Use of novel micropeptides mp1 and mp2 in tumor diagnosis and treatment

Abstract

Provided is use of an endogenous micropeptide in cancer diagnosis or treatment. Particularly, the present invention relates to use of a micropeptide encoded on a long non-coding RNA as a tumor marker, a tumor treatment drug, and / or a tumor treatment target. Specifically, two micropeptides and variants thereof have been discovered for the first time. Moreover, provided are nucleotide sequences encoding the described micropeptides and recombinant vectors containing these nucleotide sequences, and also provided are use thereof in the preparation of a reagent or a drug for diagnosing, preventing, alleviating, or treating a tumor, and use of a drug or a reagent for an interfering micropeptide or a coding nucleic acid thereof in the preparation of a medicament or a pharmaceutical composition for preventing, alleviating, or treating a disease. The micropeptides not only provide biomarkers for the diagnosis of tumors, but also provide therapeutic drugs or therapeutic targets for the treatment of tumors.

Description

Application of novel micropeptides MP1 and MP2 in tumor diagnosis and treatment

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 2024114182422, filed with the China National Intellectual Property Administration on October 11, 2024, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This application belongs to the field of biomedical technology, specifically involving the application of novel endogenous micropeptides in cancer diagnosis and treatment. Background Technology

[0004] Approximately 98% of human gene transcripts are non-coding RNAs (ncRNAs). These ncRNAs form a complex family, including long non-coding RNAs (lncRNAs), microRNAs (miRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small interfering RNAs (siRNAs), and circular RNAs (circRNAs). Long non-coding RNAs (lncRNAs) are RNA molecules longer than 200 nucleotides, produced in large quantities during transcription, and were initially considered "noise" in gene transcription. In recent years, lncRNA research has attracted widespread attention, revealing their important roles in gene expression regulation, cell biological processes, and various diseases. With the continuous development of biotechnology, lncRNAs have gradually become a research hotspot in the life sciences. With the advancement of ribosome mapping technology, gene editing technology, and bioinformatics prediction methods, more and more studies have discovered that some lncRNAs can encode micropeptides (MPs) of less than 100 amino acids. These micropeptides are widely involved in multiple developmental processes and the occurrence and development of certain diseases. These micropeptides play important roles in a wide range of biological systems. They are typically generated from short open reading frames (sORFs) less than 300 nucleotides in length, arranged with NUG as the start and stop codons. Micropeptides can be classified into different categories based on the type of transcript they encode. Compared to traditional large proteins, micropeptides are significantly different in length and have different origins and characteristics from traditional small bioactive peptides.

[0005] Currently, research on non-coding RNA translation mainly focuses on lncRNAs, pri-miRNAs, and circRNAs, and most studies employ bioinformatics tools to predict coding sORFs, screen for potential coding lncRNAs using ribosome mapping, and identify potential translational micropeptides using mass spectrometry. For example, in 2011, American scientist Jonathan S. Weissman discovered ribosome binding sites on a large number of lncRNAs using ribosome display technology, indicating their potential translational capabilities. In 2015, Eric Olson's team at the University of Texas Southwestern Medical Center discovered a 46-amino acid micropeptide encoded by long non-coding RNA in skeletal muscle, named Myoregulin. This research directly demonstrated the coding capabilities of long non-coding RNAs and was published in the journal *Cell*. In 2016, John G. Clohessy and Pier Paolo Pandolfi published a study in Nature entitled "mTORC1 and muscle regeneration are regulated by the LINC00961-encoded SPAR polypeptide", which found that the long non-coding RNA LINC00961 has the ability to encode the micropeptide SPAR, which can inhibit the activity of mTORC1.

[0006] pcRNA (potential-coding RNA) refers to a class of non-coding RNAs with coding potential. These non-coding RNAs, originally thought to be non-coding, actually contain short open reading frames (sORFs) and can encode bioactive peptides, or micropeptides. The discovery of pcRNA challenged the traditional view that the genome contains only long protein-coding genes, revealing a large amount of unexplored coding potential within the genome. Currently, micropeptides and their encoded nucleic acids (such as pcRNA) are attracting significant attention in cancer research, as these bioactive molecules play crucial roles in tumorigenesis and development. They significantly influence the biological behavior of cancer by affecting important processes such as cell proliferation, migration, and invasion. Furthermore, they interact with other cellular components in the tumor microenvironment, altering tumor immune escape mechanisms and thus promoting tumor growth and spread. Recent studies have found that pcRNA-encoded micropeptides exhibit aberrant expression in various cancer types. These micropeptides can not only serve as tumor markers for early diagnosis but also as therapeutic targets for developing novel anticancer drugs. However, in recent years, there have been few reports on micropeptides encoded by pcRNA both domestically and internationally. Therefore, the discovery of novel micropeptides and further elucidation of their biological functions and exploration of molecular mechanisms are of great significance for the clinical diagnosis and treatment of diseases.

[0007] It should be noted that the methods described in this section are not necessarily methods that had been previously conceived or adopted. Unless otherwise specified, no method described in this section should be assumed to be prior art simply because it is included in this section. Similarly, unless otherwise specified, the issues mentioned in this section should not be considered to be accepted in any prior art. Summary of the Invention

[0008] To address the aforementioned technical challenges, this application identifies and defines two coding open reading frames (ORFs) from long non-coding RNAs. These ORFs encode two active micropeptides that can be used for the diagnosis, prevention, alleviation, or treatment of various cancers, such as cholangiocarcinoma, colorectal cancer, liver cancer, head and neck squamous cell carcinoma, testicular cancer, melanoma, lung cancer, leukemia, kidney cancer, glioma, invasive breast cancer, cervical cancer, esophageal cancer, gastric cancer, prostate cancer, thyroid cancer, ovarian cancer, pancreatic cancer, and uterine sarcoma. This discovery not only reveals the crucial role of micropeptides in tumorigenesis and development but also provides new insights and targets for the clinical diagnosis and treatment of tumors.

[0009] A first aspect of this application provides a polypeptide comprising at least one of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:1.

[0010] According to one embodiment of this application, a polynucleotide is also provided, said polynucleotide encoding the polypeptide described in this application.

[0011] According to one embodiment of this application, a recombinant vector is also provided, the recombinant vector comprising the polynucleotide described in this application.

[0012] A second aspect of this application provides a drug or reagent for intervening in a target polypeptide or its encoded nucleic acid, characterized in that the target polypeptide comprises the amino acid sequence shown in SEQ ID NO:1.

[0013] A third aspect of this application provides a polypeptide comprising at least one of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:2.

[0014] According to one embodiment of this application, a polynucleotide is also provided, said polynucleotide encoding the polypeptide described in this application.

[0015] According to one embodiment of this application, a recombinant vector is also provided, the recombinant vector comprising the polynucleotide described in this application.

[0016] According to one embodiment of this application, the use of the polypeptide, polynucleotide, or recombinant vector described in this application in the preparation of a medicament or pharmaceutical composition for the prevention, relief, or treatment of a disease is also provided.

[0017] According to one embodiment of this application, the use of the medicament or reagent described in this application for intervening in a target polypeptide or its encoded nucleic acid in the preparation of a medicament or pharmaceutical composition for the prevention, relief or treatment of a disease is also provided.

[0018] According to one embodiment of this application, the use of a substance for intervening in the polypeptide or polynucleotide described in this application in the preparation of a medicament or pharmaceutical composition for the prevention, relief or treatment of a disease is also provided.

[0019] According to one embodiment of this application, the use of a substance for detecting the polypeptide or polynucleotide described in this application in the preparation of a reagent or kit for diagnosing or assisting in the diagnosis of a disease is also provided.

[0020] According to one embodiment of this application, a method for preventing, alleviating, or treating a disease is also provided, the method comprising administering to a subject in need the polypeptide, polynucleotide, or recombinant vector described in this application.

[0021] According to one embodiment of this application, a method for preventing, alleviating, or treating a disease is also provided, the method comprising administering to a subject in need the drug or reagent described in this application for intervening in the expression level of a target polypeptide or its encoded nucleic acid.

[0022] According to one embodiment of this application, a method for preventing, alleviating, or treating a disease is also provided, the method comprising administering to a subject in need a substance for intervening in the polypeptide or polynucleotide described in this application.

[0023] According to one embodiment of this application, a method for diagnosing or assisting in the diagnosis of a disease is also provided, the method comprising detecting the expression levels of the polypeptide or polynucleotide described in this application in a clinical sample derived from a subject.

[0024] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0025] The accompanying drawings exemplify embodiments and form part of the specification, serving together with the textual description to explain exemplary implementations of the embodiments. The illustrated embodiments are for illustrative purposes only and do not limit the scope of the claims. Throughout the drawings, the same reference numerals refer to similar but not necessarily identical elements.

[0026] Figure 1 shows the vector element map of the pSpCas9(BB)-2A-GFP plasmid used in Example 1 to verify the endogenous expression ability of the micropeptide.

[0027] Figure 2 shows the expression level of the target protein in the Hep3B cell line overexpressing the micropeptide MP1 as detected by Western Blot in Example 1.

[0028] Figure 3 shows the expression level of the target protein detected by q-PCR in the cell line overexpressing the micropeptide MP2 in Example 1.

[0029] Figure 4 shows the expression level of the target protein in the Hep3B cell line with MP1 knocked out in Example 2, as detected by Western Blot.

[0030] Figure 5 shows the results of the effect of knockout or overexpression of the micropeptide MP1 on the proliferation activity of liver cancer cells in Example 3.

[0031] Figures 6A and 6B show the results of the effects of knockout (Figure 6A) or overexpression (Figure 6B) of the micropeptide MP1 on the migration activity of liver cancer cells in Example 4.

[0032] Figure 7 shows the effect of knockout or overexpression of the micropeptide MP1 on the clonogenic activity of liver cancer cells in Example 5.

[0033] Figure 8 is a schematic diagram of nude mouse xenografts of Hep3B cells overexpressing MP1 (denoted as Hep3B-MP1-OE), Hep3B cells knocked out of MP1 (denoted as Hep3B-MP1-KO), and wild-type Hep3B cells (denoted as Hep3B) in Example 7.

[0034] Figure 9 shows the volume changes of xenografts in nude mice from Hep3B cells overexpressing MP1 (denoted as Hep3B-MP1-OE), Hep3B cells knocked out MP1 (denoted as Hep3B-MP1-KO), and wild-type Hep3B cells (denoted as Hep3B) in Example 7.

[0035] Figure 10 shows the proliferation activity results of the tumor cell line overexpressing the micropeptide MP2 in Example 8.

[0036] Figure 11 shows the purity analysis results of the artificially synthesized micropeptide MP2 in Example 9.

[0037] Figures 12A and 12B show the results of inhibiting the proliferation of gastric cancer cells by exogenous administration of different concentrations of artificially synthesized micropeptide MP2 in Example 10. Figure 12A shows the gastric cancer cell line MGC-823, and Figure 12B shows the gastric cancer cell line AGS.

[0038] Figures 13A and 13B show the results of inhibiting the migration activity of gastric cancer cells by exogenous administration of different concentrations of artificially synthesized micropeptide MP2 in Example 11. Figure 13A shows the gastric cancer cell line MGC-823, and Figure 13B shows the gastric cancer cell line AGS.

[0039] Figure 14 shows the expression analysis results of micropeptide MP1 in different types of tumor tissues in Example 13.

[0040] Figure 15 shows the relationship between micropeptide MP1 and the prognosis of different types of tumors in Example 14.

[0041] Figure 16 shows the results of qPCR detection of MP1 expression levels in liver cancer Hep3B, HepG2, 7721 and normal liver cells LO2 in Example 15. Detailed Implementation

[0042] Unless otherwise stated, all figures used in this specification and claims to represent content, concentration, proportion, mass, volume, time, temperature, thickness, technical effect, etc., should in any instance be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. They can vary for those skilled in the art depending on the desired properties and effects sought through this disclosure, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or in a manner understood by those skilled in the art.

[0043] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​presented in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain some errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range falling within that wider range, as if these narrower numerical ranges were explicitly stated herein.

[0044] Unless otherwise stated or contradicted by the context, the terms or expressions used herein should be read in the context of the document and as understood by one of ordinary skill in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0045] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0046] In this application, the terms "polypeptide" and "peptide" are used interchangeably, referring to a polymer of amino acids of any length. Therefore, polypeptides, oligopeptides, micropeptides, proteins, antibodies, and enzymes are all included in the definition of polypeptide.

[0047] The terms “nucleic acid” and “polynucleotide” used in this application are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogues.

[0048] It should be noted that, in the context of this application, upstream refers to the 5' end of the nucleic acid or the N-terminus of the polypeptide, and downstream refers to the 3' end of the nucleic acid or the C-terminus of the polypeptide. The direction from upstream to downstream is from the 5' end to the 3' end or from the N-terminus to the C-terminus.

[0049] The term "vector" as used in this application refers to a self-replicating DNA molecule that transfers a foreign target gene into a host organism, and is often in the form of a circular double-stranded DNA molecule. Typical vectors include plasmids, viruses, bacteriophages, kinases, and mini-chromosomes. Among these, plasmids are the most common form of vector, referring to circular double-stranded DNA that can accept foreign nucleic acid fragments and replicate in prokaryotic or eukaryotic cells.

[0050] The terms "expression vector" and "recombinant vector" used in this application are interchangeable and refer to a vector containing a foreign gene, and also containing regulatory elements that are expressed in a specified host organism. Introducing the expression vector into a suitable host organism enables it to express the inserted target gene (e.g., the nucleotide sequence encoding a micropeptide provided in this application).

[0051] The terms "exogenous" and "heterogeneous" used in this application are used interchangeably and refer to sources different from the native (original) organism, such as organisms derived from another species. The terms "heterogeneous gene" or "exogenous gene" used in this application refer to genes that do not naturally exist in the host organism and are introduced into the host organism through gene transfer.

[0052] The terms "relief" and "treatment" as used in this application, and their synonyms, refer to the improvement of a disease, symptom, and / or condition. "Relief" and "treatment" can be an improvement in at least one measurable physical parameter, which is not necessarily identifiable by the patient. "Relief" and "treatment" can also be the physical (e.g., stabilizing identifiable symptoms), physiological (e.g., stabilizing physical parameters), or both, suppression of the progression of a disease, symptom, and / or condition. "Relief" and "treatment" can also be the slowing of or reversal of the progression of a disease, symptom, and / or condition.

[0053] The term “prevention” as used in this application and its synonyms refer to delaying the onset of a particular disease, condition and / or symptom or related symptoms of such disease, condition and / or symptom or reducing the risk of acquiring such disease, condition and / or symptom.

[0054] The term "sample" as used in this application can refer to biological samples, typically clinical samples, including, for example, blood and other bodily fluids, including but not limited to peripheral blood, serum, plasma, urine, and saliva; it also includes solid tissue samples, such as biopsy specimens, especially those containing cancer cells. In some embodiments, samples containing cancer cells, such as tumor tissue or cancer cell lines, are the most preferred sample type to be used in this method.

[0055] The “primer” mentioned in this application refers to a nucleic acid fragment containing a certain length (e.g., 5-100 nucleotides), preferably containing 15-30 nucleotides capable of initiating an enzymatic reaction (e.g., an enzymatic amplification reaction).

[0056] The "probe" described in this application refers to a nucleic acid sequence comprising at least 5 nucleotides (e.g., containing 5-100 nucleotides), which can hybridize with the expression product of a target gene or the amplification product of that expression product under specified conditions to form a complex. The hybridization probe may also include a marker for detection. The marker includes, but is not limited to, markers used in quantitative real-time PCR or fluorescence in situ hybridization. In some non-limiting embodiments, the marker may be biotin, digoxigenin, etc.

[0057] The term "antibody" as used in this application refers to a specific immunoglobulin or its antigen-binding fragment that targets an antigen site. The antibody in this application refers to an antibody that specifically binds to the biomarker protein of this application, and can be manufactured according to methods known in the art. 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 an antigen-binding site, and any other modified immunoglobulin molecule containing an antigen-binding site, provided that the antibody exhibits the desired biobinding activity.

[0058] The terms "double-stranded ribonucleic acid," "double-stranded RNA," or "dsRNA" as used herein refer to a complex of one or more ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands. These two strands forming the double-stranded structure can be different parts of a larger RNA molecule, or they can be separate RNA molecules. In the case of separate RNA molecules, such dsRNA is generally referred to as "short interfering RNA (siRNA)"; when the two strands are part of a larger molecule, and thus linked by an uninterrupted nucleotide chain between the 3' end of the first strand and the 5' end of the second strand to form the double-stranded structure, this linking RNA chain is referred to as "short hairpin RNA (shRNA)." Those skilled in the art will understand that siRNA or shRNA is responsible for RNA interference, a pathway for sequence-specific posttranscriptional gene silencing in animals and plants. siRNA is produced by ribonuclease III from the cleavage of a longer double-stranded RNA (dsRNA) homologous to the silenced gene, or by delivering synthetic RNA to the cell. Techniques for designing such molecules for targeted gene expression repression are well known to those skilled in the art.

[0059] To make the above-mentioned objectives, features and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below.

[0060] Micropeptides

[0061] This application provides a polypeptide comprising at least one of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:1.

[0062] In some embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO:1.

[0063] In some preferred embodiments, the polypeptide has an amino acid sequence as shown in SEQ ID NO:1.

[0064] Drugs or reagents used to intervene in target peptides or their encoded nucleic acids

[0065] This application provides a drug or reagent for intervening in a target polypeptide or its encoded nucleic acid, wherein the target polypeptide has the amino acid sequence shown in SEQ ID NO:1.

[0066] In some embodiments, the intervention target polypeptide or its encoded nucleic acid includes at least one of the following: inhibiting the activity of the target polypeptide; causing deletion and / or mutation of the encoded nucleic acid; inhibiting transcription and / or translation of the encoded nucleic acid; and / or degrading the target polypeptide or its encoded nucleic acid.

[0067] In some embodiments, the type of the drug or reagent includes at least one of the following: protein, polypeptide, nucleic acid, polynucleotide, polynucleotide, antibody or derivative thereof, organic compound, inorganic compound and / or natural product.

[0068] Those skilled in the art can select appropriate types of drugs or reagents as needed to intervene in the target polypeptide or its encoded nucleic acid. For example, when intervention in the target polypeptide is required, common methods for inhibiting protein expression levels or protein activity can be used, including but not limited to antibodies, small molecule inhibitors, and chimeric proteins that target protein degradation. If intervention in the encoded nucleic acid of the target polypeptide is required, common methods for inhibiting nucleic acid expression levels or gene regulation can be used, including but not limited to gene knockout, antisense nucleic acids, ribozymes, or interfering RNA technology. Those skilled in the art can prepare the above-mentioned types of drugs or reagents using methods known in the art based on the sequence of the target polypeptide for intervention in the target polypeptide or its encoded nucleic acid.

[0069] Interfering RNA technology can be used to intervene in target polypeptides or their encoded nucleic acids. In some embodiments, the drug or reagent is a polynucleotide. In some embodiments, the drug or reagent is double-stranded ribonucleic acid.

[0070] In some embodiments, the double-stranded ribonucleic acid (BRNA) is used to inhibit the expression of a target polypeptide comprising SEQ ID NO:1 or its encoded nucleic acid. In some embodiments, the BRNA comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are selected from any one of the following:

[0071] (1) The sense strand contains the nucleotide sequence shown in SEQ ID NO:12, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:13;

[0072] (2) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:14, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:15; or

[0073] (3) The sense strand contains the nucleotide sequence shown in SEQ ID NO:16 and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:17.

[0074] Gene knockout can also be used to intervene in the target polypeptide or its encoding nucleic acid. For example, the gene encoding the target polypeptide can be deleted using a CRISPR (clustered regularly spaced short palindromic repeats) / Cas system or components of such systems, thereby inhibiting the transcription of the target polypeptide's encoding nucleic acid into mRNA. In some embodiments, the target polypeptide's encoding nucleic acid is cleaved at specific sites using a CRISPR complex containing a single-guide RNA (sgRNA) complexed with a Cas protein.

[0075] In some embodiments, the drug or reagent comprises sgRNA containing the nucleotide sequence shown in SEQ ID NO:7. In some embodiments, the drug or reagent comprises sgRNA containing the nucleotide sequence shown in SEQ ID NO:10 or 11.

[0076] Any suitable Cas protein known in the art can be used in this application, and no limitation is made herein. In some preferred embodiments, the Cas protein is the Cas9 protein.

[0077] In some embodiments, the drug or reagent is used to treat a disease.

[0078] In some implementations, the disease includes tumors or cancer.

[0079] In some embodiments, the diseases include bile duct cancer, colorectal cancer, liver cancer, head and neck squamous cell carcinoma, testicular cancer, melanoma, lung cancer, leukemia, kidney cancer, glioma, invasive breast cancer, cervical cancer, esophageal cancer, gastric cancer, prostate cancer, thyroid cancer, ovarian cancer, pancreatic cancer, uterine sarcoma, colon cancer, or bladder cancer.

[0080] Micropeptides

[0081] This application provides a polypeptide comprising at least one of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:2.

[0082] In some embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO:2.

[0083] In some preferred embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO:2.

[0084] Polynucleotides, recombinant vectors, and pharmaceutical compositions

[0085] According to one embodiment of this application, a polynucleotide is also provided, said polynucleotide encoding the polypeptide described in this application.

[0086] In some embodiments, the sequence of the polynucleotide is shown in SEQ ID NO:3.

[0087] In some embodiments, the sequence of the polynucleotide is shown in SEQ ID NO:4.

[0088] Due to the degeneracy of codons, those skilled in the art will understand that the nucleic acid sequences capable of encoding the polypeptides provided in this application are not limited to those shown in SEQ ID NO:3 or SEQ ID NO:4, and are not limited thereto. Those skilled in the art can select appropriate nucleic acid sequences for expressing the polynucleotides provided in this application, depending on the intended use or the host.

[0089] According to one embodiment of this application, a recombinant vector is also provided, the recombinant vector comprising the polynucleotide described in this application.

[0090] In some embodiments, the recombinant vector comprises a nucleotide sequence as shown in SEQ ID NO:3.

[0091] In some embodiments, the recombinant vector comprises a nucleotide sequence as shown in SEQ ID NO:4.

[0092] The recombinant vector can be any suitable vector, such as plasmids, viruses, bacteria, bacteriophages, and insertable DNA fragments, etc., and is not limited thereto. In some preferred embodiments, the recombinant vector includes, but is not limited to, recombinant plasmid vectors or recombinant viral vectors. Those skilled in the art can select appropriate recombinant vectors for expressing the polynucleotides provided in this application, depending on the expression purpose or the host.

[0093] The recombinant vector of this application can be constructed using methods known in the art. For example, appropriate restriction enzyme sites can be added to both ends of the polynucleotide of this application according to the restriction enzyme sites contained in the backbone vector used, and then the vector can be inserted into the backbone vector.

[0094] In some embodiments, the polynucleotide or the recombinant vector further includes a promoter. The promoter can be any suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by a host cell expressing a nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates polypeptide expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins or polypeptides that are homologous or heterologous to those of the host cell.

[0095] In some embodiments, the polynucleotide or the recombinant vector further includes a transcription termination sequence. The transcription termination sequence is a sequence that can be recognized by the host cell to terminate transcription. In some embodiments, the transcription termination sequence is operatively attached to the 3' end of a nucleic acid sequence encoding a protein or polypeptide. Any terminator that can function in a selected host cell can be used in this application.

[0096] According to one embodiment of this application, a pharmaceutical composition is also provided, the pharmaceutical composition comprising the polypeptide, the polynucleotide, or the recombinant vector described in this application.

[0097] In some embodiments, the pharmaceutical composition further includes a pharmaceutically or physiologically acceptable carrier. The carrier may be any compatible, physiologically acceptable, non-toxic substance suitable for delivering the polypeptide, polynucleotide, or recombinant vector provided in this application into a mammal (e.g., a human).

[0098] "Pharmaceutically acceptable carrier" refers to a carrier, diluent, or adjuvant used in the formulation or administration of the polypeptide, polynucleotide, or recombinant carrier provided in this application, which is not an essential active ingredient and does not cause excessive toxicity after administration. Suitable pharmaceutically acceptable carriers are well known to those skilled in the art.

[0099] "Physiologically acceptable carrier" refers to a carrier, diluent, or adjuvant that does not cause significant irritation to an organism and does not eliminate the pharmaceutical activity and properties of the peptide, polynucleotide, or recombinant carrier provided in this application. Suitable physiologically acceptable carriers are also well known to those skilled in the art.

[0100] In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients. In some embodiments, the excipients include at least one selected from solubilizers, disintegrants, wetting agents, stabilizers, thickeners, diluents, buffers, and flavoring agents.

[0101] In some non-limiting embodiments, the carrier and / or excipients used in the pharmaceutical compositions of this application may comprise, for example, liquid, gel or solid carriers, aqueous mediators, non-aqueous mediators, antimicrobial agents, isotonic agents, buffers, antioxidants, suspending agents, dispersants, chelating agents, diluents, adjuvants, excipients or non-toxic excipients, other components known in the art, or various combinations thereof.

[0102] Uses or methods for preventing, alleviating or treating diseases

[0103] According to one embodiment of this application, the use of the polypeptide, polynucleotide, or recombinant vector described in this application in the preparation of a medicament or pharmaceutical composition for the prevention, relief, or treatment of a disease is also provided.

[0104] According to one embodiment of this application, a method for preventing, alleviating, or treating a disease is also provided, characterized in that the method includes administering the polypeptide, the polynucleotide, or the recombinant vector described in this application to a subject in need of the treatment.

[0105] In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO:2.

[0106] In some embodiments, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO:4.

[0107] In some embodiments, the recombinant vector comprises a nucleotide sequence as shown in SEQ ID NO:4.

[0108] According to one embodiment of this application, the use of the drug or reagent described in this application for intervening in a target polypeptide or its encoded nucleic acid in the preparation of a drug or drug composition for preventing, alleviating or treating a disease is also provided.

[0109] According to one embodiment of this application, the use of a substance for intervening in the polypeptide or polynucleotide described in this application in the preparation of a medicament or pharmaceutical composition for the prevention, relief or treatment of a disease is also provided.

[0110] According to one embodiment of this application, a method for preventing, alleviating, or treating a disease is also provided, the method comprising administering to a subject in need of the drug or reagent described in this application for reducing the expression level of a target polypeptide or its encoded nucleic acid.

[0111] According to one embodiment of this application, a method for preventing, alleviating, or treating a disease is also provided, the method comprising administering to a subject in need a substance for intervening in the polypeptide or polynucleotide described in this application.

[0112] In some embodiments, the amino acid sequence of the target polypeptide is shown in SEQ ID NO:1.

[0113] In some embodiments, the nucleotide sequence encoding the nucleic acid of the target polypeptide is shown in SEQ ID NO:3.

[0114] In some embodiments, the intervention target polypeptide or its encoded nucleic acid includes at least one of the following: inhibiting the activity of the target polypeptide; causing deletion and / or mutation of the encoded nucleic acid; inhibiting transcription and / or translation of the encoded nucleic acid; and / or degrading the target polypeptide or its encoded nucleic acid.

[0115] In some embodiments, the type of the drug or reagent includes at least one of the following: protein, polypeptide, nucleic acid, polynucleotide, polynucleotide, antibody or derivative thereof, organic compound, inorganic compound and / or natural product.

[0116] In some implementations, the disease includes tumors or cancer.

[0117] In some embodiments, the diseases include bile duct cancer, colorectal cancer, liver cancer, head and neck squamous cell carcinoma, testicular cancer, melanoma, lung cancer, leukemia, kidney cancer, glioma, invasive breast cancer, cervical cancer, esophageal cancer, gastric cancer, prostate cancer, thyroid cancer, ovarian cancer, pancreatic cancer, uterine sarcoma, colon cancer, or bladder cancer.

[0118] In the uses or methods described in this application, the dosage of the polypeptide, polynucleotide, recombinant vector, or composition provided in this application may depend on several factors, including the severity and responsiveness of symptoms, the route of administration, the duration of treatment (from days to months to years), and the time to symptom improvement. Those skilled in the art can adjust the dosage regimen to provide a therapeutic response based on the patient's specific circumstances. For example, a single dose may be administered, several separate doses may be administered over a predetermined time period, or the dose may be reduced or increased as indicated by the treatment outcome. The dosage specification is determined by the specific therapeutic effect to be achieved. The dosage value may also vary depending on the type and severity of the condition to be alleviated. For any particular subject, the specific dosage regimen may be adjusted over time according to individual needs and the professional judgment of the treating clinician.

[0119] Uses or methods for diagnosing or assisting in the diagnosis of diseases

[0120] According to one embodiment of this application, the use of a substance for detecting the polypeptide or polynucleotide described in this application in the preparation of a reagent or kit for diagnosing or assisting in the diagnosis of a disease is also provided.

[0121] According to one embodiment of this application, a method for diagnosing or assisting in the diagnosis of a disease is also provided, characterized in that the method includes detecting the expression levels of the polypeptide or polynucleotide described in this application in a clinical sample derived from a subject.

[0122] In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO:1. In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO:2.

[0123] In some embodiments, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO:3. In some embodiments, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO:4.

[0124] In some implementations, the disease includes tumors or cancer.

[0125] In some embodiments, the diseases include bile duct cancer, colorectal cancer, liver cancer, head and neck squamous cell carcinoma, testicular cancer, melanoma, lung cancer, leukemia, kidney cancer, glioma, invasive breast cancer, cervical cancer, esophageal cancer, gastric cancer, prostate cancer, thyroid cancer, ovarian cancer, pancreatic cancer, uterine sarcoma, colon cancer, or bladder cancer.

[0126] In some embodiments, the detection methods include transcriptome sequencing, polymerase chain reaction (PCR), in situ hybridization, gene chip analysis, proteomics, flow cytometry, enzyme-linked immunosorbent assay (ELISA), Western blotting, and / or immunostaining. Those skilled in the art can select appropriate methods as needed. For example, when it is necessary to detect the expression level of the polypeptide described in this application in clinical samples, commonly used methods for detecting protein expression levels can be used, including but not limited to proteomics, ELISA, Western blotting, immunostaining, and flow cytometry. If it is necessary to detect the expression level of the polynucleotide described in this application in clinical samples, commonly used methods for detecting nucleic acid expression levels can be used, including but not limited to in situ hybridization, transcriptome sequencing, and polymerase chain reaction (PCR). The implementation steps of these detection methods are all known in the art. Those skilled in the art can determine the occurrence and / or progression of the disease by detecting the expression level of the polypeptide or polynucleotide described in this application in clinical samples derived from subjects.

[0127] In some embodiments, the substances used to detect the polypeptides or polynucleotides described in this application include probes, antibodies or antigen-binding fragments, and nucleic acid primers. Those skilled in the art can prepare the substances for detection as needed. For example, based on the sequence of the polynucleotide to be detected, those skilled in the art can design and synthesize nucleic acid primers for polymerase chain reaction (PCR) and detect the expression level of the polynucleotide using PCR. The design and synthesis methods of these substances for detection are known in the art.

[0128] For example, if a polymerase chain reaction (PCR) method can be used for detection, then the substance used to detect the polypeptide or polynucleotide described in this application is a nucleic acid primer.

[0129] In some embodiments, the nucleic acid primer is used to detect polynucleotides, the nucleotide sequences of which are shown in SEQ ID NO:4. In some embodiments, the nucleic acid primer includes an upstream primer and a downstream primer, the upstream primer comprising the nucleotide sequence shown in SEQ ID NO:5, and the downstream primer comprising the nucleotide sequence shown in SEQ ID NO:6.

[0130] In some embodiments, the nucleic acid primer is used to detect polynucleotides, the nucleotide sequences of which are shown in SEQ ID NO:3. In some embodiments, the nucleic acid primer includes an upstream primer and a downstream primer, the upstream primer containing the nucleotide sequence shown in SEQ ID NO:8, and the downstream primer containing the nucleotide sequence shown in SEQ ID NO:9.

[0131] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory), and all embodiments formed by such combinations are considered as part of the disclosure of this application.

[0132] The exemplary embodiments of this application will now be described with reference to the accompanying drawings, including various details of the embodiments to aid understanding. It should be understood that these are merely exemplary and are in no way intended to limit the scope of protection of this application. The scope of protection of this application is defined only by the claims. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0133] Example

[0134] Unless otherwise specified in this embodiment, the techniques or conditions described in the literature in this field or in accordance with the product instructions shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0135] Example 1: Construction of cell lines overexpressing MP1 and MP2

[0136] In this embodiment, a cell line for endogenous stable overexpression of micropeptide MP1 was constructed using a lentiviral system. The amino acid sequence and the encoding nucleic acid sequence are shown in Table 1 below.

[0137] Table 1 Micropeptide-related sequences

[0138] First, the plvx-mCMV-zsGreen-Puro plasmid (constructed by Universal Biotechnology) was constructed. The element map of the plasmid is shown in Figure 1, where the nucleotide sequences of the "gene sequence" element are shown in Table 1 under "encoding nucleic acid sequence" (SEQ ID NO:3 or SEQ ID NO:4). HEK-293T cells (Shanghai Institute of Life Sciences, Chinese Academy of Sciences, Cell Resource Center) were selected for lentiviral packaging. MP1 peptide-overexpressing lentivirus was used to infect Hep3B cells (Wuhan Pronosei Biotechnology Co., Ltd.) to construct an MP1-overexpressing cell line. MP2 peptide-overexpressing lentivirus was used to infect a series of target cells to construct an MP2-overexpressing cell line (color cancer HT29 cells, thyroid cancer BCPAP cells, liver cancer Hep3B cells, gastric cancer MGC-823 and AGS cells, and lung cancer Calu-3 cells, all purchased from Wuhan Pronosei Biotechnology Co., Ltd.). The experimental procedure is briefly described below:

[0139] HEK-293T was passaged into 100mm culture dishes. Lentiviral packaging was performed when the density reached 60-70%. Two 1.5mL EP tubes were prepared. In tube 1, 500μL of DMEM basal medium, 10μg of expression vector, 7.5μg of psPAX2 (purchased from General Biotechnology (Anhui) Co., Ltd.), and 2.5μg of pMD2.G (purchased from General Biotechnology (Anhui) Co., Ltd.) were added. In tube 2, 500μL of DMEM basal medium and 72μL of EZ were added. Transfection reagent (AC04L091, Shanghai Liji Biotechnology Co., Ltd.): Add tube 2 to tube 1, mix by pipetting and aspiration, incubate for 10 min, add transfection complex along the tube wall, gently shake, replace with fresh culture medium after 12 h, collect the culture medium supernatant at 48 h and 72 h after transfection, centrifuge the virus supernatant collected twice at 1000 rpm for 5 min, remove cell debris, filter the supernatant through a 0.45 μm filter into an ultracentrifuge tube, add virus concentrate (AC04L441, Shanghai Liji Biotechnology Co., Ltd.) at a virus solution: virus concentrate = 4:1, incubate overnight at 4℃, centrifuge the virus solution at 4℃ at 4000g for 15 min, discard the supernatant to obtain the virus.

[0140] The overexpressing lentivirus was resuspended in 1 mL of culture medium, and 3 μL of polybrene (TA003, General Biotechnology (Anhui) Co., Ltd.) was added. After gentle mixing, the cells were infected with the aforementioned different cell lines. The culture medium was replaced with fresh complete medium 24 h after infection. Drug screening for the resistance gene was initiated 72 h after infection to construct a stable cell line overexpressing the micropeptide. The overexpression efficiency of MP1 was verified by Western blotting, and the results are shown in Figure 2. The overexpression efficiency of MP2 was verified by q-PCR, and the results are shown in Figure 3. The antibodies used in the Western blotting were Anti-GAPDH Rabbit pAb (AB-P-R001, Wuhan Sanying Biotechnology Co., Ltd.) and Anti-MP1 Rabbit mAb (General Biotechnology (Anhui) Co., Ltd.). The q-PCR primers for detecting MP2 were SEQ ID NO:5 (upstream primer) and SEQ ID NO:6 (downstream primer).

[0141] The results are shown in Figures 2 and 3. Western blot results showed that the expression level of micropeptide MP1 in overexpressing cells plvx-MP1 (the axes and lanes in Figure 2 are all labeled "MP1") was significantly higher than that in control cells (the axes and lanes in Figure 2 are all labeled "plvx"), indicating that the overexpressing MP1 cell line was successfully constructed. q-PCR results showed that the expression level of micropeptide MP2 in overexpressing cells plvx-MP2 (labeled "MP2" in Figure 3, cell lines were: colon cancer HT29 cells, thyroid cancer BCPAP cells, liver cancer Hep3B cells, gastric cancer MGC-823 cells and AGS cells, and lung cancer Calu-3 cells, all purchased from Wuhan Pronosei Biotechnology Co., Ltd.) was significantly higher than that in control cells (labeled "plvx" in Figure 3).

[0142] Example 2: Construction of MP1 knockout cell lines

[0143] To achieve knockout (KO) of the micropeptide MP1 gene in Hep3B cells (Wuhan Pronosai Biotechnology Co., Ltd.), RNP complex-mediated CRISPR / Cas9 technology was employed. Twenty-four hours prior to the experiment, cells were seeded into 6-well plates, ensuring a cell density of approximately 80% and good growth status at transfection. sgRNA (synthesized by General Biotech Co., Ltd., sequence shown in SEQ ID NO:7) and Cas9 protein (purchased from Genscript Biotech Co., Ltd., catalog number Z03389-100) were added to the RNP complex formation buffer, gently mixed, and incubated at room temperature for 10 minutes to form the RNP complex. Subsequently, the formed RNP complex was added to pre-chilled, serum-free electroporation buffer and gently mixed. The well-grown cells in the 6-well plates were digested, and the cells were resuspended in this mixture. The RNP complex was then electroporated into the cells using an electroporator. After overnight culture at 37°C and 5% CO2, the original medium was replaced with serum-containing complete medium. Twenty-four hours later, these cells were seeded into 96-well plates at a density of 1 cell / well and cultured for another 2-3 weeks until monoclonal colonies formed. Western blot analysis was used to confirm the knockout effect of the target gene expression level, and the results are shown in Figure 4.

[0144] Western blot results showed that MP1 was not expressed in MP1 knockout Hep3B cells (the axes and lanes in Figure 4 are labeled "MP1-KO"), while MP1 expression was normal in control cells (the axes and lanes in Figure 4 are labeled "Hep3B"), indicating that the MP1 knockout Hep3B cell line was successfully constructed.

[0145] Example 3: MP1 regulates the proliferation of human hepatocellular carcinoma Hep3B cells.

[0146] Hep3B cells that successfully overexpressed MP1 in Example 1 (labeled MP1-OE in Figure 5), Hep3B cells that successfully knocked out MP1 in Example 2 (labeled MP1-KO in Figure 5), and normal Hep3B cells (control group, labeled control in Figure 5) were cultured in an incubator at 37°C and 5% CO2 until the cell density reached over 80%. The cells were then digested with trypsin, centrifuged at 1000 rpm for 5 min to remove the supernatant, resuspended in culture medium, and counted under a microscope. The cell concentration was adjusted to 3.0 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates and incubated at 37°C in a 5% CO2 incubator for 48 h. Then, 10 μL of CCK8 reagent was added to each well under dark conditions, and the plate was incubated at 37°C in the dark for 4 h. The microplate reader was preheated 30 min before incubation. After incubation, the absorbance at 450 nm was measured in each well. Data analysis was performed. The experiment was independently repeated three times. Results are expressed as mean ± SD and statistical t-tests were conducted. *P < 0.05 was considered statistically significant, and ***P < 0.001 was considered highly statistically significant.

[0147] As shown in Figure 5, compared with the control group, overexpression of MP1 significantly promoted the proliferation of Hep3B hepatocellular carcinoma, while knockout of MP1 significantly inhibited the proliferation of Hep3B hepatocellular carcinoma. This indicates that the novel micropeptide MP1 can exert a pro-tumor effect by promoting the proliferation of hepatocellular carcinoma cells, and thus it can serve as a target for the diagnosis and treatment of hepatocellular carcinoma. By detecting the expression level of MP1 in patients, the prognosis of patients can be predicted. At the same time, inhibiting the expression of MP1 using chemotherapy drugs, gene therapy and other means can treat hepatocellular carcinoma.

[0148] Example 4: MP1 regulates the migration ability of Hep3B cells in human liver cancer

[0149] Hep3B cells that successfully overexpressed MP1 in Example 1 (labeled MP1-OE), Hep3B cells that successfully knocked out MP1 in Example 2 (labeled MP1-KO), and normal Hep3B cells (control group, labeled control) were seeded into transwell chambers (purchased from Corning, catalog number CLS3415), 100 μL per well, with 10,000 cells per well. Then, 0.6 mL of complete culture medium containing 10% FBS was added to the lower chamber of the transwell to stimulate cell migration, and the cells were cultured at 37°C in 5% CO2 for 48 h. The culture medium in the wells was discarded, and the cells were fixed with methanol at room temperature for 30 min, stained with 0.1% crystal violet at room temperature for 10 min, rinsed with water, and the supernatant of unmigrated cells was wiped off with a cotton swab. The cells were observed under a microscope, and four fields of view were photographed for cell counting. The experiment was independently repeated three times. The results are expressed as mean ± SD, and statistical t-tests were performed. *P < 0.05 was considered statistically significant, and **P < 0.01 was considered highly statistically significant.

[0150] The results are shown in Figures 6A and 6B. Compared with the control group, overexpression of MP1 significantly promoted the migration of Hep3B hepatocellular carcinoma, while knockout of MP1 significantly inhibited the migration of Hep3B hepatocellular carcinoma. This indicates that the novel micropeptide MP1 can exert a pro-tumor effect by promoting the metastasis of hepatocellular carcinoma, and thus it can serve as a target for the diagnosis and treatment of hepatocellular carcinoma. By detecting the expression level of MP1 in patients, the prognosis of patients can be predicted. At the same time, inhibiting the expression of MP1 using chemotherapy drugs, gene therapy and other means can treat hepatocellular carcinoma.

[0151] Example 5: MP1 regulates Hep3B clone formation in human hepatocellular carcinoma

[0152] Hep3B cells that successfully overexpressed MP1 in Example 1 (labeled MP1-OE), Hep3B cells that successfully knocked out MP1 in Example 2 (labeled MP1-KO), and normal Hep3B cells (control group, labeled control) were seeded into six-well plates. Each well contained 1 mL of complete culture medium with 10% FBS, with 2000 cells / well and 3 replicates per group. Cells were cultured at 37°C with 5% CO2 for approximately 14 days until visible monoclonal cell clusters appeared. The culture medium in the wells was discarded, and the cells were fixed with methanol at room temperature for 30 min, stained with 0.1% crystal violet at room temperature for 20 min, rinsed with water, and the six-well plates were inverted to air dry. The cells were photographed and counted. The experiment was independently repeated three times. The results are expressed as mean ± SD and statistically analyzed using a t-test. *P < 0.05 was considered statistically significant, and ***P < 0.001 was considered highly statistically significant.

[0153] As shown in Figure 7, compared with the control group, overexpression of MP1 significantly promoted the clonal formation of Hep3B hepatocellular carcinoma, while knockout of MP1 significantly inhibited the clonal formation of Hep3B hepatocellular carcinoma. This indicates that the novel micropeptide MP1 is closely related to the malignancy of hepatocellular carcinoma, suggesting that it can serve as a target for the diagnosis and treatment of hepatocellular carcinoma. The prognosis of patients can be predicted by detecting the expression level of MP1 in their bodies. At the same time, inhibiting the expression of MP1 using chemotherapy drugs, gene therapy and other methods can treat hepatocellular carcinoma.

[0154] Example 6: MP1siRNA inhibits the proliferation of various tumor cells

[0155] Multiple specific siRNAs were designed based on the MP1 sequence information, and their sequences are shown in Table 2 below.

[0156] Table 2 siRNA sequences

[0157] Different tumor cells (all purchased from Wuhan Pronosai Biotechnology Co., Ltd.) were cultured in an incubator according to the instructions. When the cell density reached over 80%, the cells were digested with trypsin and collected. The cells were centrifuged at 1000 rpm for 5 min to remove the supernatant. The cells were resuspended in culture medium and counted under a microscope. The cell concentration was adjusted to 5.0 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates and incubated at 37°C with 5% CO2 for 12 h to ensure cell adhesion. Then, siRNA-loaded lipo3000 medium (serum-free) was added, and the plates were incubated at 37°C with 5% CO2 for 16 h. The medium was changed, the supernatant was discarded, and 100 μL of fresh serum-containing medium was added. The plates were then incubated for another 32 h. In the dark, 10 μL of CCK8 reagent was added to each well, and the plates were incubated at 37°C with the dark for 1 h. The microplate reader was preheated 30 min before incubation. After incubation, the absorbance at 450 nm was measured for each well. The reading for the group without siRNA was recorded as OD1, and the reading for the group with siRNA was recorded as OD2. Data analysis was performed. The experiment was independently repeated 5 times.

[0158] Inhibition rate calculation formula: Tumor inhibition rate (%) = (1-OD2 / OD1)*100%.

[0159] As shown in Table 3, interfering with MP1 expression significantly inhibited the proliferation of various tumor cells, indicating that tumors can be treated by interfering with MP1 expression within the tumor using siRNA. Similarly, loading the gene fragments corresponding to these segments into adenoviruses and adeno-associated viruses can also achieve the goal of tumor treatment.

[0160] Table 3. Tumor inhibition rate of MP1 siRNA against different tumor cells.

[0161] Example 7: MP1 knockout significantly inhibited the growth of Hep3B cell xenografts in nude mice.

[0162] This experiment used Hep3B cells overexpressing MP1 (denoted as Hep3B-MP1-OE) from Example 1, and Hep3B cells with MP1 knocked out (denoted as Hep3B-MP1-KO) and Hep3B cells (denoted as Hep3B) from Example 2. After expansion culture, the above cells were subcutaneously inoculated into the right axilla of BALB / c nude mice (Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.) under aseptic conditions, with a cell seeding volume of 1×10⁶ cells / mL. 7 One. The diameter of the xenograft tumor in nude mice was measured using calipers. The tumor was considered complete when it reached 80 mm. 3 Recording began around 10:00 AM. For each group of nude mice, the long and short diameters of the tumor were measured every other day, along with their body weight. The tumor volume was calculated (V = 0.5 × L × W²), and the tumor growth was dynamically observed. Measurements were taken and recorded continuously for 23 days. The results are shown in Figures 8 and 9.

[0163] The results showed that overexpression of MP1 significantly promoted the in vivo proliferation of Hep3B cells in liver cancer, while MP1 knockout significantly inhibited the in vivo proliferation of Hep3B cells in liver cancer. This indicates that MP1 gene knockout or methods to reduce intratumoral MP1 levels (i.e., siRNA, shRNA, or viral gene therapy) can be used to treat tumors, suggesting that monitoring MP1 expression in tumor tissue can predict patient prognosis and serve as a diagnostic biomarker.

[0164] Example 8: Overexpression of micropeptide MP2 inhibits tumor cell proliferation

[0165] Cells overexpressing MP2 obtained in Example 2 (labeled "MP2-OE" in Figure 10) and corresponding non-overexpressing cells (labeled "control" in Figure 10) were collected, digested, and adjusted to a suitable concentration (5000-8000 cells / well). 100 μL was seeded into each well of a 96-well plate. The plates were incubated at 37°C in a 5% CO2 incubator for 48 h. Under light-protected conditions, 10 μL of CCK8 reagent was added to each well, and the plates were incubated at 37°C for 1 h in the dark. The microplate reader was preheated 30 min before incubation. After incubation, the absorbance at 450 nm was measured in each well, and the data were analyzed. The experiment was independently repeated three times. The results are expressed as mean ± SD and statistically analyzed using a t-test. *P < 0.05 was considered statistically significant, and ***P < 0.001 was considered highly significant.

[0166] The results are shown in Figure 10. Compared with the control group, overexpression of the micropeptide MP2 can significantly inhibit the proliferation of colon cancer HT29 cells, thyroid cancer BCPAP cells, liver cancer Hep 3B cells, gastric cancer MGC-823 cells and AGS cells, and lung cancer Calu-3 cells, indicating that overexpression of the micropeptide MP2 plays an anti-tumor role by inhibiting the proliferation of tumor cells.

[0167] Example 9: Preparation, purification and detection of micropeptide MP2

[0168] The micropeptide MP2 was synthesized by solid-phase synthesis method, separated and purified by preparative HPLC, and its purity was determined by analytical HPLC.

[0169] Using Fmoc-wang-resin as the starting material, then successively connecting dipeptides to pentapeptides with protected amino acids. After the synthesis work was completed, it was washed thoroughly, and then the peptide was cleaved and post-treated to obtain the crude micropeptide MP2. The crude product was dissolved, purified twice by preparative high performance liquid chromatography, concentrated and freeze-dried to obtain the pure product. This method can not only ensure the synthesis efficiency, but also improve the product purity.

[0170] (1) The steps for synthesizing micropeptide MP2 are as follows:

[0171] Weigh an appropriate amount of Fmoc-wang-resin, pour it into a glass sand core reaction column, add an appropriate amount of CH2Cl2 to fully expand the resin, and successively connect the protected amino acids to the resin according to the peptide sequence, with each amino acid reacting for 1 hour. Use a 20% piperidine in N,N-dimethylformamide (DMF) solution for 20 minutes to remove the Fmoc protective group. Use trifluoroacetic acid: phenol: water: benzyl mercaptan: EDT = 90:3:3:2:2. Cut the peptide from the resin, then precipitate it with ice-cooled ether, then centrifuge at 4000 rpm for 10 min, pour out the supernatant, and then wash the polypeptide with anhydrous ether and drain to obtain the crude polypeptide.

[0172] (2) The steps for purifying micropeptide MP2 are as follows:

[0173] Precisely weigh the crude product, add appropriate purified water to prepare a solution with a concentration of 5 - 20 g / L, and stir it ultrasonically until it becomes a clear solution without particles; filter the crude product solution through a 0.45 μm mixed filter membrane of a sand core filter; perform primary purification and secondary purification through semi-preparative high performance liquid chromatography to obtain the refined qualified product of the polypeptide. During the preparation process, the mobile phase: phase A is acetonitrile, and phase B is an aqueous solution containing. Collect the solution with an absorbance greater than 200 mV at an ultraviolet wavelength of 220 nm, and use the solution with a detected purity greater than 98.5% as qualified. Concentrate the qualified solution under reduced pressure at 37°C using a rotary evaporator to remove the residual solvent and part of the water. Finally, filter it through a 0.22 μm filter membrane, load the filtrate into a freeze-drying tray, and perform freeze-drying with a freeze dryer to obtain the pure product.

[0174] (3) Analysis of the purity of micropeptide MP2

[0175] The purity of the freeze-dried product was analyzed by analytical high-performance liquid chromatography.

[0176] The results are shown in Figure 11. The purity of the micropeptide MP2 meets the requirements for subsequent experiments.

[0177] Example 10: Exogenous administration of micropeptide MP2 inhibits the proliferation of human gastric cancer cells.

[0178] The artificially synthesized micropeptide MP2 from Example 9 was used to test its in vitro antitumor activity against gastric cancer cells. Gastric cancer cells MGC-823 and AGS (both purchased from Wuhan Pronosai Biotechnology Co., Ltd.) were cultured in an incubator according to the instructions. When the cell density reached over 80%, the cells were digested with trypsin and collected. The cells were centrifuged at 1000 rpm for 5 min to remove the supernatant. The cells were resuspended in culture medium and counted under a microscope. The cell concentration was adjusted to 3.0 × 10⁻⁶ cells / mL. 4 Cell suspension was seeded at a rate of 100 μL / mL into 96-well plates and incubated at 37°C in a 5% CO2 incubator for 12 h. Synthetic peptide MP2, diluted with serum-free medium, was added to each well at different concentrations, and the plates were incubated at 37°C in a 5% CO2 incubator for 48 h. Under light-protected conditions, 10 μL of CCK8 reagent was added to each well, and the plates were incubated at 37°C in the dark for 1 h. The microplate reader was preheated 30 min before incubation. After incubation, the absorbance at 450 nm was measured for each well. The reading for the group without MP2 peptide was recorded as OD1, and the reading for the group with MP2 peptide was recorded as OD2. Data analysis was performed. The experiment was independently repeated three times. The results are expressed as mean ± SD and statistically analyzed using a t-test. *P < 0.05 was considered statistically significant, and ***P < 0.001 was considered highly significant.

[0179] The formula for calculating the tumor inhibition rate is: Tumor inhibition rate (%) = (1-OD2 / OD1)*100%.

[0180] As shown in Table 4 and Figures 12A and 12B, the synthesized micropeptide MP2 can inhibit the growth of gastric cancer cells MGC-823 and AGS, and the effect is better than that of the positive control docetaxel.

[0181] Table 4. Tumor inhibition rate of MP2 peptide at different concentrations

[0182] Example 11: Exogenous administration of micropeptide MP2 inhibits the migration of human gastric cancer cells.

[0183] Following the instructions, gastric cancer cells (MGC-823 and AGS) were cultured in an incubator until confluence density exceeded 90%. Cells were then collected by trypsin digestion at 1000 rpm for 5 min. After resuspending the cells in culture medium and counting them under a microscope, they were seeded into Transwell chambers (Corning, catalog number CLS3415), 100 μL per well, with MP2 dilutions added according to different concentrations. Then, 0.6 mL of complete culture medium containing 10% FBS was added to the lower chamber of the Transwell to stimulate cell migration. Cells were incubated at 37°C with 5% CO2 for 48 h. The culture medium in the wells was discarded, and the cells were fixed with methanol at room temperature for 30 min, stained with 0.1% crystal violet at room temperature for 10 min, rinsed with water, and the supernatant of unmigrated cells was wiped off with a cotton swab. The cells were observed under a microscope, and four fields of view were photographed for counting. The readings for the group without MP2 micropeptide were recorded as OD1, and the readings for the group with MP2 micropeptide were recorded as OD2. The experiment was independently repeated 3 times. The results were expressed as mean ± SD and statistical t-tests were performed. *P < 0.05 was considered statistically significant, and ***P < 0.001 was considered highly statistically significant.

[0184] Formula for calculating migration inhibition ability: Migration inhibition rate (%) = (1-OD2 / OD1)*100%.

[0185] The results are shown in Table 5 and Figures 13A and 13B. Compared with the control group, micropeptide MP2 can significantly inhibit the migration of gastric cancer MGC-823 cells and AGS cells, indicating that micropeptide MP2 can exert anti-tumor effects by inhibiting gastric cancer migration, and its effect in AGS cells is better than that of the positive control Avastin.

[0186] Table 5. Inhibition rate of micropeptide MP2 on gastric cancer cell migration

[0187] Example 12: Exogenous administration of MP2 inhibits the proliferation of different tumors

[0188] Different tumor cells (all purchased from Wuhan Pronosai Biotechnology Co., Ltd.) were cultured in an incubator according to the instructions. When the cell density reached over 80%, the cells were digested with trypsin and collected. The cells were centrifuged at 1000 rpm for 5 min to remove the supernatant. The cells were resuspended in culture medium and counted under a microscope. The cell concentration was adjusted to 3.0 × 10⁻⁶ cells / mL. 4Cell suspension was seeded at a rate of 100 μL / mL into 96-well plates and incubated at 37°C in a 5% CO2 incubator for 12 h. Micropeptide MP2 diluted with serum-free medium was added at different concentrations, and the plates were incubated at 37°C in a 5% CO2 incubator for 48 h. Under light-protected conditions, 10 μL of CCK8 reagent was added to each well, and the plates were incubated at 37°C in the dark for 1 h. The microplate reader was preheated 30 min before incubation. After incubation, the absorbance at 450 nm was measured in each well. The reading for the group without MP2 micropeptide was recorded as OD1, and the reading for the group with MP2 micropeptide was recorded as OD2. Data analysis was performed. The experiment was independently repeated three times. The results are expressed as mean ± SD and statistically analyzed using a t-test. *P < 0.05 was considered statistically significant, and **P < 0.01 was considered highly statistically significant.

[0189] The formula for calculating the tumor inhibition rate is: Tumor inhibition rate (%) = (1-OD2 / OD1)*100%.

[0190] As shown in Table 6, compared with the control group, micropeptide MP2 can inhibit the proliferation activity of various tumor cells, indicating that micropeptide MP2 can achieve anti-tumor therapeutic activity by inhibiting tumor cell proliferation, and its effect in various cell lines is better than that of the positive control docetaxel, or comparable to that of docetaxel.

[0191] Table 6. Tumor inhibition rate of micropeptide MP2 on different tumor cells.

[0192] Example 13: MP1 expression levels differ significantly between human tumor tissues and normal tissues.

[0193] The recommended method for downloading RNA-seq sequencing files and clinical information of cancerous and normal tissues of 32 types of tumors, including head and neck cancer, glioma, thyroid cancer, esophageal squamous cell carcinoma, lung cancer, liver cancer, gastric cancer, kidney cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, colorectal cancer, pancreatic cancer, osteosarcoma, and skin cancer, was used. Statistical analysis was performed using R language (version 3.1.1) software, requiring the installation and loading of packages (heatmap, venndiagram, hist, etc.). Then, MP1 expression level analysis was performed using packages such as DESeq, edgeR, and limma to identify tumor types with differential expression (as shown in Table 7). The criteria for judging differential expression were: (1) |log2(expression level in cancerous tissue / expression level in adjacent tissue)|>1, (2) P<0.05.

[0194] Table 7. Analysis of the differential expression levels of MP1 in human tumor tissues and normal tissues.

[0195] As shown in Table 7 and Figure 14 (in Figure 14, "N" represents normal tissue and "T" represents tumor tissue), the expression of MP1 in cancerous and adjacent tissues was analyzed compared with normal tissues. It was found that compared with adjacent tissues, the expression level of MP1 was significantly increased in acute myeloid leukemia (LAML), head and neck squamous cell carcinoma (HNSC), hepatocellular carcinoma (LIHC), nephroblastoma (WT), rectal adenocarcinoma (READ), and cholangiocarcinoma (CHOL) tissues, while the expression level of MP1 was significantly decreased in lung squamous cell carcinoma (LUSC), melanoma (SKCM), testicular cancer (TGCT), leukemia (ALL), and renal chromophobe carcinoma (KICH) tissues. This indicates that the micropeptide MP1 can serve as a potential new indicator for tumor diagnosis. By detecting the MP1 expression level in clinical sample tissues and normal tissues, significant changes (e.g., increases or decreases) in MP1 expression levels in clinical sample tissues compared to normal tissues can be found, suggesting that the clinical sample tissue may be tumor tissue, thus determining whether the subject has cancer; and the type of cancer the subject may have can be distinguished based on the type of expression level change (e.g., increases or decreases).

[0196] Example 14: MP1 expression can serve as a prognostic indicator for cancer.

[0197] RNA-seq sequencing files and clinical information of all tumors in the TCGA database were downloaded. Statistical analysis was performed using R language (version 3.1.1) software, requiring the installation and loading of packages (heatmap, venndiagram, hist, etc.). The relationship between MP1 expression and tumor prognosis was then analyzed.

[0198] As shown in Figure 15, patients with high expression of MP1 in adrenocortical carcinoma (ACC), clear cell renal cell carcinoma (KIRC), and hepatocellular carcinoma (LIHC) have a worse prognosis, suggesting that the micropeptide MP1 can serve as a potential indicator for tumor prognostic diagnosis.

[0199] Example 15: Detection of MP1 expression in liver cancer by real-time PCR

[0200] Specific primers were designed based on the sequence information of MP1, and the sequences are as follows:

[0201] Upstream primer (SEQ ID NO.8): GACCCACACTAGGCCTCCC

[0202] Downstream primer (SEQ ID NO.9): TAGGGAGCCCAGGCCAAG

[0203] The expression of MP1 in liver cancer was detected by real-time quantitative PCR.

[0204] Total RNA was extracted and collected from Hep3B, 7721, HepG2, and LO2 cells according to Thermo Fisher Scientific's Trizol instructions. The purity and concentration of the extracted RNA were then quantified using a NanoDrop ND-1000 nucleic acid quantification instrument, and agarose gel electrophoresis was performed to ensure the integrity of the extracted RNA. cDNA was synthesized from the extracted total RNA using the TaKaRa PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time). The TaKaRa kit was used. Premix Ex Taq TM qPCR was performed using Tli RNaseH Plus. The reaction system is shown in Table 8 below:

[0205] Table 8 PCR Reaction System

[0206] After mixing the above components evenly, follow the procedure below: pre-denaturation at 95℃ for 30s, 40 cycles; 95℃ for 5s, 60℃ for 30s.

[0207] The specificity of the reaction was determined based on the melting curve, and the relative expression level of MP1 was calculated using formula 2-ΔΔCt. The results are shown in Figure 16. The expression level of MP1 in liver cancer cells (e.g., Hep3B, hepG2, 7721) was significantly lower than that in normal cells (e.g., LO2), and the expression trend was similar to that analyzed from the TCGA database, indicating that MP1 can serve as a potential biomarker for cancer diagnosis.

[0208] Example 16: Expression analysis of MP2 in human tumor and normal tissues

[0209] The recommended method for downloading RNA-seq sequencing files and clinical information of cancerous and normal tissues of 32 types of tumors, including head and neck cancer, glioma, thyroid cancer, esophageal squamous cell carcinoma, lung cancer, liver cancer, gastric cancer, kidney cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, colorectal cancer, pancreatic cancer, osteosarcoma, and skin cancer, was used. Statistical analysis was performed using R language (version 3.1.1) software, requiring the installation and loading of packages (heatmap, venndiagram, hist, etc.). Then, MP2 expression level analysis was performed using packages such as DESeq, edgeR, and limma to identify tumor types with differential expression (as shown in Table 9). The criteria for judging differential expression were: (1) |log2(expression level in cancerous tissue / expression level in adjacent tissue)|>1, (2) P<0.05.

[0210] Table 9. Analysis of MP2 expression levels in human tumor and normal tissues (cancerous / adjacent to normal)

[0211] As shown in the table above, the expression of MP2 in cancerous and adjacent tissues was analyzed compared with normal tissues. It was found that compared with adjacent tissues, the expression level of MP2 was significantly reduced in multiple morphological glioma (GBM), low-grade glioma (LGG), invasive breast cancer (BRCA), cervical squamous cell carcinoma and adenocarcinoma (CESC), lung adenocarcinoma (LUAD), esophageal cancer (ESCA), gastric and esophageal cancer (STES), papillary renal cell carcinoma (KIRP), mixed renal cell carcinoma (KIPAN), colon cancer (COAD), colorectal cancer (COADREAD), prostate cancer (PRAD), gastric cancer (STAD), clear cell renal cell carcinoma (KIRC), melanoma (SKCM), thyroid cancer (THCA), ovarian serous cystadenocarcinoma (OV), pancreatic cancer (PAAD), testicular cancer (TGCT), uterine sarcoma (UCS), and adrenocortical carcinoma (ACC). This indicates that the micropeptide MP2 can serve as a potential new indicator for tumor diagnosis.

[0212] It should be noted that the above are merely preferred embodiments of this application and are not intended to limit the application. Various modifications and variations are possible for those skilled in the art. Although specific embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents of the above embodiments may exist or be unforeseeable to the applicant or other those skilled in the art. Therefore, the appended claims and any possible amendments to the claims are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents. Importantly, as technology evolves, many elements described herein can be replaced by equivalent elements appearing after this application.

Claims

1. A polypeptide, characterized in that, The polypeptide includes at least one of the amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

3. A polynucleotide, characterized in that, The polynucleotide encodes the polypeptide of claim 1 or 2.

4. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide of claim 3.

5. A drug or reagent for intervening in a target polypeptide or its encoded nucleic acid, characterized in that, The target polypeptide includes the amino acid sequence shown in SEQ ID NO:

1.

6. The drug or reagent according to claim 5, wherein the target polypeptide or its encoded nucleic acid comprises at least one of the following: Inhibit the activity of the target polypeptide; Induce deletion and / or mutation of the encoded nucleic acid; Inhibit the transcription and / or translation of the encoded nucleic acid; and / or The target polypeptide or its encoded nucleic acid is degraded.

7. The drug or reagent according to claim 6, wherein the type of the drug or reagent includes at least one selected from proteins, polypeptides, nucleic acids, polynucleotides, polynucleotides, antibodies or derivatives thereof, organic compounds, inorganic compounds and / or natural products.

8. The drug or reagent according to claim 7, wherein the drug or reagent comprises double-stranded ribonucleic acid, the double-stranded ribonucleic acid comprising a sense strand and an antisense strand, and the double-stranded ribonucleic acid is selected from at least one of the following: (1) The sense strand contains the nucleotide sequence shown in SEQ ID NO:12, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:13; (2) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:14, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:15; or (3) The sense strand contains the nucleotide sequence shown in SEQ ID NO:16 and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:

17.

9. The drug or reagent according to claim 7, wherein the drug or reagent comprises a single-guide RNA (sgRNA), the sgRNA comprising the nucleotide sequence shown in any one of SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:

11.

10. The drug or reagent according to claim 9, wherein the drug or reagent further comprises Cas protein.

11. The drug or reagent according to claim 5, wherein the drug or reagent is used to treat a disease, the disease including tumors or cancers, the tumors or cancers including bile duct cancer, colorectal cancer, liver cancer, head and neck squamous cell carcinoma, testicular cancer, melanoma, lung cancer, leukemia, kidney cancer, glioma, invasive breast cancer, cervical cancer, esophageal cancer, gastric cancer, prostate cancer, thyroid cancer, ovarian cancer, pancreatic cancer, uterine sarcoma, colon cancer, or bladder cancer.

12. A polypeptide, characterized in that, The polypeptide includes at least one of the amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

2.

13. The polypeptide according to claim 12, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO:

2.

14. A polynucleotide, characterized in that, The polynucleotide encodes the polypeptide of claim 12 or 13.

15. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide of claim 14.

16. Use of the polypeptide of claim 12 or 13, the polynucleotide of claim 14, or the recombinant vector of claim 15 in the preparation of a medicament or medicament composition for the prevention, relief or treatment of a disease.

17. The use of any one of the medicaments or reagents for intervening in a target polypeptide or its encoded nucleic acid, as described in any one of claims 5-11, in the preparation of a medicament or medicament composition for the prevention, relief or treatment of a disease.

18. Use of a substance for intervening in the polypeptide of claim 1 or 2, or the polynucleotide of claim 3, in the preparation of a medicament or medicament composition for the prevention, relief or treatment of a disease.

19. Use of a substance for detecting the polypeptide of any one of claims 1, 2, 12 or 13, or the polynucleotide of claim 3 or 14, in the preparation of a reagent or kit for diagnosing or assisting in the diagnosis of a disease.

20. The use according to claim 19, wherein the substance comprises a probe, an antibody or antigen-binding fragment, or a nucleic acid primer.

21. The use according to claim 20, wherein the substance is a nucleic acid primer, the nucleic acid primer comprising an upstream primer and a downstream primer, and the nucleic acid primer is selected from at least one of the following: (1) The upstream primer contains the nucleotide sequence shown in SEQ ID NO:5, and the downstream primer contains the nucleotide sequence shown in SEQ ID NO:6; or (2) The upstream primer contains the nucleotide sequence shown in SEQ ID NO:8 and the downstream primer contains the nucleotide sequence shown in SEQ ID NO:

9.

22. The use according to any one of claims 16-21, wherein the disease includes a tumor or cancer, and the tumor or cancer includes bile duct cancer, colorectal cancer, liver cancer, head and neck squamous cell carcinoma, testicular cancer, melanoma, lung cancer, leukemia, kidney cancer, glioma, invasive breast cancer, cervical cancer, esophageal cancer, gastric cancer, prostate cancer, thyroid cancer, ovarian cancer, pancreatic cancer, uterine sarcoma, colon cancer, or bladder cancer.

23. A method for preventing, alleviating, or treating a disease, characterized in that, The method includes administering to a subject in need the polypeptide of claim 12 or 13, the polynucleotide of claim 14, or the recombinant vector of claim 15.

24. A method for preventing, alleviating, or treating a disease, characterized in that, The method comprises administering to a subject in need the drug or reagent according to any one of claims 5-11 for intervening in the expression level of the target polypeptide or its encoded nucleic acid.

25. A method for preventing, alleviating, or treating a disease, characterized in that, The method includes administering to a subject in need of intervention a polypeptide of claim 1 or 2, or a polynucleotide of claim 3.

26. A method for diagnosing or assisting in the diagnosis of a disease, characterized in that, The method includes detecting the expression level of the polypeptide of any one of claims 1, 2, 12 or 13, or the polynucleotide of claim 3 or 14, in a clinical sample derived from a subject.

27. The method of claim 26, wherein the detection method comprises transcriptome sequencing, polymerase chain reaction, in situ hybridization, gene chip method, proteomics, flow cytometry, enzyme-linked immunosorbent assay (ELISA), Western blotting, and / or immunostaining.

28. The method according to any one of claims 23-27, wherein the disease includes a tumor or cancer, and the tumor or cancer includes bile duct cancer, colorectal cancer, liver cancer, head and neck squamous cell carcinoma, testicular cancer, melanoma, lung cancer, leukemia, kidney cancer, glioma, invasive breast cancer, cervical cancer, esophageal cancer, gastric cancer, prostate cancer, thyroid cancer, ovarian cancer, pancreatic cancer, uterine sarcoma, colon cancer, or bladder cancer.

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