CN117085113A - TMEM25 protein and application thereof in cancer treatment - Google Patents

Abstract

The application relates to the technical field of tumor treatment, in particular to TMEM25 protein and application thereof in cancer treatment, and particularly provides wild TMEM25 protein, a mutant TMEM25 protein or isomer thereof, polynucleotide molecules, nucleic acid constructs, vectors, host cells and pharmaceutical compositions, and application thereof in cancer treatment.

Description

TMEM25 protein and its application in cancer treatment

This application claims priority for the invention patent with the subject name "TMEM25 protein and applications" and the application number "2023100677139" submitted on February 6, 2023.

Technical field

This application relates to the technical field of tumor treatment, and in particular to the TMEM25 protein and its application in cancer treatment.

Background technique

Breast cancer is the most common malignancy among women worldwide. Breast cancer is considered a heterogeneous disease due to the constant changes in genomic and epigenetic factors across differentiation, subtypes, and stages. For the treatment of in situ breast cancer, surgery and radiotherapy are still the main treatments. For migratory malignant breast cancer, chemotherapy, hormone therapy and targeted therapy are gradually emerging, but they still have a high fatality rate. Currently, most of the drugs used to treat breast cancer are cytotoxic chemotherapy drugs, followed by hormonal drugs, and only a few are drugs that target tumors and the tumor microenvironment. Although cytotoxic drugs are highly effective in the treatment of orthotopic breast cancer, they have some significant side effects because they target all replicating and proliferating cells, including normal and tumor cells. Similar to chemotherapy drugs, hormone therapy is also used systemically. Its efficacy in metastatic breast cancer is limited, and long-term use can lead to drug resistance. Due to the high incidence of breast cancer and the limitations of current treatment options, it is particularly important to find new targets and treatment options for breast cancer.

Compared with other anti-tumor treatment options, gene therapy provides many new possibilities for tumor treatment. There are currently some targeted gene therapy options for breast cancer undergoing clinical trial evaluation, but there are still no relevant gene therapies approved for breast cancer treatment. Therefore, it is even more important to find new targets for gene therapy of breast cancer. In addition, protein drugs are also an important direction in current tumor treatment. Protein drugs have the characteristics of high activity, strong specificity, low toxicity, clear biological functions, and are conducive to clinical application.

Transmembrane protein 25 (TMEM25) is a member of the immunoglobulin superfamily and is a single-pass transmembrane protein. Its amino acids at positions 42-112 are homologous to C2-type immunoglobulin domains such as Hemicentin and Titin. There are currently only a few studies on TMEM25. For example, in neurons, TMEM25 can affect epilepsy by regulating the level of neuronal excitation. In colorectal cancer, the hypermethylation of the TMEM25 promoter leads to a decrease in its expression level, making it possible to become a tumor marker. In breast cancer, studies have shown that the expression level of TMEM25 is related to patient prognosis and may be related to the resistance of breast cancer cells to paclitaxel. However, the expression of TMEM25 or its mutants in breast cancer and the role it plays in the progression of breast cancer are still unclear.

Contents of the invention

During the in-depth study of TMEM25 protein, the inventor of the present application unexpectedly discovered that TMEM25 protein, as well as TMEM25 protein containing specific mutations or its isomers, have a significant inhibitory effect on the growth of tumor cells, and based on this, they completed this application.

The first aspect of the present application provides wild-type or mutated TMEM25 proteins or isomers thereof, polynucleotide molecules encoding the wild-type or mutated TMEM25 proteins or isomers thereof, and nucleic acids comprising the polynucleotide molecules. Construct or vector, host cell comprising said polynucleotide molecule, said nucleic acid construct or vector, said wild-type or mutant TMEM25 protein or isoforms thereof, said polynucleotide sequence, said nucleic acid The use of the pharmaceutical composition of the construct, the vector or the host cell for the preparation of a medicament for the treatment of cancer, or for the treatment of cancer; and a method of treating cancer comprising administering wild-type Type or mutant TMEM25 protein or isomers thereof, polynucleotide molecules encoding said wild-type or mutant TMEM25 proteins or isomers thereof, nucleic acid constructs or vectors comprising said polynucleotide molecules, said Polynucleotide molecules, host cells of said nucleic acid constructs or vectors, said wild-type or mutant TMEM25 proteins or isoforms thereof, said polynucleotide sequences, said nucleic acid constructs, said vectors or their Pharmaceutical compositions of the host cells.

The second aspect of the present application provides a mutant TMEM25 protein or an isomer thereof, in which the tyrosine residue at position 13 and/or position 15 from the C terminus is replaced with a glutamic acid residue or aspartic acid. Residues.

The third aspect of the present application provides a polynucleotide molecule encoding the mutated TMEM25 protein or isoform thereof of the second aspect of the present application.

The fourth aspect of the present application provides a nucleic acid construct, which includes the polynucleotide molecule provided by the third aspect of the present application, and a promoter operably linked to the polynucleotide molecule.

The fifth aspect of the present application provides a vector, which contains the polynucleotide molecule provided by the third aspect of the present application or the nucleic acid construct provided by the fourth aspect of the present application; preferably, the vector is selected from the group consisting of plasmid vectors and lentivirus vectors. Viral vectors or related adenoviral vectors.

The sixth aspect of the present application provides a host cell, which contains at least the polynucleotide molecule provided by the third aspect of the present application, the nucleic acid construct provided by the fourth aspect of the present application, or the vector provided by the fifth aspect of the present application. One; preferably, the vector expresses the mutated TMEM25 protein or its isomer provided in the second aspect of the application.

The seventh aspect of this application provides the mutated TMEM25 protein or its isomer provided by the second aspect of this application, the polynucleotide molecule provided by the third aspect of this application, the nucleic acid construct provided by the fourth aspect of this application, The vector provided in the fifth aspect of this application and the host cell provided in the sixth aspect of this application are used as medicines.

The eighth aspect of this application provides a pharmaceutical composition, which contains the mutated TMEM25 protein or its isomer provided by the second aspect of this application, the polynucleotide molecule provided by the third aspect of this application, and the fourth aspect of this application. At least one of the nucleic acid construct provided in the aspect, the vector provided in the fifth aspect of this application, and the host cell provided in the sixth aspect of this application.

The ninth aspect of this application provides the mutated TMEM25 protein or its isomer provided by the second aspect of this application, the polynucleotide molecule provided by the third aspect of this application, the nucleic acid construct provided by the fourth aspect of this application, The vector provided in the fifth aspect of this application, the host cell provided in the sixth aspect of this application, and the pharmaceutical composition provided in the eighth aspect of this application are used for preparing drugs for treating cancer.

The tenth aspect of this application provides the mutated TMEM25 protein or its isomer provided by the second aspect of this application, the polynucleotide molecule provided by the third aspect of this application, the nucleic acid construct provided by the fourth aspect of this application, The vector provided by the fifth aspect of this application, the host cell provided by the sixth aspect of this application, and the pharmaceutical composition provided by the eighth aspect of this application are used to treat cancer.

The eleventh aspect of the present application provides a method for treating cancer, which includes administering to an individual in need an effective dose of the mutated TMEM25 protein or isomer thereof provided in the second aspect of the present application, or the mutant TMEM25 protein provided in the third aspect of the present application. The polynucleotide molecule provided, the nucleic acid construct provided by the fourth aspect of this application, the vector provided by the fifth aspect of this application, the host cell provided by the sixth aspect of this application, or the pharmaceutical combination provided by the eighth aspect of this application things.

In some embodiments, the cancer is selected from breast cancer, colon cancer, cervical cancer, and osteosarcoma.

Description of the drawings

Figure 1A and Figure 1B show the growth curves of MDA-MB-231 and 4T1 cells overexpressing or knocking out TMEM25.

Figure 1C and Figure 1D show the ability of colony formation in soft agar of MDA-MB-231 and 4T1 overexpressing or knocking out TMEM25.

Figures 2A-2C show the effects of transgenic high expression of TMEM25 and knockout of TMEM25 on mouse tumor growth rate, tumor size and mouse survival in the context of primary breast cancer mice (MMTV; PyMT). Figure 2A shows that in TMEM25 knockout mice, the tumor mass was significantly higher than that of the control group, while in transgenic mice, the tumor mass was significantly lower than that of the control group. Figure 2B shows that knocking out TMEM25 significantly accelerated tumor growth, while transgenic high expression of TMEM25 inhibited tumor growth. Figure 2C shows that TMEM25 knockout shortens mouse survival, while high expression of TMEM25 prolongs mouse survival.

Figure 3A shows the effect of orthotopic injection of adeno-associated virus carrying the TMEM25 gene on mouse tumor growth in primary breast cancer mice MMTV; PyMT.

Figure 3B shows the impact on survival of mice after orthotopic injection of adeno-associated virus carrying the TMEM25 gene in primary breast cancer mice MMTV; PyMT.

Figures 4A to 4E respectively show the effects of overexpression of TMEM25 wild type and mutants in different types of tumor cell lines on cell proliferation and the expression of TMEM25 wild type or its mutants in cells.

Figure 5 shows photomicrographs of cells overexpressing TMEM25 mutants in different types of tumor cell lines.

Detailed ways

In order to explain the embodiments of the present application or the technical solutions in the prior art more clearly, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only This is an embodiment of the present application. For those of ordinary skill in the art, other embodiments can be obtained based on these drawings.

In this application, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used in this article are terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand this application, definitions and explanations of relevant terms are provided below.

It will also be understood that in certain methods described herein that include more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are recited, unless the context dictates otherwise. .

definition

As used herein, the terms "a" and "an" as well as "the" and similar referents refer to the singular and the plural unless otherwise indicated herein or otherwise clearly contradicted by context.

As used herein, the terms "about," "substantially," and "similar to" mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which error range may depend in part on the measurement of the value Either the way it is determined, or it depends on the limitations of the measurement system. As used herein, reference to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, descriptions that refer to "about X" include descriptions of "X."

As used herein, the term "isoform" has its general meaning, meaning that the same gene encodes proteins with different structures but the same source due to alternative splicing of mRNA; in this application, it can be understood as encoding TMEM25 protein The gene encodes a protein homologous to TMEM25 formed due to alternative splicing of the gene.

The term "polynucleotide molecule" or "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single, double or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or DNA containing purine and pyrimidine bases or other natural, chemical or biochemical modifications, non-natural or derivatized A polymer of nucleotide bases. The backbone of a nucleic acid may contain sugar and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the nucleic acid backbone may comprise synthetic subunits such as polymers of phosphoramidates and thus may be oligodeoxynucleoside phosphoramidates (P- _NH2 ) or mixed phosphoramidate-phosphodiester oligomers. In addition, double-stranded nucleic acids can be obtained from single-stranded polynucleotide products of chemical synthesis (by synthesizing the complementary strand under appropriate conditions and annealing the strand, or de novo synthesis of the complementary strand using a DNA polymerase with appropriate primers).

The term "nucleic acid construct" as used herein means a single- or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified to contain nucleic acid segments in a manner that would not otherwise occur in nature, or is synthetic . The term nucleic acid construct is synonymous with the term "expression cassette" or "expression vector" when the nucleic acid construct contains regulatory sequences required for expression of the coding sequence of the invention.

The term "vector" refers to any vehicle that delivers nucleic acid into a cell or organism. Examples include plasmid vectors, viral vectors, liposomes or cationic lipids. The term also refers to a construct comprising genetic material designed to directly transform a target cell by delivering a nucleic acid sequence into the cell. The vector may contain multiple genetic elements oriented positionally and sequentially with other necessary elements such that the contained nucleic acid cassette can be transcribed and, if necessary, translated in the transfected cell. These components are effectively connected.

One type of vector is a "plasmid," which generally refers to a circular double-stranded DNA circle into which additional DNA segments (foreign genes) can be ligated, but may also include linear double-stranded molecules, such as those derived from polymerase chains. Amplification by reaction (PCR) or treatment of circular plasmid with restriction enzymes yields linear double-stranded molecules. Plasmid vectors include vector backbone (i.e. empty vector) and expression framework. The term "expression framework" refers to a sequence with the potential to encode a protein. Another type of vector is a viral vector in which virus-derived DNA or RNA sequences are present in the vector for packaging into viruses (e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses and adeno-associated virus (AAV)). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) integrate into the host cell's genome when introduced into the host cell, thereby replicating with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors."

As used herein, the term "host cell" refers to a cell into which a vector is introduced, including many cell types such as prokaryotic cells such as E. coli or Bacillus subtilis, yeast cells or fungal cells such as Aspergillus, S2 Drosophila cells, or Insect cells such as Sf9, or animal cells such as HEK 293T cells, MDA-MB-231 cells, 4T1 cells, etc.; in this application, the "host cells" particularly refer to mammalian cells, more specifically, cells of human origin.

As used herein, the term "treatment" generally refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms; and/or may be therapeutic in terms of partially or completely stabilizing or curing the disease and/or side effects due to the disease. As used herein, "treatment" encompasses any treatment of a disease in a patient, including: (a) preventing the disease or symptoms in a patient who is susceptible to the disease or symptoms but has not yet been diagnosed with the disease; (b) suppressing the symptoms of the disease, i.e., arresting its progression; or (c) alleviating the symptoms of a disease, i.e., causing regression of the disease or symptoms.

As used herein, the term "disease and/or disorder" refers to a physical state of the subject that is associated with the disease and/or disorder of the invention. As used herein, the term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, who receives a pharmaceutical composition of the present invention to treat, prevent, alleviate and/or alleviate a disease or condition described in the present invention. Dogs, monkeys, cows, horses, etc.

The term "individual in need" refers to an individual who is in need of treatment or prevention as determined by a researcher, veterinarian, physician, or other clinician. In one embodiment, the individual in need thereof is a mammal, such as a human.

The term "cancer" refers to a disease or disorder resulting from the proliferation of cells that undergo oncogenic transformation. "Cancer" shall include any one or more of a wide range of tumors, benign or malignant, including those capable of growing and metastasizing through the human or animal body or parts thereof, for example, via invasion of the lymphatic system and/or bloodstream. Although the present invention is particularly directed to the diagnosis or detection of malignant tumors and solid cancers, as used herein, the term "tumor" encompasses both benign and malignant tumors or solid masses. Cancer further includes, but is not limited to, carcinoma, lymphoma or sarcoma, such as ovarian cancer, colon cancer, breast cancer, pancreatic cancer, lung cancer, prostate cancer, urinary tract cancer, uterine cancer, acute lymphoblastic leukemia, Hodgkin's Hodgkin's disease, small cell lung cancer, melanoma, neuroblastoma, glioma (such as glioblastoma), and soft tissue sarcoma, lymphoma, melanoma, sarcoma and adenocarcinoma.

In one aspect, the application provides wild-type or mutated TMEM25 proteins or isomers thereof, polynucleotide molecules encoding said wild-type or mutated TMEM25 proteins or isomers thereof, and polynucleotide molecules comprising said polynucleotide molecules. Nucleic acid constructs or vectors, host cells comprising the polynucleotide molecule, the nucleic acid construct or vector, the wild-type or mutant TMEM25 protein or isomers thereof, the polynucleotide sequences, the The use of the nucleic acid construct, the vector or the pharmaceutical composition of the host cell for preparing a medicament for treating cancer; or for treating cancer.

In another aspect, the present application provides a method of treating cancer, which includes regulating the expression level of wild-type or mutated TMEM25 protein or isoforms thereof in tumor cells.

In some embodiments, the methods of regulating TMEM25 include, but are not limited to, gene editing, lentiviral overexpression, adenoviral overexpression, CRISPR/CAS9 knockout, etc.

In some embodiments, the methods include administering to a subject with cancer or a subject at high risk for cancer a drug that includes or targets TMEM25. In some embodiments, the drugs may be, but are not limited to, genetic vaccines, nucleic acid drugs, targeted drugs and nanocarrier drugs.

In some embodiments, the method includes administering to an individual in need thereof a wild-type or mutated TMEM25 protein or an isoform thereof, a polynucleotide molecule encoding the wild-type or mutated TMEM25 protein or an isoform thereof, A nucleic acid construct or vector comprising the polynucleotide molecule, a host cell comprising the polynucleotide molecule, the nucleic acid construct or vector, a wild-type or mutant TMEM25 protein or an isomer thereof, the The polynucleotide sequence, the nucleic acid construct, the vector or the pharmaceutical composition of the host cell.

In some embodiments, the drugs containing or targeting TMEM25 of the present application can be administered to individuals in need by systemic administration, such as oral administration, intravenous injection, etc.

In some embodiments, the drug containing or targeting TMEM25 of the present application can be directly administered to the tumor tissue of an individual in need through local administration, such as in situ injection.

In this application, the TMEM25 protein or its isomer includes, but is not limited to, murine TMEM25 (for example, according to the amino acid sequence shown in NCBI protein database Accession: NP_001344313) or its isomer and human TMEM25 (for example, according to NCBI Protein database Accession: the amino acid sequence shown in NP_116169) or its isomer.

Exemplarily, the wild-type human TMEM25 protein sequence can be: MALPPGPAALRHTLLLLPALLSSGWG ELEPQIDGQTWAERALRENERHAFTCRVAGGPGTPRLAWYLDGQLQEASTSRLLSVGGEAFSGGTSTFTVTAHRAQHELNCSLQDPRSGRSANASVILNVQFKPEIAQVGAKYQEAQGPGLLVVLFALVRANPPANVTWIDQDGPVTVNTSDFLVLDA QNYPWLTNHTVQLQLRSLAHNLSVVATNDVGVTSASLPAPGLLATRVEVPLLGIVVAAGLALGTLVGFSTLVACLVCRKEKKTKGPSRHPSLISSDSNNLKLNNVRLPRENMSLPSNLQLNDLTPDSRAVKPADRQMAQNNSRPELLDPEPGGLLTSQGFIRLPVLGYIYRVSSVSSDEIWL(SEQ ID NO.1)

Exemplarily, the wild mouse TMEM25 protein sequence can be: MELPLSQATLRHTLLLLPALLSSGQGEL APQIDGQTWAERALRENEHHAFTCRVAGGSATPRLAWYLDGQLQEATTSRLLSVGGDAFSGGTSTFTVTAQRSQHELNCSLQDPGSGRPANASVILNVQFKPEIAQVGAKYQEAQGPGLLVVLFALVRANPPANVTWIDQDGPVTVNASDFLVLDAQ NYPWLTNHTVQLQLRSLAHNLSVVATNDVGVTSASLPAPGLLATRIEVPLLGIVVAGGLALGTLVGFSTLVACLVCRKEKKTKGPSRRPSLISSDSNNLKLNNVRLPRENMSLPSNLQLNDLTPDLRGKATERPMAQHSSRPELLEAEPGGLLTSRGFIRLPMLGYIYRVSSVSSDEIWL(SEQ ID NO.2)

The mutated TMEM25 protein or its isomer described in this application can be understood as having at least one amino acid substitution, deletion or insertion based on the wild-type TMEM25 protein or its isomer. PCR technology can be used to perform site-directed mutation of the TMEM25 gene.

In some embodiments, the mutated TMEM25 protein or isoform thereof is an artificially engineered mutant.

In some embodiments, the mutation includes the replacement of at least one tyrosine residue with a glutamic acid residue or an aspartic acid residue. For example, Tyr (Y) in the key amino acid sequence, that is, the amino acid sequence "YIYRVSSVSSDEIWL" (SEQ ID NO. 9) at the C-terminus of the TMEM25 protein, is each replaced with Glu (E) or Asp (D).

In some embodiments, the tyrosine residue (Y) at position 13 and/or 15 from the C terminus of the mutated TMEM25 protein or its isomer is replaced with a glutamic acid residue (E) or Aspartic acid residue (D).

In some embodiments, the mutated TMEM25 protein or isomers thereof are selected from human TMEM25 proteins or isomers thereof comprising at least one of Y352E, Y352D, Y354E, and Y354D mutations; preferably, they comprise Y352E, Y352D, Y354E, Y354D, Y352,354E, Y352,354D, Y352D/Y354E or Y352E/Y354D mutations, etc.; preferably, it contains Y352E, Y354E or Y352,354E mutation; preferably, it has SEQ ID NO. 3-5 The amino acid sequence shown in any one of them.

In some embodiments, the mutated TMEM25 protein or isomers thereof are selected from the group consisting of murine TMEM25 proteins or isomers thereof comprising at least one of Y351E, Y351D, Y353E, and Y353D mutations; preferably, they comprise Y351E, Y351D, Y353E, Y353D, Y351,353E, Y351,353D, Y351D/Y353E or Y351E/Y353D mutations, etc.; Preferably, it contains Y351E, Y353E or Y351,353E mutation; Preferably, it has SEQ ID NO.6-8 The amino acid sequence shown in any one of them.

In some embodiments, the TMEM25 protein inhibits the growth of tumor cells.

In some embodiments, the TMEM25 protein inhibits clonogenesis of tumor cells.

In some embodiments, the TMEM25 protein inhibits tumor growth.

In some embodiments, the TMEM25 protein prolongs survival of tumor-bearing mice.

In some embodiments, the cancer is selected from at least one of breast cancer, colon cancer, cervical cancer, and osteosarcoma.

In one aspect, the application provides a mutant TMEM25 protein or an isomer thereof, in which the tyrosine residue at position 13 and/or position 15 from the C terminus is replaced with a glutamic acid residue or aspartate. acid residue.

In some embodiments, the mutated TMEM25 protein or isoform thereof, wherein the TMEM25 protein or isoform thereof is a human TMEM25 protein or isoform thereof.

In some embodiments, the mutated TMEM25 protein or isoform thereof contains at least one of Y352E, Y352D, Y354E, and Y354D mutations; preferably, it contains Y352E, Y352D, Y354E, Y354D, Y352,354E , Y352,354D, Y352D/Y354E or Y352E/Y354D mutations, etc.; Preferably, it contains Y352E, Y354E or Y352,354E mutation; Preferably, it has the amino acid sequence shown in any one of SEQ ID NO.3-5 .

In some embodiments, the mutated TMEM25 protein or isomers thereof are selected from the group consisting of murine TMEM25 proteins or isomers thereof containing at least one of Y351E, Y351D, Y353E, and Y353D mutations; preferably, Contains Y351E, Y351D, Y353E, Y353D, Y351,353E, Y351,353D, Y351D/Y353E or Y351E/Y353D mutations, etc.; preferably, it has the amino acid sequence shown in any one of SEQ ID NO. 6-8.

In another aspect, the application provides a polynucleotide molecule encoding a mutated TMEM25 protein of the application or an isoform thereof.

In one aspect, the present application provides a nucleic acid construct comprising a polynucleotide molecule of the present application, and a promoter operably linked to the polynucleotide molecule.

A vector comprising a polynucleotide molecule or nucleic acid construct of the present application.

In some embodiments, the vector includes, but is not limited to, a plasmid, a phagemid, a cosmid, an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC); phage Such as lambda phage or M13 phage and animal viruses. In other embodiments, animal virus species used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus, cytomegalovirus (CMV)), poxviruses , baculovirus, papillomavirus, papillomavirus (such as SV40).

Preferably, the vector is selected from plasmid vectors, lentiviral vectors or related adenoviral vectors.

In some embodiments, the adeno-associated virus (AAV) serotypes include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, etc. Preferably the serotype is AAV9.

In one aspect, the present application provides a host cell comprising at least one of the polynucleotide molecule, nucleic acid construct and vector of the present application; preferably, the host cell expresses the mutated TMEM25 protein of the present application or its isomer.

In one aspect, the present application provides a pharmaceutical composition comprising at least one of the mutated TMEM25 protein of the present application or an isomer thereof, a polynucleotide molecule, a nucleic acid construct, a vector and a host cell.

In another aspect, the application provides the mutated TMEM25 protein of the application or isoforms thereof, polynucleotide molecules encoding the same, nucleic acid constructs, vectors, host cells and pharmaceutical compositions for preparing drugs for treating cancer. Purpose; Preferably, the cancer is selected from breast cancer, colon cancer, cervical cancer and osteosarcoma.

This application also includes kits for applying the methods of this application. The kit contains one or more reagents for detecting TMEM25 levels, optionally, an expression plasmid expressing TMEM25, optionally, a host cell expressing TMEM25, optionally, a TMEM25 protein and a Its target detection reagent, optionally, the detection or treatment method adopts any of the aforementioned methods of the present invention. The kit may also include appropriate containers, instructions for use, etc. The detection of TMEM25 levels can be at the DNA, mRNA or protein level. For the detection of DNA, mRNA or protein levels, other technologies and systems implemented in the art can be used, such as, for example, DNA sequencing, which uses one or more designed primers; RT - PCR assays using one or more designed primers; immunoassays, such as enzyme-linked immunosorbent assay (ELISA); immunoblotting, for example, Western blotting or in situ hybridization and similar techniques.

The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the field shall be followed (for example, refer to "Molecular Cloning Experimental Guide" translated by J. Sambrook et al., Huang Peitang et al., third edition, Science Press) or follow the product instructions. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

According to the method of the present application, the "expressing transgenic mouse PyMT; TMEM25 ^wt/tg " is to use the transposase TMGI system to insert the TMEM25 cDNA fragment into the mouse genome.

In this application, the detection method can be a conventional molecular level detection method in the art, including but not limited to microarray, ELISA, mass spectrometry, flow cytometry, Northern blot, Southern blot, Western blot and PCR.

In this application, the functional analysis methods in the detection can be conventional in vitro detection and in vivo biological function methods in this field, including but not limited to MTT, growth curve, CCK8, colony formation, transplanted tumor experiments and spontaneous tumor formation experiments. .

Cell line:

Human breast cancer cell MDA-MB-231 (cat.HTB-26), mouse breast cancer cell 4T1 (cat.CRL-2539), human embryonic kidney cell HEK293T (cat.CRL-3216), human cervical cancer cell HeLa (cat .CCL-2), human osteosarcoma cell U-2OS (cat.HTB-96), and human colon cancer cell HCT-116 (cat.CCL-247) were purchased from ATCC.

Example 1: Exogenous expression of TMEM25 in MDA-MB-231 and 4T1 cells can reduce cell growth ability

1. Experimental materials and main reagents

Plasmids: pAX2, pMD2G, pBobi-TMEM25-Flag, pLenti-CRISPR TMEM25-gRNA are all constructed in the laboratory. The human and mouse TMEM25 cDNA used were kindly donated by Professor Han Jiahuai of Xiamen University.

After linearizing the pBOBI-CMV vector using Kpn I and Xba I, the TMEM25 fragment was inserted into the vector using the same restriction sites to obtain the pBobi-TMEM25-Flag plasmid containing the human or mouse TMEM25 gene.

The gRNA sequences targeting human and mouse TMEM25 were designed through the Zhang Feng laboratory website (https://zlab.bio/guide-design-resources). The two gRNA sequences targeting human TMEM25 are 5'-CCACGCCTTCACCTGCCGGG-3' (SEQ ID NO. 10) and 5'-TCCAGGTGACA TTGGCCGGC-3' (SEQ ID NO. 11); the two gRNA sequences targeting mouse TMEM25 The gRNA sequences are 5'-CTTGGCACACAACCTCTCGG-3' (SEQ ID NO. 12) and 5'-TCCAGGTACCAGGCTAATCG-3' (SEQ ID NO. 13). Use the annealing method to match the two corresponding gRNA primers into double-stranded fragments; then use BsmBI to linearize the pLenti-CRISPR V2 vector, and use T4 DNA ligase to connect the linearized vector and gRNA double-stranded fragments to obtain pLenti-CRISPR TMEM25-gRNA plasmid targeting human or mouse TMEM25.

Transfection reagent: PEI (cat.11668-027), purchased from Invitrogen.

Dulbecco's modified medium (DMEM, Gibco, cat. 11965), purchased from ThermoFisher.

RPMI 1640 medium (Gibco, cat. 31800022), purchased from ThermoFisher.

Agar powder (cat.A100637) was purchased from Sangon.

Cell Counting Kit-8 (CCK8, cat.HY-K0301), purchased from MCE.

0.45 μm syringe filter purchased from Millipore.

2. Experimental methods

a. Lentivirus packaging

1) The density of HEK293T cells in a 6cm cell culture dish is 80%, and two 6cm dishes are used to transfect pBobi-GFP and pBobi-TMEM25-Flag plasmids respectively.

2) PEI transfection: For the lentivirus used to overexpress TMEM25, the packaging plasmid pBobi-GFP or pBobi-TMEM25-Flag: 3 μg, the viral packaging factor pAX2: 2 μg, and pMD2G: 1 μg were co-transfected into HEK293T cells. Change the medium 6 hours after transfection.

For the lentivirus used for TMEM25 knockout, HEK293T cells were co-transfected with pLenti-CRISPR empty vector or pLenti-CRISPR TMEM25-gRNA:pAX2:pMD2G at a ratio of 3:2:1.

3) 48 hours after changing the medium, collect the culture medium supernatant and filter it through a 0.45 μm filter to obtain the lentivirus liquid.

b. Lentivirus infects target cells

The lentivirus liquids carrying different genes collected in the above experiments were used to infect human breast cancer cells MDA-MB-231 (human TMEM25) or mouse breast cancer cells 4T1 (mouse TMEM25) to obtain exogenous expression or knockout. cell.

1) Take 200 mL of lentivirus liquid, add polyethylene infection reagent polybrane (Sigma-Aldrich, 10 mg/ml) to a 6-well plate, add 300,000 cells to each well, and finally fill it up with cell culture medium until the total volume of liquid in each well is 2 ml. .

2) After 18 hours of infection, after the cells have adhered, aspirate the cell culture medium, replace with fresh culture medium and continue culturing for 24 hours.

3) When the infection efficiency of the GFP expression group is greater than 95% under fluorescence microscopy, exogenously expressed TMEM25 cells (TMEM25-Flag) are obtained. To construct knockout cells, TMEM25 knockout cells (TMEM25 ^-/- ) can be obtained after lentivirus-infected cells are continuously screened with puromycin (Sigma-Aldrich, 2mg/ml) for one week. The constructed overexpression or knockout cells will be used in subsequent experiments.

c. Cell growth curve measurement

1) MDA-MB-231 or 4T1 cells with exogenous expression of TMEM25 (TMEM25-Flag), pBobi-GFP (control) or TMEM25 knockout (TMEM25 ^-/- ), pLenti-CRISPR empty vector (TMEM25 ^+/+ ) In a 96-well plate, plate 1,000 cells per well for MDA-MB-231 and 500 cells per well for 4T1.

2) After the cells adhere, add CCK-8, 10 μl/well, react at 37°C for two hours, and read OD 450.

3) Take readings at the same time every day for 6 consecutive days. The cell growth curve was drawn, and the results are shown in Figure 1A and Figure 1B.

d. Determination of cell tumorigenic ability

1) The bottom agar configuration concentration is 1%. When used, mix it with 2×DMEM/FBS in a volume ratio of 1:1 to prepare 0.5% bottom agar. Spread into 12-well plate, 500μl per well.

2) For cell counting, 2000 cells were plated in each well of MDA-MB-231 and 1000 cells were plated in each well of 4T1.

3) The final concentration of the upper agar is 0.3%, which is obtained by mixing and diluting 1% agar with DMEM/FBS. Add the corresponding number of cells, blow evenly with a pipette, and add to the well plate in step 1), 500ml per well.

4) Place the cell well plate in a cell culture incubator at 37°C and 5% _CO2 for 7-15 days. Add fresh culture medium every 3-6 days, 200 μl per well.

5) Observe the formation of cell colonies in the agar every day. When the size of the cell clones is enough to take pictures, take pictures and record them. Observe and compare the number of cell colonies and the size of the colonies between the experimental group and the control group. The results are shown in Figure 1C and As shown in Figure 1D.

3.Experimental results

Figure 1A shows MDA-MB-231 cells overexpressing TMEM25 (TMEM25-Flag), MDA-MB-231 cells knocking out TMEM25 (TMEM25 ^-/- ), cells transfected with pBobi-GFP (control) and transfection Growth curve of cells with pLenti-CRISPR empty vector (TMEM25 ^+/+ ). Figure 1B shows 4T1 cells overexpressing TMEM25 (TMEM25-Flag), 4T1 cells knocking out TMEM25 (TMEM25 ^-/- ), cells transfected with pBobi-GFP (control) and cells transfected with pLenti-CRISPR empty vector ( Growth curve of TMEM25 ^+/+ ). As can be seen from the figure, after knocking out TMEM25 (TMEM25 ^-/- ), the growth rate of tumor cells is significantly accelerated, while after overexpressing TMEM25 (TMEM25-Flag), the growth rate of tumor cells is the slowest, indicating that excessive changes in tumor cells Expressing TMEM25 can significantly inhibit the growth rate of tumor cells.

Figure 1C shows the control group transfected with pBobi-GFP (GFP), overexpressed TMEM25 (TMEM25-Flag), knocked out TMEM25 (TMEM25 ^-/- ), and MDA- transfected with pLenti-CRISPR empty vector (TMEM25 ^+/+ ) Colonies formed by MB-231 cells on soft-agar. Figure 1D shows the control group transfected with pBobi-GFP (GFP), overexpressed TMEM25 (TMEM25-Flag), knocked out TMEM25 (TMEM25 ^-/- ), and 4T1 transfected with pLenti-CRISPR empty vector (TMEM25 ^+/+ ). Colonies formed by cells on soft-agar. It can be seen from the results that after knocking out TMEM25 (TMEM25 ^-/- ), the ability of tumor cells to form colonies in soft agar is significantly increased, while exogenous expression of TMEM25 (TMEM25-Flag) can significantly inhibit the colony forming ability of tumor cells. Colonies are marked with arrows in the figure.

The above results show that exogenous expression of TMEM25 in breast cancer cells can inhibit the cell growth rate and colony formation ability.

Example 2: Transgenic mouse PyMT; TMEM25 ^wt/tg tumor growth rate and survival time determination

1. Experimental materials and main reagents

TMEM25 transgenic heterozygous mice on FVB background, TMEM25 ^wt/tg mice, were ordered from Shanghai Southern Model Animal Company;

Breast cancer model MMTV; PyMT mice, ordered from Suzhou Saiye Biotechnology.

MMTV; PyMT male mice and TMEM25 ^wt/tg female mice were caged together, and positive mice were determined through PCR genotype identification. PyMT; TMEM25 ^wt/tg female mice were selected for experiments, and they were about 8 weeks old.

PyMT; TMEM25 ^+/- male mice on FVB background were caged with TMEM25 ^+/- female mice. Positive mice were determined by PCR genotyping, and PyMT; TMEM25 ^+/+ mice and PyMT; TMEM25 ^-/- mice were selected for testing. Experiment, about 8 weeks old.

2. Experimental methods

2.1 Starting from about 8 weeks of age, tumors were measured every two days in PyMT; TMEM25 ^wt/tg mice, PyMT; TMEM25 ^+/+ mice and PyMT; TMEM25 ^-/- mice. Tumor volume = (length × width × Width/2)mm ³ , and the survival ratio was calculated.

2.2 Take another PyMT; TMEM25 ^+/+ mice and PyMT; TMEM25 ^-/- mice about 110 days after birth, sacrifice them to obtain tumors, weigh them and use them for other tests.

3.Experimental results

Figure 2A shows the mass statistical results of tumors in TMEM25 knockout mice (PyMT; TMEM25 ^-/- ) and TMEM25 transgenic mice (PyMT; TMEM25 ^wt/tg ) compared to mice without TMEM25 knockout (PyMT; TMEM25 ^+/+ ) is the control. Figure 2B shows the growth curves of tumors in transgenic mice and knockout mice. Figure 2C shows survival statistics of transgenic mice. The results showed that in mice transgenicly expressing TMEM25, tumor mass and tumor growth rate were significantly reduced, and mouse survival was significantly prolonged.

Example 3: Effects of expressing TMEM25 adeno-associated virus on tumor growth rate and survival in breast cancer model mice

1. Experimental materials and main reagents

Cell line: human embryonic kidney cell HEK293T (cat.CRL-3216), purchased from ATCC.

The vector plasmid pAAV-CMV-EGFP and the plasmid pAAV-CMV-TMEM25-Flag were constructed in the laboratory.

After the vector plasmid pAAV-CMV was linearized with BamHI and EcoR I, the EGFP fragment linearized with the same restriction endonuclease was inserted into the vector using T4 ligase to obtain the vector plasmid pAAV-CMV-EGFP.

Using pBobi-TMEM25-Flag plasmid as a template, the TMEM25/Flag fragment with BamHI and EcoR I enzyme cutting sites was amplified by PCR, and the TMEM25/Flag fragment was digested with restriction endonucleases BamHI and EcoR I. Linearize the TMEM25/Flag fragment. After the vector plasmid pAAV-CMV-EGFP is linearized with BamH I and EcoR I, the linearized TMEM25/Flag fragment is inserted into the vector pAAV-CMV-EGFP vector using T4 ligase to obtain the plasmid pAAV. -CMV-TMEM25-Flag.

AAV virus loading plasmids AAV helper plasmid (Rep/Cap) and pHGTI-adenol were kindly donated by Professor Lin Shengcai of Xiamen University.

Transfection reagent: PEI (cat.11668-027), purchased from Invitrogen.

Dulbecco's modified medium (DMEM, Gibco, cat. 11965), purchased from ThermoFisher.

Iodixanol (Sigma-Aldrich, cat. D1556) was purchased from Sigma-Aldrich.

Polyethylene glycol 8000 (cat.A100159) was purchased from Sangon.

FVB background MMTV; PyMT female mice 8 weeks old were purchased from Suzhou Saiye Biotechnology.

2. Experimental methods

a. Adeno-associated virus (AAV) packaging and extraction

1) Prepare cells: Spread HEK293T cells into 10 15cm cell culture dishes and transfect after 24 hours. The cell density reaches 80% during transfection.

2) PEI transfection: 70m g AAV helper plasmid (Rep/Cap), 210 μg AAV vector (pAAV-CMV-EGFP or pAAV-CMV-TMEM25-Flag) and 210 mg pHGTI-adenol.

3) 72 hours after transfection, collect cells and culture medium, centrifuge at 2000 rpm for 5 minutes, and store the supernatant in a 4°C refrigerator.

4) Resuspend cells in lysis buffer.

5) Use liquid nitrogen and a 37°C water bath to freeze and thaw cells repeatedly.

6) Centrifuge at 5500 rpm and 4°C for 10 min. The virus is in the supernatant.

7) Add 5×PEG (40% PEG8000, 2.5M NaCl, autoclaved) to a final concentration of 8% PEG8000 and 0.5M NaCl. Mix well and incubate at 4°C overnight.

8) Centrifuge at 3000 rpm and 4°C for 5 minutes, discard the supernatant, and add lysis buffer to dissolve the precipitate.

9) Use iodixanol density gradient centrifugation to concentrate the virus. The Iodixanol concentrations are 17%, 25%, 40%, and 60% respectively.

10) Add 60%, 40%, 25%, 17% Iodixanol in the Backman ultracentrifuge tube from bottom to top, and finally add the virus solution.

11) Use Backman ultracentrifuge to centrifuge at 16°C and 60,000 rpm for 75 minutes.

12) Obtain viruses from 40% of the components and obtain AAV viral vectors containing pAAV-CMV-EGFP or pAAV-CMV-TMEM25-Flag respectively. Then use a 100K ultrafiltration tube to centrifuge to concentrate the virus. Finally, the viral titer was determined by RT-PCR.

b.AAV in situ injection

Using MMTV on FVB background; PyMT female mice, about 8 weeks old, the tumor volume of each mouse was measured before AAV injection and divided into two groups equally. The tumor volume at this time was the initial tumor volume.

Select multiple sites in the mouse mammary fat pad to inject AAV virus, 10ml/site, once every two weeks. The control group (expressing GFP,) and the experimental group TMEM25-Flag (expressing TMEM25) were injected with the same amount of virus.

c. Mouse tumor volume measurement and survival rate statistics

After the mice were injected with the virus, the tumor length and width were measured every two days, and the volume = (length × width × width/2) mm ³ . The total tumor volume of the mice was calculated. The results are shown in Figure 3A. At the same time, the mouse survival curve was calculated, and the results are shown in Figure 3B.

3.Experimental results

Figure 3A shows the volume change curve of tumors in breast cancer model mice after injection of adeno-associated virus. It can be seen from the figure that after the adeno-associated virus vector expressing TMEM25 was injected into the tumor tissue of breast cancer model mice, the tumor growth was significantly improved. There was no inhibition, indicating that TMEM25 has the effect of inhibiting tumor growth. Figure 3B shows the survival rate of breast cancer model mice after injection of adeno-associated virus. It can be seen that the survival period of mice is significantly prolonged after injection of TMEM25. The results of this example show that after injection of adeno-associated virus expressing TMEM25, the tumor growth rate of mice was significantly reduced, and the survival period of mice was significantly prolonged.

Example 4: Exogenous expression of TMEM25 mutants in different tumor cells reduces cell growth ability

1. Experimental methods

When the mutation site is known, the method for obtaining the gene encoding the TMEM25 mutant can be obtained by those skilled in the art based on the amino acid sequence of the mutant, which will not be described in detail here.

In this example, a HA tag gene is added to the 3' end of the human or mouse TMEM25 mutant encoding gene to obtain a TMEM25 mutant protein with a C-terminal HA tag fused (TMEM25/HA), which is used for Westernblot of TMEM25 mutant protein. detection.

The same lentiviral vector construction method and transfection method as in Example 1 were used to obtain overexpression of human TMEM25 mutants (Y352E, Y354E, Y352,354E) or mouse TMEM25 mutants (Y351E, Y353E, Y351,353E). Tumor cells, use Western blot method to detect the expression of TMEM25 wild-type or mutant protein in different tumor cells (the primary antibody is an anti-HA rat monoclonal antibody, cat#11867431001), and use the same cell growth curve as in Example 1 Determination method: Determine the growth curve of cells after exogenously expressing TMEM25 mutants in different tumor cells, in which the human or mouse TMEM25 mutants are respectively transferred into the breast cancer cell line MDA-MB-231. The results are shown in Figures 4A and 4B, and the measurement results after transfection of human TMEM25 mutants into other types of tumor cell lines are shown in Figures 4C to 4E.

2.Experimental results

Figure 4A shows the cell growth curve detected by CCK8 (left picture) and the protein expression level detected by Western blot (right picture) after expressing different human TMEM25 (hTMEM25) mutants in the MDA-MB-231 cell line. Figure 4B shows the cell growth curve detected by CCK8 (left picture) and the protein expression level detected by Western blot (right picture) after expressing different mouse TMEM25 (mTMEM25) mutants in the MDA-MB-231 cell line. Figure 4C shows the cell growth curve detected by CCK8 (left picture) and the protein expression level detected by Western blot (right picture) after expressing different human TMEM25 mutants in the Hela cell line. Figure 4D shows the cell growth curve detected by CCK8 (left picture) and the protein expression level detected by Western blot (right picture) after expressing different human TMEM25 mutants in the U-2OS cell line. Figure 4E shows the cell growth curve detected by CCK8 (left picture) and the protein expression level detected by Western blot (right picture) after expressing different human TMEM25 mutants in HCT116 cell line. In each Western blot result picture, "-" represents cells transfected with empty vector pBobi-GFP, "WT" represents cells that overexpress wild-type human TMEM25, "Y352E" represents cells that overexpress Y352E mutant human TMEM25, and "Y354E" represents Overexpressing Y354E mutant human TMEM25 cells, "2YE" represents overexpressing Y352, 354E mutant human TMEM25 cells, and "GFP" represents overexpressing GFP protein in the cells.

Experimental results show that compared with wild-type TMEM25, cells exogenously expressing TMEM25 mutants can more significantly inhibit tumor cell proliferation, especially the 2YE mutant, which has the most significant inhibitory effect; at the same time, human or mouse TMEM25 mutants All can significantly inhibit tumor cell proliferation. In addition, it can also be observed from the WB results that the TMEM25 mutant with 2YE mutation has higher stability and cellular expression.

Figure 5 shows micrographs of tumor cells after overexpression of wild-type or different mutant TMEM25 proteins, where “Vector” represents transfection of empty vector pBobi-GFP. It can be seen from the figure that compared with wild-type TMEM25, exogenous expression of TMEM25 mutants is more likely to cause tumor cell death, especially the 2YE mutant, which has the most significant effect.

Different murine TMEM25 mutants showed similar effects in murine or human tumor cells, indicating that compared with wild-type TMEM25, cells exogenously expressing murine TMEM25 mutants can also significantly inhibit tumor cell proliferation.

Although specific embodiments of the invention have been described in detail, they will be understood by those skilled in the art. According to all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are within the protection scope of the present invention.

Claims (17)

1. Wild-type or mutated TMEM25 protein or isomers thereof, polynucleotide molecules encoding said wild-type or mutated TMEM25 protein or isomers thereof, nucleic acid constructs or vectors comprising said polynucleotide molecules, A host cell comprising the polynucleotide molecule, the nucleic acid construct or vector, the wild-type or mutant TMEM25 protein or an isoform thereof, the polynucleotide sequence, the nucleic acid construct, the The pharmaceutical composition of the carrier or the host cell is used to prepare a drug for treating cancer; preferably, the cancer is selected from at least one of breast cancer, colon cancer, cervical cancer and osteosarcoma. 2. The use according to claim 1, wherein the TMEM25 protein or its isomer is human or mouse TMEM25 protein or its isomer. 3. The use according to claim 1 or 2, wherein the mutated TMEM25 protein or isomer thereof contains at least one tyrosine residue replaced with a glutamic acid residue or an aspartic acid residue. 4. The use according to any one of claims 1 to 3, wherein the wild-type TMEM25 protein has the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2. 5. The use according to claim 3, wherein the tyrosine residue at position 13 and/or position 15 from the C terminus of the mutated TMEM25 protein or its isomer is replaced with a glutamic acid residue. or aspartic acid residues. 6. The use according to claim 3 or 5, wherein the mutated TMEM25 protein or isomers thereof are selected from the group consisting of human TMEM25 proteins or isomers thereof comprising at least one of Y352E, Y352D, Y354E, Y354D mutations. body; preferably, it contains Y352E, Y354E or Y352,354E mutation; preferably, it has the amino acid sequence shown in any one of SEQ ID NO. 3-5. 7. The use according to claim 3 or 5, wherein the mutated TMEM25 protein or isomer thereof is selected from the group consisting of mouse TMEM25 protein or isomer thereof comprising at least one of Y351E, Y351D, Y353E, Y353D mutations. body; preferably, it has the amino acid sequence shown in any one of SEQ ID NO. 6-8. 8. A mutant TMEM25 protein or an isomer thereof, in which the tyrosine residue at position 13 and/or position 15 from the C terminus is replaced with a glutamic acid residue or an aspartic acid residue. 9. The mutated TMEM25 protein or isomer thereof according to claim 8, wherein the TMEM25 protein or isomer thereof is a human TMEM25 protein or isomer thereof. 10. The mutated TMEM25 protein or isoform thereof according to claim 9, which comprises at least one of Y352E, Y352D, Y354E, Y354D mutations; preferably, it comprises Y352E, Y354E or Y352,354E mutations; preferably , which has the amino acid sequence shown in any one of SEQ ID NO. 3-5. 11. The mutated TMEM25 protein or isomer thereof according to claim 8, which is selected from the group consisting of mouse TMEM25 protein or isomer thereof comprising at least one of Y351E, Y351D, Y353E, Y353D mutations; preferably, it It has the amino acid sequence shown in any one of SEQ ID NO. 6-8. 12. A polynucleotide molecule encoding the mutated TMEM25 protein of any one of claims 8-11 or an isoform thereof. 13. A nucleic acid construct comprising the polynucleotide molecule of claim 12 and a promoter operably linked to the polynucleotide molecule. 14. A vector comprising the polynucleotide molecule of claim 12 or the nucleic acid construct of claim 13; preferably, the vector is selected from plasmid vectors, lentiviral vectors or related adenovirus vectors. 15. A host cell comprising at least one of the polynucleotide molecule of claim 12, the nucleic acid construct of claim 13, and the vector of claim 14; preferably, the host cell expresses The mutated TMEM25 protein or isomer thereof according to any one of claims 8-11. 16. A pharmaceutical composition comprising the mutated TMEM25 protein or isomer thereof according to any one of claims 8-11, the polynucleotide molecule according to claim 12, and the nucleic acid according to claim 13 At least one of the construct, the vector of claim 14 and the host cell of claim 15. 17. The mutated TMEM25 protein or isomer thereof according to any one of claims 8-11, the polynucleotide molecule according to claim 12, the nucleic acid construct according to claim 13, or the nucleic acid construct according to claim 14 The use of the vector, the host cell of claim 15 and the pharmaceutical composition of claim 16 for preparing a drug for treating cancer; preferably, the cancer is selected from the group consisting of breast cancer, colon cancer, cervical cancer and osteosarcoma.