleic acid sequence is complementary to a fragment of a nucleic acid sequence.

[0015] Unless otherwise indicated, in the present invention, a probe generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as 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 the present invention include gene-specific DNA oligonucleotide probes, such as microarray probes fixed on a microarray substrate, quantitative nuclease protection test probes, probes connected to molecular barcodes, and probes fixed on beads.

[0016] The stringency of hybridization reaction can be easily determined by those of ordinary skill in the art, and is usually calculated empirically based on probe length, washing temperature and salt concentration. Generally speaking, longer probes require higher temperatures to anneal correctly, while shorter probes require lower temperatures. Hybridization usually depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below its melting temperature. The higher the degree of expected homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it is inferred that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures are also less stringent.

[0017] 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.

[0018] In some embodiments, the antibody is well known in the art, and the antibody refers to a specific immunoglobulin against an antigenic site. The antibody described in the present invention refers to an antibody that specifically binds to the protein encoded by MARCHF6 described in the present invention, and the antibody can be prepared according to conventional methods in the art.

[0019] The form of the antibody includes polyclonal antibodies 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 comprising the antigen binding site of the antibody, and any other modified immunoglobulin molecules comprising the antigen binding site, as long as the antibody exhibits the desired biological binding activity.

[0020] In some embodiments, the peptide has the ability to highly bind to the target substance (protein encoded by MARCHF6 of the present invention) and does not denature during heat treatment or chemical treatment. Moreover, due to its small size, it can be used as a fusion protein by attaching it to other proteins. Specifically, because it can be specifically attached to a polymer protein chain, it can be used as a diagnostic kit and a drug delivery substance.

[0021] 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), which has a stable tertiary structure and has the property of being able to bind to the target molecule (the protein encoded by MARCHF6 of the present invention) with high affinity and specificity. As mentioned above, since the aptamer can specifically bind to antigenic substances like an antibody, but is more stable than a protein, has a simpler structure, and is composed of a polynucleotide that is easy to synthesize, it can be used instead of an antibody.

[0022] In some embodiments, the reagents for detecting the number of MARCHF6-positive expressing cells by immunohistochemistry experiments include but are not limited to any reagents required for detecting the number of MARCHF6-positive expressing cells by immunohistochemistry experiments.

[0023] In some embodiments, the reagents required for detecting the number of MARCHF6-positive expression cells by immunohistochemistry include, but are not limited to, fixatives, buffers, colorimetric solutions, adhesives, mounting agents, enzyme digestion solutions, and sucrose solutions.

[0024] In some embodiments, the fixative includes, but is not limited to, formaldehyde, glutaraldehyde, paraformaldehyde, ethanol, HneFIX, and acetone.

[0025] In some embodiments, the buffer includes, but is not limited to, PBS buffer, citrate buffer, EDTA buffer, TBS buffer.

[0026] In some embodiments, the color developing solution includes but is not limited to: DAB color developing solution, 4-chloro-1-naphthol (4-Cl-1-Naphthol) color developing solution, 3-amino-9-ethylcarbozole (3-amino-9-ethylcarbozole, AEC) color developing solution, TMB color developing solution, NBT color developing solution.

[0027] In some embodiments, the adhesive includes, but is not limited to, gelatin, resin glue, polylysine, and commercial adhesives.

[0028] In some embodiments, the mounting medium includes, but is not limited to, skim milk powder, BSA, serum, and Fab fragment single-chain secondary antibody.

[0029] In some embodiments, the enzyme digestion solution includes, but is not limited to, trypsin digestion solution and pepsin digestion solution.

[0030] In some embodiments, the expression level described in the present invention refers to the absolute amount or relative amount of the MARCHF6. The expression level of the MARCHF6 described in the present invention can be determined by a variety of techniques well known to those skilled in the art. In particular, the absolute amount or relative amount of the MARCHF6 described in the present invention can be detected by using immunohistochemical detection methods well known to those skilled in the art.

[0031] In some embodiments, the sample refers to a composition obtained or derived from a subject of interest that 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.

[0032] Further, the sample may be obtained from a tissue sample, a blood sample, or other fluid sample of biological origin, such as a biopsy tissue sample or a tissue culture or cell derived therefrom, from a subject. The source of the tissue sample may be solid tissue, such as a fresh, frozen and / or preserved organ or tissue sample, biopsy tissue, or aspirate; blood or any blood component; body fluid; cells from any time of pregnancy or development of an individual; or plasma. The term sample includes biological samples that have been processed in any way after they have been obtained, such as 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.

[0033] 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 washing fluid, secretions (e.g., breast secretions), oral wash fluid, swabs (e.g., oral swabs), touch preparations, fine needle aspirations, cell extracts, and combinations thereof. In a specific embodiment of the present invention, the sample is preferably a subject-derived tissue sample.

[0034] In a specific embodiment of the present invention, the receiver operating characteristic (ROC) analysis method is used to evaluate the performance of MARCHF6 in differential diagnosis of esophageal cancer, or to test its accuracy in separating each group of patients.

[0035] The ROC curve is a graphical representation of the sensitivity and specificity spectrum generated using sensitivity as the y-axis and 1-specificity as the x-axis using 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 particular decision threshold. The ROC curve for a test with perfect discrimination (no overlap in the two distributions) passes through the upper left corner (sensitivity is 100% and specificity is 100%). Therefore, qualitatively speaking, the closer the plot is to the upper left corner, the better 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 plot. The closer the AUC is to 1 when it is greater than 0.5, the better the diagnostic or predictive effect of the test is.

[0036] In a specific embodiment of the present invention, the present invention has been verified to have an AUC value of 0.9113, a specificity of 85%, and a sensitivity of 100% for the MARCHF6 in the real clinical sample set collected by the present invention (40 esophageal cancer tissue samples and corresponding 40 adjacent cancer tissue samples), which proves that the MARCHF6 has extremely high diagnostic efficacy for esophageal cancer, with high accuracy, specificity and sensitivity.

[0037] A second aspect of the present invention provides a diagnostic kit or a detection chip for diagnosing or identifying esophageal cancer.

[0038] Furthermore, the diagnostic kit or detection chip comprises the reagent for detecting the expression level of MARCHF6 in a sample as described in the first aspect of the present invention.

[0039] Furthermore, the diagnostic kit comprises a primer or a probe that specifically binds to the MARCHF6; Optionally, the detection chip includes a solid phase carrier and a probe attached to the solid phase carrier that specifically recognizes the MARCHF6.

[0040] In some embodiments, the diagnostic kit also includes instructions for use or labels, positive controls, negative controls, buffers, adjuvants or solvents, and one or more containers for containing the compositions contained in the diagnostic kit. The composition may be in liquid form or may be lyophilized. Suitable containers for the composition include, for example, bottles, vials, syringes and test tubes. The container may be formed of a variety of materials, including glass or plastic. The instructions or labels describe in detail how to use the diagnostic kit to detect samples and how the diagnostic kit is used for diagnosis and differentiation or auxiliary diagnosis and differentiation of esophageal cancer.

[0041] In some embodiments, the diagnostic kit may also include a variety of different reagents suitable for practical use (such as for different detection methods), and is not limited to the reagents currently listed in the present invention. As long as the reagent is based on the detection of MARCHF6 to diagnose and identify or assist in the diagnosis and identification of esophageal cancer, it falls within the scope of protection of the present invention.

[0042] In some embodiments, the diagnostic kit includes, but is not limited to, a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry analysis kit, an immunohistochemistry detection kit, an ELISA kit, and an electrochemiluminescence detection kit.

[0043] In some embodiments, the preparation of the detection chip can adopt the conventional preparation method of biochip 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, the 5' end of the probe contains an amino-modified poly dT string, the oligonucleotide probe is prepared into a solution, and then it is spotted on the modified glass slide or silicon wafer using a spotter, arranged into a predetermined sequence or array, and then fixed by leaving it overnight to obtain the detection chip of the present invention.

[0044] In some embodiments, the primers that specifically bind to the MARCHF6 can be prepared by chemical synthesis, appropriately designed with reference to known information using methods well known to those skilled in the art, and prepared by chemical synthesis.

[0045] The third 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 a system and / or device for diagnosing or identifying esophageal cancer.

[0046] Furthermore, the system and / or device comprises the following units: Input unit: The input unit is used to input the data of the MARCHF6 expression level obtained from the subject sample into the processing unit; 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 diagnosis or identification result of the subject; Output unit: The output unit is used to output the diagnosis or identification result of the subject obtained by the processing unit.

[0047] The fourth aspect of the present invention provides the use of an agent for inhibiting the expression of MARCHF6 in the preparation of a medicament for treating and / or preventing esophageal cancer.

[0048] Furthermore, the agent for inhibiting the expression of MARCHF6 includes siRNA targeting the MARCHF6 gene, shRNA targeting the MARCHF6 gene, an agent for inhibiting the transcription activity of the MARCHF6 gene, an agent for inhibiting the transcription level of MARCHF6 mRNA, an agent for promoting the degradation of MARCHF6 mRNA, or an agent for inhibiting the translation of MARCHF6 mRNA; Optionally, the agent for inhibiting the expression of MARCHF6 is siRNA targeting the MARCHF6 gene; Optionally, the sequence of the siRNA is as shown in SEQ ID NO: 1-2 or as shown in SEQ ID NO: 3-4; Optionally, the dosage form of the drug is selected from a dosage form for enteral administration and a dosage form for parenteral administration; Optionally, the dosage form for administration through the gastrointestinal tract is selected from solutions, granules, tablets, capsules, suspensions, powders, sustained-release agents, effervescent agents, emulsions, syrups, drops, and chewables; Optionally, the non-intestinal administration dosage form is selected from an injection dosage form, a respiratory tract administration dosage form, a cavity administration dosage form, a mucosal administration dosage form, and a skin administration dosage form.

[0049] In some embodiments, the agents for inhibiting the expression of MARCHF6 of the present invention include but are not limited to: agents that inhibit the expression of the gene encoding the MARCHF6 protein or reduce the expression level of the gene, and reduce the activity of the MARCHF6 protein, for example, agents that inhibit the expression of the MARCHF6 gene or reduce its expression level, these agents include but are not limited to: agents that inhibit the transcriptional activity of the MARCHF6 gene, agents that inhibit the transcriptional level of MARCHF6 mRNA, agents that promote the degradation of MARCHF6 mRNA, siRNA against the MARCHF6 gene, shRNA against the MARCHF6 gene, agents that inhibit the translation of MARCHF6 mRNA, agents that specifically recognize the guide nucleic acid of the MARCHF6 gene and cut it to reduce its expression level, dsRNA against the MARCHF6 gene, micro RNA against the MARCHF6 gene, and antisense nucleic acid against the MARCHF6 gene.

[0050] In other embodiments, the whole MARCHF6 gene can be knocked out by administering a targeting vector, thereby achieving inhibition or reduction of MARCHF6 gene expression. In other embodiments, the agent that reduces the activity of the MARCHF6 protein can be, for example, a specific antibody targeting MARCHF6 or a small molecule compound that inhibits the activity of the MARCHF6 protein. In a specific embodiment of the present invention, the agent that inhibits the expression of MARCHF6 is a siRNA whose coding sequence is shown in SEQ ID NO: 1-2 or SEQ ID NO: 3-4.

[0051] In some embodiments, the activity of MARCHF6 protein can also be reduced by introducing mutations into the protein. In other embodiments, a mutation is introduced into the functional domain of MARCHF6 protein that causes the corresponding activity to be weakened or lost. The mutation can be the insertion, deletion or substitution of one or several or even more (e.g., more than 10, more than 20, more than 30) amino acids. By administering an agent that acts on the MARCHF6 gene, a mutation that causes the relevant biological activity to be weakened or lost can be present in the functional domain of the MARCHF6 protein encoded by it. Such agents can change the sequence of the MARCHF6 gene, resulting in the presence of corresponding mutations in the MARCHF6 protein encoded by it, thereby having weakened activity or loss of activity. For example, the wild-type MARCHF6 gene can be replaced by a mutant MARCHF6 gene by homologous recombination technology, resulting in the expression of a weakly active or inactive MARCHF6 protein.

[0052] In the present invention, the siRNA refers to small interfering ribonucleic acid, i.e. a relatively short-length double-stranded nucleic acid or optionally a longer precursor thereof. In some embodiments, the length of the available siRNA in the present invention is preferably a length of about 20-50 bp. However, there is no particular restriction on the length of the available siRNA in the present invention. For example, the siRNA may initially be present in the cell in a precursor form, and the final or processed form of the siRNA that performs and exerts gene silencing activity when or after the precursor form is delivered to the target cell is substantially different. For example, the precursor form of the siRNA may include a precursor sequence element, which may be processed, degraded, changed or cut when or after delivery, to produce an siRNA that mediates gene silencing activity in the cell.

[0053] In the present invention, the shRNA refers to short hairpin RNA, which includes two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector includes two short inverted repeat sequences separated by a stem-loop sequence in the middle, forming a hairpin structure, which is controlled by the polⅢ promoter. Subsequently, 5-6 Ts are connected as the transcription terminator of RNA polymerase Ⅲ. shRNA can be stably integrated into the genome of the cell, allowing long-term gene knockout.

[0054] In the present invention, the dsRNA refers to a double-stranded ribonucleic acid, an RNA molecule formed by the renaturation of two complementary chains, which can be cleaved by the Dicer enzyme to form siRNA. dsRNA inhibits gene expression through RNA interference (RNAi), and 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.

[0055] In the present invention, the microRNA refers to microribonucleic acid (miRNA), which is a non-coding RNA of about 22 nt in length and widely exists in various organisms from viruses to humans. The main function of mature miRNA is to negatively regulate the post-transcriptional level of genes, and participate in many life processes such as cell proliferation, apoptosis, immunity, neuroendocrine and stem cell differentiation by causing the degradation of its target mRNA or interruption of the translation process.

[0056] In the present invention, the antisense nucleic acid refers to a nucleic acid containing a sequence complementary to that encoding MARCHF6. The antisense nucleic acid may be composed of DNA, RNA or both. The antisense nucleic acid may contain non-complementary bases as long as it can specifically hybridize under stringent conditions. When the antisense nucleic acid is introduced into a cell, it binds to the target polynucleotide and inhibits transcription, RNA processing, or stability. In addition to antisense polynucleotides, antisense nucleic acids also include polynucleotide mimics, which contain a modified main chain, and 3' and 5' end portions. Such antisense nucleic acids can be appropriately designed based on the MARCHF6 sequence information and prepared using methods known to those skilled in the art.

[0057] A fifth aspect of the present invention provides any of the following products: (1) A pharmaceutical composition comprising the agent for inhibiting MARCHF6 expression as described in the fourth aspect of the present invention; (2) A pharmaceutical preparation comprising the agent for inhibiting MARCHF6 expression as described in the fourth aspect of the present invention.

[0058] In some embodiments, the pharmaceutical composition or pharmaceutical preparation may also include pharmaceutically acceptable carriers and / or excipients, which are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used to help the stability of the formulation or to help improve its activity or biological effectiveness as needed. 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; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); preservatives; and any other pharmaceutically or physiologically acceptable carriers or excipients reported in any prior art that can be used in pharmaceutical compositions.

[0059] In some embodiments, the dosage form of the pharmaceutical composition or pharmaceutical preparation includes, but is not limited to: a dosage form for parenteral administration or an oral preparation. Exemplary dosage forms for parenteral administration include injections, aerosols, suppositories, or subcutaneous dosage forms; exemplary oral preparations include tablets, capsules, pills, granules, microcapsules, suspensions, pellets, and oral liquid preparations.

[0060] A sixth aspect of the present invention provides a method for screening candidate drugs for treating and / or preventing esophageal cancer.

[0061] Further, the method comprises the following steps: (1) Treating a system expressing or containing MARCHF6 with a test substance; (2) detecting the expression of MARCHF6 in the system; (3) Selecting test substances that can inhibit the expression of MARCHF6 as candidate drugs.

[0062] In some embodiments, the system is selected from a cellular system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

[0063] In some embodiments, the test substance includes, but is not limited to, interfering molecules designed for MARCHF6, nucleic acid inhibitors, small molecule compounds, etc.

[0064] In some embodiments, the method for detecting the expression of MARCHF6 in the detection system includes but is not limited to reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, ribonuclease protection assay (RPA), Northern blotting and DNA chip.

[0065] In some embodiments, the reagents for detecting the expression of MARCHF6 include, but are not limited to, primers, probes or antisense nucleotides. Those skilled in the art can design primers, probes or antisense nucleotides that can specifically bind to MARCHF6 based on the sequence information of MARCHF6.

[0066] In some embodiments, the selected candidate drug is a test substance that can inhibit the expression level of MARCHF6 in the system when the candidate drug is present, compared to the expression level of MARCHF6 detected in the absence of the candidate drug.

[0067] In addition, the present invention also provides a method for diagnosing or identifying esophageal cancer, which comprises detecting the expression level of MARCHF6 in a sample derived from a subject in need, and judging whether the subject is an esophageal cancer patient, the risk of esophageal cancer or the specific type of esophageal cancer based on the expression level of MARCHF6.

[0068] In addition, the present invention also provides a method for treating and / or preventing esophageal cancer, the method comprising administering a therapeutically and / or preventively effective amount of the agent for inhibiting MARCHF6 expression described in the fourth aspect of the present invention, the pharmaceutical composition described in the fifth aspect of the present invention, or the pharmaceutical preparation described in the fifth aspect of the present invention to a subject in need thereof.

[0069] In some embodiments, the subject of the present invention refers to any animal, and also refers to humans and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human.

[0070] In some embodiments, the administration route of the agent, pharmaceutical composition or pharmaceutical preparation of the present invention is not limited, as long as it can exert the desired therapeutic effect or preventive effect, and the administration route of the agent, pharmaceutical composition or pharmaceutical preparation includes but is not limited to: topical, through the skin, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, inhalation, through the pleura, through the mucosa, skin, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, ​​intrahepatic. In some cases, it can be administered systemically, and in some cases, it can be administered locally.

[0071] In some embodiments, the dosage of the agent, pharmaceutical composition or pharmaceutical preparation of the present invention is not limited, as long as the desired therapeutic effect or preventive effect can be obtained, and can be appropriately determined according to the subject's symptoms, gender, age, etc. The dosage of the agent, pharmaceutical composition or pharmaceutical preparation of the present invention can be determined in detail using, for example, the therapeutic effect or preventive effect on the disease as an indicator.

[0072] In some embodiments, the treatment and / or prevention refers to slowing down, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of existing symptoms, disorders, conditions, or diseases (e.g., esophageal cancer). Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

[0073] In some embodiments, the effective amount refers to a therapeutically effective amount, and the effective amount used herein refers to the amount of the pharmaceutical composition sufficient to treat a specified disorder, condition or disease (e.g., improve, alleviate, weaken 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 the recurrence of a disease. The effective dose can be administered in one or more administrations. The pharmaceutically effective amount depends on a variety of factors, including but not limited to: characteristic factors of the subject (subject) being treated (such as height, weight, sex, age and medication history), the severity of the disease, etc.

[0074] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses MARCHF6 for the diagnosis and treatment of esophageal cancer for the first time. After verification in real clinical samples collected by the present invention, it is found that the MARCHF6 has good diagnostic efficacy for the diagnosis of esophageal cancer, with high accuracy, sensitivity and specificity, and can be used for the effective diagnosis of esophageal cancer. Knocking down the expression of MARCHF6 can significantly inhibit the proliferation of esophageal cancer cells. In addition, the present invention also provides an esophageal cancer diagnostic kit that is simple to operate, rapid, and has high sensitivity and specificity, which can be used for the early diagnosis and screening of esophageal cancer, improve the survival rate of patients, and is easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 :Gene expression matrix and clinical information of different esophageal cancer datasets; Figure 2 : Gene expression characteristics of MARCHF6 in esophageal cancer and normal tissues, including: A: Unannotated UMAP map of 8 esophageal cancer datasets; B: UMAP map of the integrated esophageal cancer dataset; CD: Gene expression characteristics of MARCHF6 in esophageal cancer tissues and normal tissues in esophageal cancer datasets (training set and validation set); EF: ROC curve shows a discriminatory effect between esophageal cancer tissues and normal tissues (training set and validation set); Figure 3 : Gene expression characteristics of MARCHF6 in esophageal squamous cell carcinoma and normal tissues, where AB graphs: Gene expression characteristics of MARCHF6 in esophageal squamous cell carcinoma tissues and normal tissues in esophageal cancer datasets (training set and validation set); CD graphs: ROC curves showing a discriminatory effect between esophageal squamous cell carcinoma tissues and normal tissues (training set and validation set); Figure 4 : Gene expression characteristics of MARCHF6 in esophageal adenocarcinoma-normal tissues, including A, C, E: esophageal cancer data set (training set and validation set), MARCHF6 gene expression characteristics in esophageal adenocarcinoma cancer tissue-normal tissue, Barrett's esophagus and esophageal adenocarcinoma, Barrett's esophagus and normal tissue; B, D, F: ROC curves show discrimination effect in esophageal adenocarcinoma cancer tissue-normal tissue, Barrett's esophagus and esophageal adenocarcinoma, Barrett's esophagus and normal tissue (training set and validation set); Figure 5: Gene expression characteristics of MARCHF6 in esophageal adenocarcinoma-esophageal squamous cell carcinoma, where A: Gene expression characteristics of MARCHF6 in esophageal adenocarcinoma-esophageal squamous cell carcinoma in esophageal cancer dataset (training set and validation set); B: ROC curve shows a discriminatory effect in esophageal adenocarcinoma-esophageal squamous cell carcinoma (training set and validation set); Figure 6 : Biological function analysis of MARCHF6 gene in esophageal adenocarcinoma and esophageal squamous cell carcinoma, wherein, Figure A: In the Hallmark database, MARCHF6 activation in esophageal adenocarcinoma is associated with important cancer characteristics, such as proliferation, KRAS, and AKT activation; Figure B: In the Hallmark database, MARCHF6 in esophageal squamous cell carcinoma is also associated with various cancer characteristics, such as proliferation, KRAS, and AKT activation; Figure C: Biological function analysis of MARCHF6 gene in esophageal adenocarcinoma in the Reactome database; Figure D: Biological function analysis of MARCHF6 gene in esophageal squamous cell carcinoma in the Reactome database; Figure 7 : The results of tissue chip analysis of MARCHF6 in real clinical esophageal cancer tissue samples and adjacent tissue samples collected by the present invention; Figure 8 : Analysis of protein expression of MARCHF6 in esophageal cancer, wherein, Figure A: differential expression analysis results of MARCHF6 in real clinical samples collected by the present invention (quantitative immunohistochemical analysis of MARCHF6 in esophageal cancer tissue-normal tissue); Figure B: diagnostic efficacy analysis results of MARCHF6 in real clinical samples collected by the present invention (ROC curve presents a distinguishing effect in esophageal cancer tissue-normal tissue); Fig. 9 : MARCH1 has no diagnostic efficacy for esophageal cancer, among which, Figure A: There is no difference in the expression of MARCH1 between esophageal cancer and normal tissues; Figure B: There is no difference in ROC curve diagnosis; Figure C: There is no difference in the expression of MARCH1 between esophageal squamous cell carcinoma and normal tissues; Figure D: There is no difference in ROC curve diagnosis; Figure E: There is no difference in the expression of MARCH1 between esophageal adenocarcinoma and normal tissues; Figure F: There is no difference in ROC curve diagnosis; Fig.10 : The effect of knocking down the expression of MARCHF6 on esophageal cancer cells, wherein, Figure A: The MARCHF6 low-expression esophageal cancer cell line model was established by siRNA technology; Figure B: The plate cloning experiment detected the effect of knocking down the expression of MARCHF6 on the cloning ability of esophageal cancer cells; Figure C: CCK8 cell proliferation experiment detected the effect of knocking down the expression of MARCHF6 on the proliferation ability of esophageal cancer cells. DETAILED DESCRIPTION

[0076] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0077] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0078] Example 1 Study on the diagnosis and biological function of MARCHF6 in esophageal cancer 1. Materials and methods 1.1 Public Dataset Information The gene expression matrix and clinical information of different esophageal cancer datasets were obtained through the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ) (see Figure 1 ).

[0079] Among them, 8 esophageal cancer datasets were integrated for analyzing gene expression characteristics, including: dataset (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), GSE92396 (12 / 9).

[0080] 1.2 Patients and organizations 40 cases of esophageal cancer tissues (corresponding to 40 cases of paracancerous tissues) were obtained from the Department of Thoracic Surgery, the Second Affiliated Hospital of Air Force Medical University, and detailed clinical information was collected, sorted and analyzed at the same time. The tissues were divided into two parts, one for paraffin embedding and immunohistochemistry experiments, and one frozen tissue for protein, RNA extraction and subsequent experiments. The use of human pathological specimens involved in this study complies with the ethical requirements of the Ethics Committee of the Second Affiliated Hospital of Air Force Medical University, and also complies with the 1964 Declaration of Helsinki and its subsequent amendments and supplements. All subjects gave informed consent and signed the informed consent form.

[0081] 1.3 Immunohistochemistry After the above-mentioned esophageal cancer and adjacent tissues were collected, they were fixed with 4% paraformaldehyde, dehydrated with gradient ethanol, transparentized with xylene, immersed in wax, and embedded in paraffin. After the paraffin sections were dried, dewaxed with xylene, and hydrated with gradient ethanol, they were washed with phosphate buffer and microwave heated for antigen repair. After 3% hydrogen peroxide treatment of endogenous peroxidase, they were blocked and incubated with primary antibodies overnight. After rinsing the next day, they were incubated with enzyme-labeled secondary antibodies, washed, and then developed with DAB. The protein content in immunohistochemistry was measured by semi-quantitative histochemical method: H-score=Σ(pi*i), "pi" represents the percentage of positive cells, and "I" represents the signal intensity.

[0082] 1.4 Western-blot The frozen tissue was lysed with RIPA lysis buffer containing phosphatase and protease inhibitors, and the lysate was added with loading buffer containing sodium dodecyl sulfate (SDS) and boiled in boiling water, and then separated by SDS-polyacrylamide gel (PAGE) electrophoresis. The protein was then transferred to a nitrocellulose membrane. After blocking with 5% skim milk, it was incubated with a specific primary antibody overnight, incubated with an enzyme-labeled secondary antibody of the corresponding species, and detected by an ECL kit after washing.

[0083] 1.5 Methods and statistical analysis Gene expression data were normalized by Z-scaling. The MARCHF6 gene expression in esophageal cancer tissues and normal tissues and the MARCHF6 protein expression in esophageal cancer tissues and adjacent tissues were evaluated by Wilcoxon test. The correlation between MARCHF6 and genes was evaluated by Pearson correlation analysis, and the gene set enrichment analysis (GSEA) was used to evaluate the biological enrichment of MARCHF6 in the hallmark database and the reactome database. Graphics were drawn and statistically analyzed using R language (statistically significant: two-sided p <0.05).

[0084] 2. Experimental results 2.1 MARCHF6 shows high gene expression in esophageal cancer Since the sample size of both normal tissue and esophageal cancer tissue in the above single dataset is small, no statistically significant comparison can be made. Therefore, this example integrates the above gene set. It can be seen that there is an obvious batch effect before annotation ( Figure 2 A), the batch effect is significantly weakened after integration (through U-map, Figure 2B). We divided the dataset into training and validation sets at a 1:1 ratio according to the ratio of esophageal cancer to normal tissue (Normal tissue / Tumor tissue, 156 / 173). We can see that the MARCHF6 gene in esophageal cancer tissue is significantly more highly expressed than that in normal tissue, whether in the validation set or the training set ( Figure 2 C, training set; Figure 2 D, validation set). At the same time, the ROC curve shows a significant discrimination effect ( Figure 2 E, training set; Figure 2 F, validation set).

[0085] 2.2 MARCHF6 shows high gene expression in esophageal squamous cell carcinoma. MARCHF6 can be used to differentiate esophageal squamous cell carcinoma from normal esophageal tissue Esophageal squamous cell carcinoma is an important pathological subtype of esophageal cancer. We also divided the data set into training and validation sets in a 1:1 ratio according to 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 set and the training set, the MARCHF6 gene in the tissue showed a significantly higher expression in esophageal squamous cell carcinoma tissue compared with normal tissue ( Figure 3 A, training set; Figure 3 B, validation set). At the same time, the ROC curve shows a significant discrimination effect ( Figure 3 C, training set; Figure 3 D, validation set).

[0086] 2.3 Analysis of MARCHF6 gene expression in esophageal adenocarcinoma, Barrett's esophagus, and normal tissues Due to the small number of esophageal adenocarcinoma cases, cross-validation could not be performed. We put them all together in the overall data set to explore expression analysis. Esophageal adenocarcinoma follows the transition from normal tissue to Barrett's esophagus to esophageal adenocarcinoma. We compared the relationship between these three (Normal tissue / Barretts esophagus / adenocarcinoma, 156 / 28 / 41). We found that the MARCHF6 gene was significantly more highly expressed in esophageal adenocarcinoma tissue than in normal tissue ( Figure 4 A). At the same time, the ROC curve shows a significant discrimination effect ( Figure 4 B). There was no significant difference in MARCHF6 between Barrett's esophagus and adenocarcinoma ( Figure 4 C) and discrimination effect ( Figure 4D). Barrett's esophagus showed high expression of MARCHF6 compared with normal tissues ( Figure 4 E), while the diagnostic ROC curve showed a critical diagnostic performance ( Figure 4 F). This indicates the potential for canceration of Barrett's esophagus.

[0087] 2.4 Analysis of MARCHF6 gene expression in esophageal adenocarcinoma and esophageal squamous cell carcinoma Due to the small number of esophageal adenocarcinoma cases, cross-validation could not be performed. We put them all together in the overall data set to explore expression analysis, esophageal adenocarcinoma (AD) and esophageal squamous cell carcinoma (SQC) (adenocarcinoma / squamous cell carcinoma, 41 / 116). We found that the expression level of MARCHF6 gene in esophageal adenocarcinoma tissue was lower than that in esophageal squamous cell carcinoma ( Figure 5 A). At the same time, the ROC curve shows a significant discrimination effect ( Figure 5 B). This suggests that MARCHF6 has the function of differentially diagnosing esophageal squamous cell carcinoma and adenocarcinoma.

[0088] 2.5 Biological function analysis of MARCHF6 gene in esophageal adenocarcinoma and esophageal squamous cell carcinoma We analyzed the biological significance of MARCHF6 in tumor tissues in esophageal adenocarcinoma and esophageal squamous cell carcinoma. We used Pearson correlation to analyze the associated genes of MARCHF6 and performed GSEA analysis by sorting the correlation coefficients. We found that in the Hallmark data, MARCHF6 activation in esophageal adenocarcinoma was associated with important cancer features, such as proliferation, KRAS, and AKT activation ( Figure 6 A), and MARCHF6 in esophageal squamous cell carcinoma is also associated with various cancer characteristics, such as proliferation and KRASAKT activation ( Figure 6 B). In the Reactome database, the biological function analysis results of the MARCHF6 gene in esophageal adenocarcinoma and esophageal squamous cell carcinoma are shown in Figure 6 C and Figure 6 D.

[0089] 2.6 Analysis of MARCHF6 expression in esophageal cancer and normal tissues In this example, the tissue chip method was used to further verify the expression of MARCHF6 in the real clinical sample set, and the area under the receiver operating characteristic curve (AUC) was calculated 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-mentioned real clinical sample set.

[0090] In 40 esophageal cancer tissue samples and 40 corresponding adjacent tissue samples collected by our department (Department of Thoracic Surgery, The Second Affiliated Hospital of Air Force Medical University), immunohistochemistry revealed that MARCHF6 showed high expression of protein in cancer compared with adjacent tissues ( Figure 8 A), tissue chip analysis results Figure 7 As shown. At the same time, ROC curve analysis showed that there was a significant discrimination effect ( Figure 8 B) The above results showed that MARCHF6 had the function of accurately differentiating and diagnosing esophageal cancer, with an AUC value of 0.9113, a specificity of 85%, and a sensitivity of 100%.

[0091] Example 2 Application of MARCHF6 in the treatment of esophageal cancer 1. qPCR experiment After adding Trizol solution to the extracted cells to lyse the cells, chloroform was added to extract RNA, and after centrifugation, anhydrous ethanol was added to the supernatant to precipitate RNA. After centrifugation, the supernatant was discarded, and then washed with anhydrous ethanol, dried and dissolved RNA. The extracted RNA was measured for concentration using a Nano drop instrument. Using the extracted RNA as a template, cDNA was synthesized under the action of reverse transcriptase, and a qPCR reaction solution containing primers, templates, etc. was prepared. The plate was spotted and the reaction conditions of the PCR instrument were set for amplification. Finally, the target DNA content was obtained through data analysis.

[0092] 2. CCK8 cell proliferation assay The cells in good logarithmic growth phase were fully diluted into single cell suspension, and after counting, the cells were divided into equal groups according to the cell density (10 4 / mL), inoculated in 96-well plates, 8 wells per group, 100 μL per well. Cultured normally, tested at a fixed time every day (100 μL of 10% CCK8 reagent was added to each well, incubated for 2 h, and the absorbance value was detected at 450 nm by a microplate reader), and the relative proliferation of cells was plotted based on the absorbance value-time.

[0093] 3. Plate cloning experiment The cells in good logarithmic growth phase were fully diluted into single cell suspension, and then inoculated into 6-well plates (the cell count was kept consistent in the number of cells in each group, 500-2000). After 2-3 weeks of normal culture (visible clones were formed), they were fixed with methanol, washed, stained with crystal violet, photographed, and statistically analyzed for clone formation.

[0094] 4. Small interfering RNA transfection Liposomes (Lipofectamine 2000) and MARCHF6 small interfering RNA (si-RNA) were mixed and transfected into the esophageal cancer cell line TE1. The transfection effect was detected by WB 48-74 hours later. When conditions were ripe, it was used for subsequent functional experiments.

[0095] The sequences of si-MARCHF6-1 and si-MARCHF6-2 are as follows: si-MARCHF6-1 sense strand: 5′-GCUCUACAGUGAUGCUCCA-3′ (SEQ ID NO: 1); si-MARCHF6-1 antisense strand: 5′-UGGAGCAUCACUGUAGAGC-3′ (SEQ ID NO: 2); si-MARCHF6-2 sense strand: 5′-CCCUUACCAUAUUGGUCAU-3′ (SEQ ID NO: 3); si-MARCHF6-2 antisense strand: 5′-AUGACCAAUAUGGUAAGGG-3′ (SEQ ID NO: 4).

[0096] 5. Experimental results In view of the positive proliferation potential of MARCHF6 in esophageal cancer tissue, the present invention further analyzed the proliferation-promoting function of MARCHF6 in esophageal cancer cell lines. The present invention first established a MARCHF6 low expression model in esophageal cancer cell line (TE1) by siRNA technology. The results are as follows: Fig.10 As shown in A, the results showed that si-MARCHF6-1 and si-MARCHF6-2 significantly knocked down the expression of MARCHF6 in the esophageal cancer cell line TE1. Further plate cloning experiments found that knocking down the expression of MARCHF6 could significantly reduce the cloning ability of esophageal cancer cell lines, and si-MARCHF6-2 had a more significant inhibitory effect on the proliferation of esophageal cancer cells ( Fig.10 B). Finally, the CCK8 cell proliferation experiment showed that knocking down the expression of MARCHF6 could significantly inhibit the proliferation of esophageal cancer cell lines ( Fig.10 C).

[0097] Comparison ratio: Not all MARCH family members have the function of diagnosing esophageal cancer MARCH1 is also a member of the MARCH family. The method in the above-mentioned embodiment was used to verify that MARCH1 could not effectively diagnose esophageal cancer. There was no difference in the expression of MARCH1 in esophageal cancer and normal tissues ( Fig. 9 A), ROC curve showed no difference in diagnosis ( Fig. 9 B). There is no difference in the expression of MARCH1 between esophageal squamous cell carcinoma and normal tissues ( Fig. 9 C), ROC curve diagnosis no difference ( Fig. 9 D). There is no difference in the expression of MARCH1 between esophageal adenocarcinoma and normal tissues ( Fig. 9 E), ROC curve showed no difference in diagnosis ( Fig. 9 F).

[0098] The above results indicate that not all members of the MARCH family have the function of diagnosing esophageal cancer. The MARCHF6 discovered for the first time in the present invention has the function of accurately distinguishing and diagnosing esophageal cancer, which is a technical effect that was unexpected by those skilled in the art.

Claims

1. Use of a reagent for detecting the expression level of MARCHF6 in a sample in the preparation of a product for diagnosing or differentiating esophageal cancer.

2. The use according to claim 1, characterized in that: The reagents include: A reagent for detecting the mRNA expression level of MARCHF6 in a sample; A reagent for detecting the protein expression level of MARCHF6 in a sample; or Reagent for detecting the number of MARCHF6-positive cells in a sample; Optionally, the reagent for detecting the mRNA expression level of MARCHF6 in the sample comprises: Primers that specifically amplify MARCHF6; or A probe that specifically recognizes MARCHF6; Optionally, the reagent for detecting the protein expression level of MARCHF6 in the sample comprises: An antibody that specifically binds to a protein encoded by MARCHF6; or Affinity protein that specifically binds to the protein encoded by MARCHF6; Optionally, the reagent for detecting the number of MARCHF6-positive expressing cells in a sample includes a reagent for detecting the number of MARCHF6-positive expressing cells by immunohistochemistry.

3. The use according to claim 1, characterized in that: The identification of esophageal cancer includes identifying and differentiating esophageal squamous cell carcinoma and esophageal adenocarcinoma.

4. A diagnostic kit or detection chip for diagnosing or distinguishing esophageal cancer, characterized in that: The diagnostic kit or detection chip comprises the reagent for detecting the expression level of MARCHF6 in a sample as described in any one of claims 1 to 3.

5. The diagnostic kit or detection chip according to claim 4, characterized in that: The diagnostic kit comprises a primer or a probe that specifically binds to the MARCHF6; Optionally, the detection chip includes a solid phase carrier and a probe attached to the solid phase carrier that specifically recognizes the MARCHF6.

6. Use of a reagent for detecting the expression level of MARCHF6 in a sample in the preparation of a system and / or device for diagnosing or distinguishing esophageal cancer.

7. Use of an agent for inhibiting the expression of MARCHF6 in the preparation of a drug for treating and / or preventing esophageal cancer.

8. The use according to claim 7, characterized in that: The reagents for inhibiting the expression of MARCHF6 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; Optionally, the agent for inhibiting the expression of MARCHF6 is siRNA targeting the MARCHF6 gene; Optionally, the sequence of the siRNA is as shown in SEQ ID NO: 1-2 or as shown in SEQ ID NO: 3-4; Optionally, the dosage form of the drug is selected from a dosage form for enteral administration and a dosage form for parenteral administration; Optionally, the dosage form for administration through the gastrointestinal tract is selected from solutions, granules, tablets, capsules, suspensions, powders, sustained-release agents, effervescent agents, emulsions, syrups, drops, and chewables; Optionally, the non-intestinal administration dosage form is selected from an injection dosage form, a respiratory tract administration dosage form, a cavity administration dosage form, a mucosal administration dosage form, and a skin administration dosage form.

9. Any of the following products: (1) A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the agent for inhibiting the expression of MARCHF6 as described in claim 7 or 8; (2) A pharmaceutical preparation, characterized in that the pharmaceutical preparation comprises the agent for inhibiting MARCHF6 expression as described in claim 7 or 8.

10. A method for screening candidate drugs for treating and / or preventing esophageal cancer, characterized in that: The method comprises the following steps: (1) Treating a system expressing or containing MARCHF6 with a test substance; (2) detecting the expression of MARCHF6 in the system; (3) Selecting test substances that can inhibit the expression of MARCHF6 as candidate drugs.

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