CN117085113A

CN117085113A - TMEM25 protein and application thereof in cancer treatment

Info

Publication number: CN117085113A
Application number: CN202310978628.8A
Authority: CN
Inventors: 王洪睿; 毕竞; 吴志辉; 曾涛玲
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2023-02-06
Filing date: 2023-08-04
Publication date: 2023-11-21

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 application thereof in cancer treatment

The application claims the priority of the application patent with the application number of '2023100677139' and the topic name of 'TMEM 25 protein and application' submitted by 2023, 02 and 06.

Technical Field

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

Background

Breast cancer is the most common malignancy in women worldwide. Tumor heterogeneity is thought to occur due to the progressive changes in genome and epigenetic factors in differentiation, subtype and stage of breast cancer. For the treatment of breast cancer in situ, surgery and radiotherapy have been the dominant treatments to date. For metastatic malignant breast cancer, such as chemotherapy, hormonal therapy, targeted therapy, etc., gradually appears, but it still has a higher mortality rate. At present, most of medicines for treating breast cancer belong to cytotoxic chemotherapeutics, the second is hormonal medicines, and only a few are medicines for targeting tumors and tumor microenvironments. Although cytotoxic drugs have good efficacy for the treatment of breast cancer in situ, there are some significant side effects since they target all replicating and proliferating cells, i.e. including normal and tumor cells. Like chemotherapy drugs, hormone therapy is also a systemic use, with limited efficacy against metastatic breast cancer, and prolonged administration results in resistance. Because of the high incidence of breast cancer and the limitations of current treatment regimens, it is particularly important to find new targets and treatment regimens that target breast cancer.

Gene therapy offers many new possibilities for tumor therapy compared to other anti-tumor therapy regimens. There are several targeted gene therapy regimens for breast cancer currently being evaluated in clinical trials, but no relevant gene therapy has yet been approved for breast cancer treatment. Therefore, it is more important to find new targets for gene therapy of breast cancer. In addition, protein medicines are also an important direction of the current tumor treatment, and the protein medicines have the characteristics of high activity, strong specificity, low toxicity, definite biological functions and contribution to clinical application.

Transmembrane protein 25 (TMEM 25) is one of the immunoglobulin superfamily members, belongs to a single transmembrane protein, and amino acids 42-112 of the transmembrane protein are homologous with C2 immunoglobulin domains such as Hemicetin, titin and the like. There is currently only a small amount of research on TMEM 25. For example, in neurons, TMEM25 can affect epilepsy by modulating neuronal excitation. In colorectal cancer, the expression level of TMEM25 promoter is reduced due to its hypermethylation, which makes it possible to become a tumor marker. In breast cancer, however, studies have shown that expression levels of TMEM25 are related to patient prognosis and may be related to paclitaxel resistance in breast cancer cells. However, it is currently unclear as to the expression of TMEM25 or its mutants in breast cancer and the role played in the progression of breast cancer.

Disclosure of Invention

The present inventors have unexpectedly found that TMEM25 protein, as well as TMEM25 protein or an isomer thereof containing a specific mutation, has a remarkable inhibitory effect on tumor cell growth during intensive studies on TMEM25 protein, and have completed the present application based on this.

The present application provides in a first aspect the use of a wild-type or mutant TMEM25 protein or an isomer thereof, a polynucleotide molecule encoding said wild-type or mutant TMEM25 protein or an isomer thereof, a nucleic acid construct or a vector comprising said polynucleotide molecule, a host cell comprising said polynucleotide molecule, said nucleic acid construct or vector, a pharmaceutical composition comprising said wild-type or mutant TMEM25 protein or an isomer thereof, said polynucleotide sequence, said nucleic acid construct, said vector or said host cell for the manufacture of a medicament for the treatment of cancer, or for the treatment of cancer; and a method of treating cancer comprising administering to an individual in need thereof a wild-type or mutant TMEM25 protein or an isomer thereof, a polynucleotide molecule encoding said wild-type or mutant TMEM25 protein or an isomer thereof, a nucleic acid construct or vector comprising said polynucleotide molecule, a host cell comprising said polynucleotide molecule, said nucleic acid construct or vector, a pharmaceutical composition comprising said wild-type or mutant TMEM25 protein or an isomer thereof, said polynucleotide sequence, said nucleic acid construct, said vector or said host cell.

In a second aspect the present application provides a mutated TMEM25 protein or an isomer thereof having a tyrosine residue at position 13 and/or 15 from the C-terminal end replaced by a glutamic acid residue or an aspartic acid residue.

In a third aspect the application provides a polynucleotide molecule encoding a mutated TMEM25 protein or an isomer thereof of the second aspect of the application.

In a fourth aspect, the application provides a nucleic acid construct comprising a polynucleotide molecule provided in the third aspect of the application, and a promoter operably linked to the polynucleotide molecule.

In a fifth aspect the present application provides a vector comprising a polynucleotide molecule as provided in the third aspect of the application or a nucleic acid construct as provided in the fourth aspect of the application; preferably, the vector is selected from a plasmid vector, a lentiviral vector or a related adenoviral vector.

In a sixth aspect the application provides a host cell comprising at least one of the polynucleotide molecule provided in the third aspect of the application, the nucleic acid construct provided in the fourth aspect of the application or the vector provided in the fifth aspect of the application; preferably, the vector expresses a mutated TMEM25 protein or an isomer thereof provided in the second aspect of the present application.

The seventh aspect of the present application provides the use of a mutant TMEM25 protein or isomer thereof provided in the second aspect of the present application, a polynucleotide molecule provided in the third aspect of the present application, a nucleic acid construct provided in the fourth aspect of the present application, a vector provided in the fifth aspect of the present application and a host cell provided in the sixth aspect of the present application as a medicament.

In an eighth aspect, the application provides a pharmaceutical composition comprising at least one of a mutated TMEM25 protein or an isomer thereof provided in the second aspect of the application, a polynucleotide molecule provided in the third aspect of the application, a nucleic acid construct provided in the fourth aspect of the application, a vector provided in the fifth aspect of the application and a host cell provided in the sixth aspect of the application.

The ninth aspect of the present application provides the use of a mutant TMEM25 protein or isomer thereof provided in the second aspect of the present application, a polynucleotide molecule provided in the third aspect of the present application, a nucleic acid construct provided in the fourth aspect of the present application, a vector provided in the fifth aspect of the present application, a host cell provided in the sixth aspect of the present application, a pharmaceutical composition provided in the eighth aspect of the present application for the preparation of a medicament for the treatment of cancer.

The tenth aspect of the present application provides the use of a mutant TMEM25 protein or isomer thereof provided in the second aspect of the present application, a polynucleotide molecule provided in the third aspect of the present application, a nucleic acid construct provided in the fourth aspect of the present application, a vector provided in the fifth aspect of the present application, a host cell provided in the sixth aspect of the present application, a pharmaceutical composition provided in the eighth aspect of the present application for the treatment of cancer.

In an eleventh aspect the present application provides a method of treating cancer comprising administering to a subject in need thereof an effective amount of a mutant TMEM25 protein or isomer thereof provided in the second aspect of the application, a polynucleotide molecule provided in the third aspect of the application, a nucleic acid construct provided in the fourth aspect of the application, a vector provided in the fifth aspect of the application, a host cell provided in the sixth aspect of the application or a pharmaceutical composition provided in the eighth aspect of the application.

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

Drawings

FIGS. 1A and 1B show growth curves of MDA-MB-231 and 4T1 cells over-expressing or knocking out TMEM 25.

FIGS. 1C and 1D show the ability to clone in soft agar that overexpresses or knocks out MDA-MB-231 of TMEM25 and 4T 1.

FIGS. 2A-2C show the effect of transgene high expression TMEM25 and knockout TMEM25, respectively, on tumor growth rate, tumor size and survival of mice in the context of primary breast cancer mice (MMTV; pyMT). Fig. 2A shows that in TMEM25 knockout mice, the tumor mass is significantly higher than in the control group, while in transgenic mice, the tumor mass is significantly lower than in the control group. Fig. 2B shows that knockout of TMEM25 significantly increased tumor growth, whereas transgenic high expression of TMEM25 inhibited tumor growth. Fig. 2C shows that TMEM25 knockout shortens mouse survival, whereas high expression of TMEM25 prolongs mouse survival.

FIG. 3A shows MMTV in a primary breast cancer mouse; in PyMT, after in situ injection of adeno-associated virus carrying TMEM25 gene, effect on tumor growth of mice.

FIG. 3B shows MMTV in a primary breast cancer mouse; in PyMT, effect on survival of mice after in situ injection of adeno-associated virus carrying TMEM25 gene.

FIGS. 4A to 4E show the effect on the proliferation capacity of cells and the expression of TMEM25 wild type or mutant thereof in cells after overexpression of TMEM25 wild type and mutant, respectively, in different types of tumor cell lines.

Fig. 5 shows a cell micrograph of TMEM25 mutants overexpressed in different types of tumor cell lines.

Detailed Description

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described, and it is apparent that the drawings in the description below are only one embodiment of the present application, and other embodiments may be obtained according to these drawings by those skilled in the art.

In the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present application, definitions and explanations of related terms are provided below.

It should also be understood that in some methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited, unless the context indicates otherwise.

Definition of the definition

As used herein, the terms "a" and "an" and "the" and similar referents refer to the singular and the plural, unless the context clearly dictates otherwise.

As used herein, the terms "about," "substantially" and "similar to" refer to 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 manner in which the value is measured or determined, or on the limitations of the measurement system. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

As used herein, the term "isomer" has its ordinary meaning, meaning that the same gene encodes a protein of different structure but identical origin due to alternative splicing of mRNA; in the present application, it is understood that a protein homologous to TMEM25 is encoded by a gene encoding TMEM25 protein, which is formed by variable cleavage 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 multiple strand DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The backbone of the nucleic acid may comprise sugar and phosphate groups (as commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the nucleic acid may comprise a synthetic sub-species Polymers of units such as phosphoramidates and thus may be oligodeoxynucleoside phosphoramidates (P-NH) ₂ ) Or mixed phosphoramidate-phosphodiester oligomers. In addition, double-stranded nucleic acids can be obtained from single-stranded polynucleotide products that are chemically synthesized (by synthesizing the complementary strand under appropriate conditions and annealing the strand, or synthesizing the complementary strand from scratch 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 that is modified to contain nucleic acid segments in a manner that would not otherwise exist in nature, or that is synthetic. The term nucleic acid construct is synonymous with the term "expression cassette" or "expression vector" when the nucleic acid construct contains the regulatory sequences required for expression of a coding sequence of the present 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 constructs 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 that are oriented in position and order with other essential elements so that the contained cassettes can be transcribed and translated when desired in the transfected cells. These elements are operatively connected.

One type of vector is a "plasmid", which generally refers to a circular double-stranded DNA loop that can be ligated into an additional DNA segment (foreign gene), and may also include linear double-stranded molecules, such as those obtained from amplification by Polymerase Chain Reaction (PCR) or treatment of circular plasmids with restriction enzymes. The plasmid vector comprises a vector backbone (i.e., empty vector) and an expression framework. The term "expression cassette" refers to a sequence having the potential to encode a protein. Another type of vector is a viral vector, wherein a virus-derived DNA or RNA sequence is present in the vector for packaging into a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus (AAV)). Viral vectors also include polynucleotides carried by the virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of a host cell upon introduction 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 operatively 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, and includes many cell types such as prokaryotic cells like e.g. escherichia coli or bacillus subtilis, fungal cells like e.g. yeast cells or aspergillus, insect cells like e.g. S2 drosophila cells or Sf9, or animal cells like HEK 293T cells, MDA-MB-231 cells, 4T1 cells; in the present application, the "host cell" refers in particular to a mammalian cell, more particularly a cell 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 according to the prevention of the disease or symptoms thereof, in whole or in part; and/or may be therapeutic in terms of partial or complete stabilization or cure of 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 diseases or symptoms which occur in patients who are susceptible to the diseases or symptoms but are not yet diagnosed with the disease; (b) inhibiting the symptoms of the disease, i.e., arresting its development; or (c) alleviating a symptom of the disease, i.e., causing regression of the disease or symptom.

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 present application. As used herein, the term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives a pharmaceutical composition of the application to treat, prevent, ameliorate and/or alleviate a disease or disorder described herein.

The term "individual in need thereof" refers to an individual in need of treatment or prophylaxis as determined by a researcher, veterinarian, medical doctor 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 caused by proliferation of oncogenically transformed cells. "cancer" shall include any one or more of a wide range of benign or malignant tumors, including those capable of growing and metastasizing through the human or animal body or portion thereof, e.g., via lymphatic system and/or blood flow invasion. Although the present invention is directed in particular to the diagnosis or detection of malignant and solid cancers, the term "tumor" as used herein encompasses benign and malignant tumors or solid tumors. Cancers further include, but are not limited to, carcinomas, lymphomas or sarcomas such as ovarian, colon, breast, pancreatic, lung, prostate, urinary tract, uterine, acute lymphoblastic leukemia, hodgkin's disease, small cell lung cancer, melanoma, neuroblastoma, glioma (e.g., glioblastoma), and soft tissue sarcomas, lymphomas, melanomas, sarcomas, adenocarcinomas, and the like.

In one aspect, the present application provides the use of a wild-type or mutant TMEM25 protein or an isomer thereof, a polynucleotide molecule encoding said wild-type or mutant TMEM25 protein or an isomer thereof, a nucleic acid construct or a vector comprising said polynucleotide molecule, a host cell comprising said polynucleotide molecule, said nucleic acid construct or vector, a pharmaceutical composition comprising said wild-type or mutant TMEM25 protein or an isomer thereof, said polynucleotide sequence, said nucleic acid construct, said vector or said host cell for the preparation of a medicament for the treatment of cancer; or for the treatment of cancer.

In another aspect, the application provides a method of treating cancer comprising modulating the expression level of a wild-type or mutated TMEM25 protein or an isomer thereof in a tumor cell.

In some embodiments, the methods of modulating TMEM25 include, but are not limited to, gene editing, lentiviral overexpression, adenovirus overexpression, CRISPR/CAS9 knockout, and the like.

In some embodiments, the methods comprise administering a drug comprising or targeting TMEM25 to a subject suffering from cancer or to a subject at high risk of cancer. In some embodiments, the drug may be, but is not limited to, a genetic vaccine, a nucleic acid drug, a targeting drug, and a nanocarrier drug.

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

In some embodiments, the medicament of the application comprising or targeting TMEM25 may be administered to an individual in need thereof by systemic administration, e.g., oral, intravenous, etc.

In some embodiments, the medicament of the application comprising TMEM25 or targeted thereto may be administered directly into the tumor tissue of an individual in need thereof by means of local administration, such as in situ injection, or the like.

In the present application, the TMEM25 protein or an isomer thereof includes, but is not limited to, a murine TMEM25 (for example, an amino acid sequence shown in NCBI protein database Accession: np_ 001344313) or an isomer thereof and a human TMEM25 (for example, an amino acid sequence shown in NCBI protein database Accession: np_ 116169) or an isomer thereof.

Illustratively, the wild-type humanized TMEM25 protein sequence may be: MALPPGPAALRHTLLLLPALLSSGWG ELEPQIDGQTWAERALRENERHAFTCRVAGGPGTPRLAWYLDGQLQEASTSRLLSVGGEAFSGGTSTFTVTAHRAQHELNCSLQDPRSGRSANASVILNVQFKPEIAQVGAKYQEAQGPGLLVVLFALVRANPPANVTWIDQDGPVTVNTSDFLVLDAQNYPWLTNHTVQLQLRSLAHNLSVVATNDVGVTSASLPAPGLLATRVEVPLLGIVVAAGLALGTLVGFSTLVACLVCRKEKKTKGPSRHPSLISSDSNNLKLNNVRLPRENMSLPSNLQLNDLTPDSRAVKPADRQMAQNNSRPELLDPEPGGLLTSQGFIRLPVLGYIYRVSSVSSDEIWL (SEQ ID NO. 1)

Illustratively, the wild-type murine TMEM25 protein sequence may be: MELPLSQATLRHTLLLLPALLSSGQGEL APQIDGQTWAERALRENEHHAFTCRVAGGSATPRLAWYLDGQLQEATTSRLLSVGGDAFSGGTSTFTVTAQRSQHELNCSLQDPGSGRPANASVILNVQFKPEIAQVGAKYQEAQGPGLLVVLFALVRANPPANVTWIDQDGPVTVNASDFLVLDAQNYPWLTNHTVQLQLRSLAHNLSVVATNDVGVTSASLPAPGLLATRIEVPLLGIVVAGGLALGTLVGFSTLVACLVCRKEKKTKGPSRRPSLISSDSNNLKLNNVRLPRENMSLPSNLQLNDLTPDLRGKATERPMAQHSSRPELLEAEPGGLLTSRGFIRLPMLGYIYRVSSVSSDEIWL (SEQ ID NO. 2)

The mutant TMEM25 protein or an isomer thereof according to the present application is understood to be a substitution, deletion or insertion of at least one amino acid based on the wild-type TMEM25 protein or an isomer thereof. Site-directed mutagenesis of the TMEM25 gene can be performed using PCR techniques.

In some embodiments, the mutant TMEM25 protein or isomer thereof is an engineered mutant.

In some embodiments, the mutation comprises a substitution of at least one tyrosine residue with a glutamic acid residue or an aspartic acid residue. For example, tyr (Y) in the amino acid sequence of key, i.e., amino acid sequence "YIYRVSSVSSDEIWL" (SEQ ID NO. 9) at the C-terminus of 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 mutant TMEM25 protein or isomer thereof is replaced with a glutamic acid residue (E) or an aspartic acid residue (D).

In some embodiments, the mutant TMEM25 protein or isomer thereof is selected from a human TMEM25 protein or isomer thereof comprising at least one Y352E, Y352D, Y354E, Y354D mutation; preferably, it comprises Y352E, Y352D, Y354E, Y D, Y352,354E, Y352,354D, Y352D/Y354E or Y352E/Y354D mutations or the like; preferably, it comprises a 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 mutant TMEM25 protein or isomer thereof is selected from a murine TMEM25 protein or isomer thereof comprising at least one Y351E, Y351D, Y353E, Y353D mutation; preferably, it comprises Y351E, Y351D, Y353E, Y353D, Y351,353E, Y351,353D, Y351D/Y353E or Y351E/Y353D mutation or the like; preferably, it comprises a Y351E, Y353E or Y351,353E mutation; preferably, it has the amino acid sequence shown in any one of SEQ ID NO. 6-8.

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

In some embodiments, the TMEM25 protein inhibits clonal formation of a tumor cell.

In some embodiments, the TMEM25 protein inhibits tumor growth.

In some embodiments, the TMEM25 protein extends 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 isomer thereof having a substitution of a tyrosine residue at position 13 and/or 15 from the C-terminus with a glutamic acid residue or an aspartic acid residue.

In some embodiments, the mutant TMEM25 protein or an isomer thereof, wherein the TMEM25 protein or isomer thereof is a human-derived TMEM25 protein or isomer thereof.

In some embodiments, the mutant TMEM25 protein or isomer thereof comprises at least one of the Y352E, Y352D, Y354E, Y354D mutations; preferably, it comprises Y352E, Y352D, Y354E, Y D, Y352,354E, Y352,354D, Y352D/Y354E or Y352E/Y354D mutations or the like; preferably, it comprises a 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 mutant TMEM25 protein or isomer thereof is selected from a murine TMEM25 protein or isomer thereof comprising at least one of the Y351E, Y351D, Y353E, Y353D mutations; preferably, it comprises Y351E, Y351D, Y353E, Y353D, Y351,353E, Y351,353D, Y351D/Y353E or Y351E/Y353D mutation or the like; 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 mutant TMEM25 protein of the application or an isomer thereof.

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

A vector comprising a polynucleotide molecule or nucleic acid construct of the 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); phages such as lambda phage or M13 phage, animal viruses, etc. In other embodiments, animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus, cytomegalovirus (CMV)), poxviruses, baculoviruses, papillomaviruses, papilloma virus (e.g., SV 40).

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

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

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

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

In another aspect, the present application provides the use of the mutant TMEM25 protein or isomer thereof, polynucleotide molecules encoding the same, nucleic acid constructs, vectors, host cells and pharmaceutical compositions of the application for the preparation of a medicament for the treatment of cancer; preferably, the cancer is selected from breast cancer, colon cancer, cervical cancer and osteosarcoma.

The application also includes kits for use in the methods of the application. The kit comprises one or more reagents for detecting the level of TMEM25, optionally further comprising an expression plasmid expressing TMEM25, optionally further comprising a host cell expressing TMEM25, optionally further comprising TMEM25 protein and a target detection reagent therefor, optionally the detection or treatment method is a method employing any of the preceding claims. The kit may also include suitable containers, instructions for use, and the like. Detecting TMEM25 levels can be at the DNA, mRNA, or protein level, for which detection can be performed using other techniques and systems practiced in the art, such as, for example, DNA sequencing, which uses one or more designed primers; an RT-PCR assay using primers in one or more designs; immunoassays, such as enzyme-linked immunosorbent assays (ELISA); immunoblotting, e.g., western blotting or in situ hybridization, and the like.

Embodiments of the present application will be described in detail below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present application and should not be construed as limiting the scope of the application. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, ind. Molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The method according to the application, wherein the "express transgenic mouse pyrt; TMEM25 ^wt/tg "the transposase TMGI system was used to insert the TMEM25 cDNA fragment into the mouse genome.

In the present application, the method of detection may be a method of detecting molecular level conventional in the art, including but not limited to microarray, ELISA, mass spectrometry, flow cytometry, RNA blotting, southern blotting, western blotting, and PCR.

In the present application, the method of functional analysis in the assay may be a method of in vitro assay, in vivo biological function conventional in the art, including but not limited to MTT, growth curve, CCK8, clonogenic, transplantable tumor assay, and spontaneous tumor formation assay.

Cell lines:

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

Example 1: exogenous expression of TMEM25 in MDA-MB-231 and 4T1 cells reduces cell growth capacity

1. Experimental materials and primary reagents

Plasmid: pAX2, pMD2G, pBobi-TMEM25-Flag, pLenti-CRISPR TMEM-gRNA were all laboratory constructs. TMEM25 cDNA of human and murine origin was used as a benefit of the teaching of Xiamen university Han Guhuai.

After linearizing the pBOBI-CMV vector with Kpn I and Xba I, the TMEM25 fragment was inserted into the vector using the same cleavage site to obtain pBob I-TMEM25-Flag plasmid containing the human or murine TMEM25 gene.

The gRNA sequences targeting both human and murine TMEM25 were designed via 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-derived TMEM25 are 5'-CTTGGCACACAACCTCTCGG-3' (SEQ ID No. 12) and 5'-TCCAGGTACCAGGCTAATCG-3' (SEQ ID No. 13). Matching the two corresponding gRNA primers into a double-stranded fragment by using an annealing method; the pLenti-CRISPR V2 vector was then linearized using BsmBI, and the linearized vector and the gRNA double stranded fragment were ligated together by T4 DNA ligase to obtain pLenti-CRISPR TMEM-gRNA plasmid targeting human or mouse TMEM25, respectively.

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

Dulbecco's modified medium (DMEM, gibco, cat.11965), available from ThermoFisher.

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

Agar powder (cat.A 100637), purchased from the mill.

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

A0.45 μm needle filter was purchased from Millipore.

2. Experimental method

a. Lentivirus package

1) HEK293T cells were 80% dense in 6cm cell culture dishes and two 6cm plates were used to transfect pBobi-GFP and pBobi-TMEM25-Flag plasmids, respectively.

2) PEI transfection: for lentiviruses used to overexpress TMEM25, the packaging plasmids pBobi-GFP or pBobi-TMEM25-Flag were used: 3 μg, viral packaging factor pAX2:2 μg, pMD2G: HEK293T cells were co-transfected with 1. Mu.g of the three plasmids. The liquid was changed 6h after transfection.

For lentiviruses used for TMEM25 knockdown, either pLenti-CRISPR empty vector or pLenti-CRISPR TMEM25-gRNA: pAX2 pMD2G HEK293T cells were co-transfected in a 3:2:1 ratio.

3) After 48h of liquid exchange, collecting culture medium supernatant, and filtering by a 0.45 mu m filter to obtain slow virus liquid.

b. Lentivirus infects cells of interest

And (3) infecting human breast cancer cells MDA-MB-231 (humanized TMEM 25) or mouse breast cancer cells 4T1 (murine TMEM 25) by using the lentiviral fluid carrying different genes collected in the experiment to obtain exogenous expression or knockout cells.

1) 200mL of a slow virus solution, polyethylene infectious agent polybrane (Sigma-Aldrich, 10 mg/mL) was added to the 6-well plate, 30 ten thousand cells were added per well, and finally the cells were filled with cell culture medium to a total volume of 2mL per well.

2) After the cells are attached to the wall after 18 hours of infection, the cell culture medium is sucked away, and the fresh culture medium is replaced for continuous culture for 24 hours.

3) Fluorescent microscopeWhen the infection efficiency of GFP expression group is observed to be more than 95%, the exogenous expression TMEM25 cell (TMEM 25-Flag) is obtained. For constructing knockout cells, cells infected with lentivirus are continuously screened for one week by Puromycin (Sigma-Aldrich, 2 mg/ml) to obtain TMEM25 knockout cells (TMEM 25) ^-/- ). The constructed overexpressing or knockdown cells will be used in subsequent experiments.

c. Cell growth curve assay

1) Exogenous expression of TMEM25 (TMEM 25-Flag), pBobi-GFP (control) or TMEM25 knockout (TMEM 25) ^-/- ) pLenti-CRISPR empty vector (TMEM 25) ^+/+ ) MDA-MB-231 or 4T1 cells of (A) were plated into 96-well plates, 1000 cells per well of MDA-MB-231, and 500 cells per well of 4T 1.

2) After cell attachment, CCK-8, 10. Mu.l/well, was added and reacted at 37℃for two hours, OD 450 reading.

3) Readings were taken at the same time each day for 6 consecutive days. Cell growth curves were plotted and the results are shown in fig. 1A and 1B.

d. Cell tumorigenicity assay

1) The bottom agar was prepared at a concentration of 1% and 0.5% of the bottom agar was prepared by mixing with 2 XDMEM/FBS at a volume ratio of 1:1 when used. Spread on 12-well plates, 500 μl per well.

2) Cell count, 2000 cells per well of MDA-MB-231, 1000 cells per well of 4T 1.

3) The final concentration of the upper agar was 0.3%, diluted by mixing 1% agar with DMEM/FBS, the corresponding cell number was added, the pipette was used for blow-down, and 500ml of each well was added to the well plate of step 1).

4) The cell well plate was placed at 37℃with 5% CO ₂ Is cultured in the cell culture box for 7-15 days, fresh culture medium is added every 3-6 days, and 200 mu l of culture medium is added in each hole.

5) The cell clone formation in the agar was observed daily, and when the cell clone size was sufficiently photographed, it was photographed and recorded, and the difference in the cell clone formation number and clone size between the comparative experiment group and the control group was observed, and the results are shown in fig. 1C and 1D.

3. Experimental results

FIG. 1A shows that TMEM25 overexpressing MDA-MB-231 cells (TMEM 25-Flag), TMEM25 knocked out MDA-MB-231 cells (TMEM 25) ^-/- ) Cells transfected with pBobi-GFP (control) and cells transfected with pLenti-CRISPR empty vector (TMEM 25) ^+/+ ) Is a growth curve of (a). FIG. 1B shows that TMEM25 overexpressing 4T1 cells (TMEM 25-Flag), TMEM25 knocked out 4T1 cells (TMEM 25) ^-/- ) Cells transfected with pBobi-GFP (control) and cells transfected with pLenti-CRISPR empty vector (TMEM 25) ^+/+ ) Is a growth curve of (a). As can be seen from the figure, after the TMEM25 is knocked out (TMEM 25 ^-/- ) The growth rate of the tumor cells is obviously accelerated, and after the TMEM25 is over-expressed (TMEM 25-Flag), the growth rate of the tumor cells is the slowest, which indicates that the over-expression of the TMEM25 in the tumor cells can obviously inhibit the growth rate of the tumor cells.

FIG. 1C shows control group transfected with pBobi-GFP (GFP), overexpressing TMEM25 (TMEM 25-Flag), knocking out TMEM25 (TMEM 25) ^-/- ) Transfection of pLenti-CRISPR empty vector (TMEM 25) ^+/+ ) Colonies of MDA-MB-231 cells formed on agar (soft-agar). FIG. 1D shows control group transfected with pBobi-GFP (GFP), overexpressing TMEM25 (TMEM 25-Flag), knocking out TMEM25 (TMEM 25) ^-/- ) Transfection of pLenti-CRISPR empty vector (TMEM 25) ^+/+ ) Colonies formed on soft-agar by 4T1 cells of (E). From the results, it can be seen that after the TMEM25 was knocked out (TMEM 25 ^-/- ) The ability of tumor cells to form colonies at soft agar is significantly increased, while exogenous expression of TMEM25 (TMEM 25-Flag) significantly inhibited the colony forming ability of tumor cells. Colonies are marked with arrows in the figure.

The above results show that when TMEM25 is exogenously expressed in breast cancer cells, the growth rate and colony forming ability of the cells can be suppressed.

Example 2: transgenic mouse pyrt; TMEM25 ^wt/tg Tumor growth rate and survival determination

1. Experimental materials and primary reagents

fVB-background TMEM25 transgenic heterozygous mouse TMEM25 ^wt/tg Mice ordered from Shanghai, southwest model animal company;

MMTV model of breast cancer; pyMT mice, ordered from Suzhou racing organisms.

MMTV; pyMT Male mouse and TMEM25 ^wt/tg Female mice are caged, positive mice are determined through PCR genotyping, and PyMT is selected; TMEM25 ^wt/tg Female mice were tested for about 8 weeks of week-old.

FVB background PyMT; TMEM25 ^+/ Male mouse and TMEM25 ^+/ -female mice were caged, positive mice were determined by PCR genotyping, and PyMT was selected; TMEM25 ^+/+ Mice and PyMT; TMEM25 ^-/- Mice were tested for about 8 weeks of week.

2. Experimental method

2.1 for PyMT from about 8 weeks of age; TMEM25 ^wt/tg Mice, pyMT; TMEM25 ^+/+ Mice and PyMT; TMEM25 ^-/- Mice were measured every two days for tumor volume = (length x width/2) mm ³ And counting survival proportion.

2.2 taking PyMT additionally; TMEM25 ^+/+ Mice and PyMT; TMEM25 ^-/- Mice were born for around 110 days, sacrificed to obtain tumors, weighed and used for other assays.

3. Experimental results

FIG. 2A shows TMEM25 knockout mice (PyMT; TMEM 25) ^-/- ) And TMEM25 transgenic mice (PyMT; TMEM25 ^wt/tg ) Quality statistics of the tumors in (1) mice not knocked out TMEM25 gene (PyMT; TMEM25 ^+/+ ) Is used as a control. FIG. 2B shows the growth curves of tumors in transgenic mice and knockout mice. Figure 2C shows survival statistics for transgenic mice. The results show that in mice transgenic for TMEM25, the mass of the tumor and the growth rate of the tumor are obviously reduced, and the survival time of the mice is obviously prolonged.

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

1. Experimental materials and primary reagents

Cell lines: human embryonic kidney cells HEK293T (cat. CRL-3216) were purchased from ATCC.

The vector plasmid pAAV-CMV-EGFP, plasmid pAAV-CMV-TMEM25-Flag, was constructed in the laboratory.

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

The pBobi-TMEM25-Flag plasmid is used as a template, TMEM25/Flag fragments with BamHI and EcoRI cleavage sites are obtained through PCR amplification, the TMEM25/Flag fragments are digested by using restriction enzymes BamHI and EcoRI to obtain linearized TMEM25/Flag fragments, after the vector plasmid pAAV-CMV-EGFP is linearized by BamHI and EcoRI, the linearized TMEM25/Flag fragments are accessed into the vector pAAV-CMV-EGFP vector by using T4 ligase to obtain the plasmid pAAV-CMV-TMEM25-Flag.

AAV virus-loaded plasmids AAV helper plasmid (Rep/Cap) and pHGTI-adenol were given benefit from the university of Xiamen Lin Shengcai.

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

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

Polyethylene glycol 8000 (cat.A100159), commercially available from Producer.

FVB background MMTV; pyMT female mice were purchased from Suzhou Sier for 8 weeks.

2. Experimental method

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

1) Preparing cells: HEK293T cells were plated in 10 15cm cell culture dishes and transfected 24h later with cell densities reaching 80%.

2) PEI transfection: 70m g AAV helper plasmid (Rep/Cap), 210 μg AAV vector (pAAV-CMV-EGFP or pAAV-CMV-TMEM 25-Flag), and 210m g pHGTI-adenol.

3) After 72h of transfection, cells and medium were collected and centrifuged at 2000rpm for 5min, and the supernatant was stored in a refrigerator at 4 ℃.

4) Cells were resuspended with lysate.

5) Cells were lysed by repeated freeze thawing using liquid nitrogen and a 37℃water bath.

6) Centrifugation was performed at 5500rpm at 4℃for 10min, and the virus was located in the supernatant.

7) 5 XPEG (40% PEG8000,2.5M NaCl, autoclaved) was added to a final concentration of 8% PEG8000 and 0.5M NaCl. Mix well, overnight at 4 ℃.

8) Centrifugation was performed at 3000rpm at 4℃for 5min, the supernatant was discarded, and the pellet was dissolved by adding lysate.

9) The virus was concentrated by Iodixanol density gradient centrifugation with Iodixanol concentrations of 17%,25%,40%,60%, respectively.

10 60%,40%,25%,17% iodixanol and finally virus liquid are added into a Backman ultracentrifuge tube from bottom to top.

11 Using a Backman ultracentrifuge at 16℃at 60000rpm for 75min.

12 Virus was obtained in 40% of the fractions, and AAV viral vectors containing pAAV-CMV-EGFP or pAAV-CMV-TMEM25-Flag were obtained, respectively. The virus was concentrated by centrifugation using a 100K ultrafiltration tube. Finally, the virus titer was determined by RT-PCR.

AAV in situ injection

MMTV using FVB background; the tumor volume of each mouse is measured before AAV injection in PyMT female mice with the week age of about 8 weeks, and the tumor volume is the initial tumor volume at the time of average division into two groups.

The mammary fat pad of the mouse is selected to be injected with AAV at multiple sites, 10ml/site, and once every two weeks. Control (GFP expression) was injected with the same amount of virus as the experimental TMEM25-Flag (TMEM 25 expression).

c. Tumor volume measurement and survival statistics in mice

Tumor length and width were measured every two days after virus injection in mice, volume = (length x width/2) mm ³ The total tumor volume of the mice was counted and the results are shown in fig. 3A. The survival curves of the mice were also counted and the results are shown in FIG. 3B.

3. Experimental results

Fig. 3A shows the volume change curve of tumor of breast cancer model mice after injection of adeno-associated virus, and it can be seen from the graph that tumor growth is obviously inhibited after injection of adeno-associated virus vector expressing TMEM25 in tumor tissue of breast cancer model mice, which indicates that TMEM25 has the effect of inhibiting tumor growth. Fig. 3B shows the survival rate of breast cancer model mice after injection of adeno-associated virus, and it can be seen that the survival period of mice is significantly prolonged after injection of TMEM 25. The results of this example show that after injection of adeno-associated virus expressing TMEM25, the tumor growth rate of mice is significantly reduced, while the survival time of mice is significantly prolonged.

Example 4: exogenous expression of TMEM25 mutants in different tumor cells reduces cell growth capacity

1. Experimental method

With knowledge of the mutation site, the method for obtaining the TMEM25 mutant encoding gene can be obtained by those skilled in the art based on the mutant amino acid sequence, and the present application will not be described herein.

In the embodiment, an HA tag gene is added to the 3' end of a humanized or murine TMEM25 mutant encoding gene, so that a TMEM25 mutant protein (TMEM 25/HA) with the C-terminal fused with an HA tag is obtained, and the protein is used for Western blot detection of the TMEM25 mutant protein.

Different tumor cells which were overexpressed with the human TMEM25 mutant (Y352E, Y E, Y352,354E) or the murine TMEM25 mutant (Y351E, Y353E, Y351,353E) were obtained by the same lentiviral vector construction method and transfection method as in example 1, the expression of the TMEM25 wild-type or mutant protein in the different tumor cells was detected by Western blot method (primary antibody was anti-HA rat monoclonal antibody, cat# 11867431001), and the growth curve of the cells after exogenous expression of the TMEM25 mutant in the different tumor cells was measured by the same cell growth curve measurement method as in example 1, wherein the measurement results after transfer of the human or murine TMEM25 mutant in the breast cancer cell line MDA-MB-231 cell line respectively are shown in FIGS. 4A and 4B, and the measurement results after transfer of the human TMEM25 mutant in other types of tumor cell lines are shown in FIGS. 4C to 4E.

2. Experimental results

FIG. 4A shows the CCK8 assay cell growth curve (left panel) and Western blot detection protein expression levels (right panel) after expression of different human TMEM25 (hTMEM 25) mutants in the MDA-MB-231 cell line. FIG. 4B shows the CCK8 assay cell growth curve (left panel) and Western blot detection protein expression levels (right panel) after expression of different murine TMEM25 (mTMM 25) mutants in the MDA-MB-231 cell line. FIG. 4C shows the CCK8 assay cell growth curve (left panel) and Western blot detection protein expression levels (right panel) after expression of different humanized TMEM25 mutants in Hela cell lines. FIG. 4D shows the CCK8 assay cell growth curve (left panel) and Western blot detection protein expression levels (right panel) after expression of different humanized TMEM25 mutants in the U-2OS cell line. FIG. 4E shows the CCK8 assay cell growth curve (left panel) and Western blot detection protein expression levels (right panel) after expression of different humanized TMEM25 mutants in the HCT116 cell line. In each Western blot result plot "-" represents transfected empty vector pBob i-GFP cells, "WT" represents over-expressed wild-type human TMEM25 cells, "Y352E" represents over-expressed Y352E mutant human TMEM25 cells, "Y354E" represents over-expressed Y354E mutant human TMEM25 cells, "2YE" represents over-expressed Y352,354E mutant human TMEM25 cells, and "GFP" represents over-expressed GFP protein in the cells.

Compared with the wild TMEM25, the cell of the exogenous expression TMEM25 mutant can inhibit the proliferation of tumor cells more obviously, and particularly, the inhibition effect of the 2YE mutant is most obvious; meanwhile, both the human-derived TMEM25 mutant and the murine-derived TMEM25 mutant can obviously inhibit the proliferation of tumor cells. In addition, from WB results, it was also observed that TMEM25 mutant with 2YE mutation had higher stability and cell expression level.

FIG. 5 shows a micrograph of tumor cells after overexpression of the wild-type or different mutant TMEM25 protein, where "Vector" represents the empty Vector pBobi-GFP transfected. As can be seen from the figure, exogenously expressed TMEM25 mutants are more prone to cause tumor cell death than wild-type TMEM25, especially with the 2YE mutant most pronounced.

Different murine TMEM25 mutants show similar effects in murine or human tumor cells, indicating that cells exogenously expressing murine TMEM25 mutants can also significantly inhibit tumor cell proliferation compared to wild-type TMEM 25.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention.

Claims

1. Use of a wild-type or mutant TMEM25 protein or an isomer thereof, a polynucleotide molecule encoding said wild-type or mutant TMEM25 protein or an isomer thereof, a nucleic acid construct or vector comprising said polynucleotide molecule, a host cell comprising said polynucleotide molecule, said nucleic acid construct or vector, a pharmaceutical composition comprising said wild-type or mutant TMEM25 protein or an isomer thereof, said polynucleotide sequence, said nucleic acid construct, said vector or said host cell for the preparation of a medicament for the treatment of 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 an isomer thereof is a TMEM25 protein of human or murine origin or an isomer thereof.

3. The use according to claim 1 or 2, wherein the mutated TMEM25 protein or isomer thereof contains at least one tyrosine residue substituted by 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 an amino acid sequence shown in SEQ ID No.1 or SEQ ID No. 2.

5. The use according to claim 3, wherein the mutated TMEM25 protein or isomer thereof has a substitution of tyrosine residue at position 13 and/or 15 from the C-terminal end with glutamic acid residue or aspartic acid residue.

6. The use according to claim 3 or 5, wherein the mutated TMEM25 protein or isomer thereof is selected from a human TMEM25 protein or isomer thereof comprising at least one of the Y352E, Y352D, Y354E, Y354D mutations; preferably, it comprises a 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 an isomer thereof is selected from a murine TMEM25 protein or an isomer thereof comprising at least one of the Y351E, Y351D, Y353E, Y353D mutations; preferably, it has the amino acid sequence shown in any one of SEQ ID NO. 6-8.

8. A mutated TMEM25 protein or an isomer thereof, wherein the tyrosine residue at position 13 and/or 15 from the C-terminal end is replaced with a glutamic acid residue or an aspartic acid residue.

9. The mutant TMEM25 protein or isomer thereof of claim 8, wherein said TMEM25 protein or isomer thereof is a human-derived TMEM25 protein or isomer thereof.

10. The mutant TMEM25 protein or isomer thereof of claim 9 comprising at least one of the Y352E, Y352D, Y354E, Y354D mutations; preferably, it comprises a Y352E, Y354E or Y352,354E mutation; preferably, it has the amino acid sequence shown in any one of SEQ ID NO. 3-5.

11. The mutant TMEM25 protein or isomer thereof of claim 8 selected from a murine TMEM25 protein or isomer thereof comprising at least one Y351E, Y351D, Y353E, Y353D mutation; preferably, it has the amino acid sequence shown in any one of SEQ ID NO. 6-8.

12. A polynucleotide molecule encoding the mutant TMEM25 protein or isomer thereof of any of claims 8-11.

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 a plasmid vector, a lentiviral vector or a related adenoviral vector.

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 of any of claims 8-11.

16. A pharmaceutical composition comprising at least one of the mutant TMEM25 protein or isomer thereof of any of claims 8-11, the polynucleotide molecule of claim 12, the nucleic acid construct of claim 13, the vector of claim 14, and the host cell of claim 15.

17. Use of the mutant TMEM25 protein or isomer thereof of any of claims 8-11, the polynucleotide molecule of claim 12, the nucleic acid construct of claim 13, the vector of claim 14, the host cell of claim 15 and the pharmaceutical composition of claim 16 for the manufacture of a medicament for the treatment of cancer; preferably, the cancer is selected from breast cancer, colon cancer, cervical cancer and osteosarcoma.