KR20140042577A - Use of mael for the predicting prognosis and treatment of cancer - Google Patents

Abstract

The present invention relates to a composition containing a preparation measuring the expression level of mRNA or protein of MAEL(MAELstrom) gene for predicting the prognosis after cancer treatment, a kit comprising the composition for predicting the cancer prognosis, a method for providing information for predicting the cancer prognosis using the kit, a composition containing a substance suppressing the expression or activation of the MAEL protein for treating or preventing cancer, and a method for screening cancer drugs using the MAEL gene. The composition in the present invention is capable of: predicting the cancer prognosis by measuring the expression level of polynucleotides coding the MAEL gene in the individuals got cancer treatment; and suppressing the expression of the polynucleotides coding the MAEL gene associated with the proliferation, metastasis, and survival of cancer cells, thereby being widely used for more effective cancer treatment.

Description

Use of MAEL for the Predicting Prognosis and Treatment of Cancer}

The present invention relates to the use of MAEL for predicting and treating the prognosis of cancer, and more particularly, the present invention relates to predicting the prognosis after treatment of cancer comprising an agent for measuring the expression level of mRNA of the MAEL (MAELstrom) gene or a protein thereof. Composition, a method for predicting cancer prognosis comprising the composition, a method for providing information for predicting the prognosis of cancer using the kit, for treating or preventing cancer comprising a substance that inhibits the expression or activity of a MAEL protein. The present invention relates to a method for screening a cancer therapeutic agent using the composition and the MAEL gene.

Cancer is one of the leading causes of death in Korea, and many studies have been conducted to conquer cancer, but it is an incurable disease that has not yet been conquered. In the treatment of such cancers, the most important is the early diagnosis of cancer, which shows a very high cure rate in the early diagnosis of cancer. However, since most of the symptoms of the cancer appear physiologically after the cancer has advanced to a considerable level, the situation is generally diagnosed by using a genetic marker.

Conventional treatments for such diagnosed cancers include surgery, chemotherapy, and radiation therapy. However, since there are limitations in each method, treatments that are different from these therapies have recently been studied. Actively researched.

Gene therapy refers to the treatment of inherited or acquired gene abnormalities that are difficult to treat by conventional methods by genetic engineering methods. Specifically, for the treatment and prevention of chronic diseases such as congenital or inherited genetic defects, viral diseases, cancer or cardiovascular diseases, genetic materials such as DNA and RNA are administered into the body to express therapeutic proteins As a therapeutic method for inhibiting the expression of a specific protein, it is expected to be an alternative means of existing medical treatment methods as well as overcoming incurable diseases because it can fundamentally treat diseases by analyzing the cause of diseases at a gene level. Research into applying such gene therapy to the treatment of cancer has been actively conducted, but the method of expressing the therapeutic protein by administering genetic material such as DNA and RNA in the human body has the advantage of confirming the therapeutic effect of cancer in a short time. On the other hand, since therapeutic proteins are expressed in cells of normal tissues rather than cancer tissues, and may cause unexpected side effects, genes encoding proteins that are not expressed in normal tissues but are specifically expressed only in cells of cancer tissues. Much research is being conducted on how to inhibit expression.

In order to treat cancer by the above method, the process of discovering a gene that can be expressed in cancer tissue and can be a target of treatment should be preceded. Currently, studies are being actively conducted to discover a target gene of cancer treatment worldwide. . Target genes identified from these studies include the recently identified adenine nucleotide trnaslocator 2 (ANT2) (Patent Publication 2007-101610), pituitary tumor-transforming gene 1 (PTTG1) (Patent Publication 2006-96872), KLK. Various genes are known, including kallikrein 5 (Patent No. 2009-81289) and epidermal growth factor receptor (EGFR) (Patent No. 2003-7640). Different genes are involved in various cancers. As a result, studies to discover more diverse genes and apply them to gene therapy methods continue. As such, genes that are the targets of treatment show a general decrease in expression level as the symptoms of cancer are alleviated or treated. Thus, the target genes can be used for predicting the prognosis of cancer.

In general, different genes are used for the above-mentioned diagnosis and treatment prognosis of cancer because genes involved in the early development of cancer and genes involved in cancer proliferation, metastasis and maintenance are different from each other. Since genes involved in the diagnosis of cancer are not related to drug treatment due to the nature of diagnosis, their expression level may not be related to drug treatment, whereas genes involved in cancer proliferation, metastasis, maintenance, etc. Since levels correlate closely with drug therapy, the genes used to diagnose cancer and the genes used to predict prognosis are mostly different.

Under these backgrounds, the present inventors conducted various studies to find genes that can be used to predict the prognosis of new cancers. As a result, MAEL (MAELstrom) can be used to predict the prognosis of cancer treatment. Completed.

One object of the present invention is to provide a composition for predicting post-treatment of cancer, comprising an agent for measuring the level of mRNA or protein derived from a polynucleotide encoding MAEL (MAELstrom).

Another object of the present invention to provide a kit for predicting cancer prognosis comprising the composition.

Another object of the present invention is to provide a method for providing information for predicting the prognosis of cancer using the kit.

Still another object of the present invention is to provide a composition for treating or preventing cancer, which comprises a substance that inhibits the expression or activity of MAEL protein.

Still another object of the present invention is to provide a method for screening a cancer therapeutic agent using the MAEL gene.

In order to achieve the above object, in one aspect the present invention provides a composition for predicting the prognosis of cancer, comprising an agent for measuring the level of mRNA or protein derived from a polynucleotide encoding MAEL (MAELstrom). do.

The term "MAEL (MAELstrom) gene" of the present invention encodes a MAEL protein (GenBank Accession No. AAH34310.1), which is one of cancer / testis associated genes (CT genes) expressed in the gonads of the testes. The intracellular expression of the MAEL protein is located in the nuage structure, which is a high electron density structure around the nucleus, and is a pi-nuclear protein complex, pi-, which maintains genetic stability and plays an important role in sperm development. It is known to be associated with RNA complexes. In mammals, in particular, MAEL proteins are known to be essential for inactivation and spermatogenesis of retrotransposon in the male gonad. However, it has never been reported that the expression level of the gene is used for predicting prognosis after cancer treatment, which was first identified by the present inventors. The MAEL, which plays an important role in the formation and maintenance of cancer cells for the purposes of the present invention, can be used for predicting the prognosis after treatment of cancer or a target for cancer treatment, but is not particularly limited thereto.

As used herein, the term "cancer marker" refers to a cancer diagnostic marker or a substance capable of predicting a cancer prognosis after treating a cancer cell or an individual having a cancer disease from a normal cell or a normal individual. Predicting cancer prognosis markers. For the purposes of the present invention, the cancer marker may be a marker for predicting cancer prognosis, which may be a polypeptide, protein or nucleic acid (eg, mRNA) showing an increase or decrease in a cell or an individual after treating the cancer as compared to the previous treatment of the cancer. Etc.), lipids, glycolipids, glycoproteins or organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like, and preferably the level of the polypeptide or polynucleotide encoding the same in cells or individuals after cancer treatment. This may be a reduced MAEL, but is not particularly limited thereto.

For the purposes of the present invention, the composition for measuring the expression level of the MAEL gene can be used not only for predicting the prognosis of cancer, but also specifically overexpressed in cancer cells, so that the composition comprising an agent for measuring the expression level of the gene is It can also be used to diagnose cancer. At this time, the type of cancer that can predict the prognosis by the composition is not particularly limited, but may be cervical cancer, breast cancer, liver cancer and the like.

The term "diagnosis" of the present invention refers to a process or method for identifying the presence or characteristic of a disease associated with expression of the gene by measuring the presence or absence of the MAEL polypeptide of the present invention and the polynucleotide encoding the same in a biological sample or tissue sample. Means.

As used herein, the term "mRNA expression level measurement" refers to a process or method for confirming the presence and expression level of mRNA of a cancer marker gene in a biological sample to diagnose cancer, and generally, a method for measuring the amount of mRNA. It can be performed by. Method for measuring the amount of the mRNA is not particularly limited, but preferably RT-PCR, Competitive RT-PCR (Real-time RT-PCR), RNase protection assay ( RPA, RNase protection assay, Northern blotting, DNA chip analysis, and the like.

The term "protein expression level measurement" of the present invention is a process of confirming the presence and expression level of a protein expressed in a cancer marker gene in a biological sample in order to predict the prognosis after diagnosis or treatment of cancer, the protein of the gene The amount of the protein is determined by using an antibody that specifically binds to. Analysis methods for this include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, and rocket immunoelectrophoresis. , Tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like, but are not limited thereto. Preferably, the agent for measuring mRNA level is a primer pair, probe, or anti-sense nucleotide to a polynucleotide or fragment thereof encoding the MAEL of the present invention, the poly of the present invention Nucleotide sequences allow those skilled in the art to easily design primers, probes, or antisense nucleotide sequences.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3'-hydroxyl group, which can form complementary templates and base pairs, By a short nucleic acid sequence that serves as a starting point for. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In the present invention, PCR can be amplified using sense and antisense primers of polynucleotides encoding MAEL to diagnose cancer through the generation of desired products. The PCR conditions, the lengths of the sense and antisense primers can be suitably modified on the basis of those known in the art.

As used herein, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, which is short, from several bases to several hundred bases, capable of specific binding with mRAN, and is labeled. The presence or absence of a specific mRNA can be confirmed. Probes may be prepared in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, and the like. In the present invention, hybridization is carried out using a probe complementary to the polynucleotide encoding the MAEL of the present invention, and whether or not the hybridization can be diagnosed. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

The primer or probe may be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphotriester, phosph Modifications to poroamidates, carbamates, etc.) or charged linkers (eg, phosphorothioates, phosphorodithioates, etc.).

The agent for measuring the level of a MAEL protein or MAEL polypeptide of the invention is preferably an antibody.

As used herein, the term "antibody" refers to a specific protein molecule directed to an antigenic site as is known in the art. For the purposes of the present invention, an antibody means an antibody that specifically binds to a MAEL polypeptide, which is a marker of the present invention, which is encoded by the marker gene by cloning each gene into an expression vector according to a conventional method. The resulting protein can be obtained and prepared by conventional methods from the obtained protein. Also included are partial peptides that can be made from the protein, and the partial peptides of the invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited and a part thereof is included in the antibody of the present invention and all immunoglobulin antibodies are included as long as they are polyclonal antibodies, monoclonal antibodies or antigen-binding agents. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies. Such antibodies against MAEL proteins of the invention can be any antibody that can be prepared by methods known in the art. For example, the antibodies used in the detection of cancer diagnostic markers of the present invention may include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. The functional fragment of the antibody molecule refers to a fragment having at least antigen binding function, and may be Fab, F (ab '), F (ab') 2, Fv, and the like, but is not particularly limited thereto.

According to one embodiment of the present invention, MAEL is expressed in somatic cancer cells (FIGS. 3A and 3C), overexpressed in cancer tissues and various cancer cells (FIGS. 3B, 4A and 4C), and can be used for prognostic prediction after liver cancer treatment. (FIG. 5), Myc / Ras was confirmed to play an essential role in the transformation into cancer cells (FIG. 9).

In addition, when MAEL was not expressed, cancer cell survival signal pathways were inactivated, transcription of reverse potential factors was induced (FIG. 7A), and natural DNA damage and senescence were induced in cancer cells (FIGS. 8 and 9).

As another aspect, the present invention provides a kit for predicting cancer prognosis comprising the composition.

The kit of the present invention can be used to predict the prognosis of the onset cancer by identifying the expression level of the MAEL polypeptide or polynucleotide encoding the marker for predicting cancer prognosis. The kit of the present invention may be a primer, a probe, or an antibody that recognizes a marker, or a fragment thereof that retains an antigen-binding ability, as well as a fragment for maintaining a marker for predicting a cancer prognosis. And other other components, compositions or devices. For example, a diagnostic kit for quantitative detection of a polynucleotide or gene of the present invention may comprise one or more oligonucleotides that specifically bind to a polynucleotide encoding a MAEL polypeptide, wherein the polynucleotide sequence of the MAEL or part thereof RT-PCR kits may include primers capable of binding to sequences, reverse transcriptase, Taq polymerase, PCR primers, dNTPs, and the like.

Meanwhile, the kit for predicting cancer prognosis for measuring the level of the MAEL protein of the present invention may be an ELISA kit or a protein chip kit including an antibody that specifically binds to the MAEL protein of the present invention. As described above, protein expression using an antibody is measured by forming an antigen-antibody complex between the MAEL protein and its antibody, and can be detected quantitatively by measuring the amount of formation of the complex by various methods.

As used herein, the term “antigen-antibody complex” means a combination of a MAEL protein, which is a marker for predicting cancer prognosis provided in the present invention, and an antibody specific thereto, and the amount of antigen-antibody complex formed is a detection label. Quantitative measurement is possible through the magnitude of the signal. For example, by comparing the amount of antigen-antibody complex formation before and after treatment in individuals with cancer, a significant change in the expression level of the MAEL protein can be determined to predict the prognosis after treatment in a particular individual. Treatment of a sample of an individual treated with an antibody against the MAEL protein of the present invention results in the formation of an antigen-antibody complex between the MAEL protein and the antibody, which is described above for ELISA, RIA, sandwich assay, western blot, and radioimmunity. The amount can be determined by a kit including diffusion method, oukteroni, immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip and immunoblot method. In addition, by comparing the analysis results with the quantitative results of the individual before cancer treatment, it is possible to predict the cancer prognosis according to the expression change of the MAEL protein of the present invention.

In another aspect, the present invention provides a method of providing information for predicting the prognosis of a cancer using the kit.

The method of providing information for predicting the prognosis of a cancer of the present invention comprises the steps of: (i) measuring the level of mRNA of the MAEL gene or protein encoded by the gene from a biological sample isolated from a subject undergoing treatment of the cancer; And (ii) determining that the cancer has been treated or improved when the level of mRNA of the measured gene or the protein encoded by the gene decreases than that of the sample prior to cancer treatment. In this case, the method for measuring the mRNA level is not particularly limited, but reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time transcriptase polymerase reaction (real time quantitative) RT-PCR), RNase protection method, Northern blotting, DNA chip technology, and the like. In addition, the method for measuring the expression level of the protein is not particularly limited, Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoimmunififfusion (radial immunodiffusion) , Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence immunofluorescence, immunochromatography, FACS analysis, protein chip technology, and the like.

The term "individual" of the present invention refers to a living organism in which a cancer is developed and MAEL is expressed in a cell or tissue, but may be a mammal, but is not particularly limited thereto.

As used herein, the term "biological sample" refers to a sample capable of detecting a polynucleotide or protein expression level encoding a MAEL, for example, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine. Samples such as, but are not particularly limited thereto.

As another aspect, the present invention provides a composition for treating or preventing cancer, comprising a substance that inhibits the expression or activity of MAEL protein.

The material for inhibiting the expression of the MAEL protein is not particularly limited, but may preferably be siRNA, shRNA, aptamer, antisense oligonucleotide, etc. for the MAEL gene.

As used herein, the term “small interfering RNA” refers to a small nucleic acid molecule of about 20 nucleotides in size that can mediate RNA interference or gene silencing, and when siRNA is introduced into a cell, dicer ( dicer) degrades the gene that is recognized by the protein and encodes the MAEL polypeptide, thereby inhibiting the expression of the gene. Such siRNAs can be synthesized directly by chemically known siRNAs (Sui G et al., (2002) Proc Natl Acad Sci USA 99: 5515-5520), and synthesis of siRNAs using transcription in a laboratory environment (Brummelkamp TR et al., (2002) Science 296: 550-553) and the like, but is not particularly limited thereto.

As used herein, the term "shRNA (short hairpin RNA)" refers to a short hairpin RNA in which the sense and antisense sequences of an siRNA target sequence are positioned between loops of 5-9 bases. . The shRNA is to overcome shortcomings such as high cost of biosynthesis of siRNA and short-term maintenance of RNA interference effect due to low cell transfection efficiency, using adenovirus, lentivirus and plasmid expression vector system from a promoter of RNA polymerase III. This shRNA can be introduced into a cell and expressed, and such shRNA can be converted into an siRNA having an accurate structure by siRNA processing enzyme (Dicer or Rnase III) present in the cell and used to induce silencing of a gene of interest.

As used herein, the term "aptamer" refers to a small RNA fragment, and refers to an oligomer molecule having a size of about 20 to 60 nt, and may have various three-dimensional structures according to sequences, and It can have a high affinity and can be effectively inhibited by reacting with a target sequence of interest.

The term "antisense" of the present invention, also referred to as an antisense oligomer, is a sequence of nucleotide bases that can hybridize with a target sequence in RNA by Watson-Crick base pairing to form mRNA and RNA: oligomeric heteroduplex within the target sequence and An oligomer having a backbone between subunits. The antisense can have exact sequence complementarity or approximate complementarity to the target sequence, block or inhibit translation of the mRNA, and alter the processing of the mRNA to produce splice variants of the mRNA. Thus, the antisense of the invention may preferably be an antisense oligomer complementary to the polynucleotide encoding the MAEL polypeptide. The antisense can be used in a manner to prevent or inhibit the expression of carcinogenic genes by administering to the subject in a conventional manner. For example, a method of mixing antisense oligodioxynucleotides with poly-L-lysine derivatives by electrostatic attraction and administering the mixture to an individual's vein (JS kim et al., J controlled Release 53, 175-182). , 1998), but is not particularly limited thereto.

In addition, the substance that inhibits the activity of the MAEL protein may be an antibody or antigen-binding fragment thereof that inhibits the activity of the MAEL protein. At this time, the antibody is not particularly limited, but may be any antibody that can specifically bind to the MAEL protein, preferably monoclonal antibodies, chimeric antibodies, humanized antibodies, Not only can it be a human antibody, but it can also be a functional fragment of the said antibody, and all include. The antibody is also a functional fragment of an antibody molecule, as well as a complete form having the full length of two heavy chains and two light chains, as long as it has the characteristic of binding specifically to recognize the MAEL protein. It includes. The functional fragment of the molecule of the antibody refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

According to one embodiment of the present invention, when MAEL is not expressed, cancer cell survival signal pathways are inactivated, transcription of reverse potential factors is induced (FIG. 7A), and natural DNA damage and senescence are induced in cancer cells. (Figures 8 and 9).

In addition, the composition provided in the present invention further includes a known therapeutic agent that exhibits cancer therapeutic activity, in addition to an oligonucleotide that inhibits the expression of a polynucleotide encoding a MAEL used as an active ingredient or an antibody that inhibits the activity of a MAEL polypeptide. can do. At this time, the known therapeutic agent is not particularly limited thereto, but may be preferably radionuclides, drugs, lymphokines, toxins, bispecific antibodies, and the like, more preferably ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, etoposide, teniposide, adriamycin, daunomycin, carminomycin, ami Nofterin, dactinomycin, mitomycin, cis-platinum and cis-platinum homologues, bleomycins, esperamicins, 5-fluorouracil, melphalan, nitrogen mustard, and the like.

Meanwhile, the composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent depending on the mode of administration. Specifically, it is possible to use a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and any of the above components, And other additives conventionally used, such as buffers. In addition, depending on the purpose of administration, diluents, dispersants, surfactants, binders, and lubricants can be added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, A target organ or tissue specific antibody or other ligand can be used in combination with the carrier. The carrier, excipient, or additive may be any conventional formulation, and the carrier, excipient, or additive is not limited by the above examples.

The composition or the mixture may be appropriately administered to a subject according to the conventional method, route of administration, and dose used in the art depending on the purpose or necessity. Examples of the administration route include oral, non-oral, subcutaneous, Intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intraperitoneally, intramuscularly, intraperitoneally, Non-oral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Appropriate dosages and times of administration may be selected according to methods known in the art, and the amount and the number of administrations of the composition comprising the antisense oligonucleotide, siRNA or shRNA of the present invention to be actually administered will depend on the symptom The route of administration, sex, state of health, diet, age and weight of the individual, and severity of the disease.

As another aspect, the present invention provides a method for screening a cancer therapeutic agent using the MAEL gene.

The method for screening a cancer therapeutic agent using the MAEL gene of the present invention comprises the steps of: (i) measuring the level of mRNA of the MAEL gene or a protein encoded by a biological sample isolated from an individual suspected of developing cancer; And (ii) determining cancer if the level of mRNA of the measured gene or the protein encoded by the gene is increased than that of the normal control sample. At this time, the method for measuring the mRNA level and the protein expression level, as described above, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting , DNA chip method, Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography , FACS method or protein chip method, but is not particularly limited thereto.

In the cancer therapeutic screening method of the present invention, when the expression of MAEL is increased by treatment of the candidate substance, the candidate substance may be determined as a substance promoting cancer disease. In addition, when MAEL expression is decreased by the candidate, it may be determined that the treated candidate may be used as a cancer therapeutic agent. In this screening method, the activity measurement can be easily determined according to the MAEL expression level.

The composition of the present invention can predict the prognosis of cancer by measuring the expression level of a polynucleotide encoding a MAEL in an individual who has undergone cancer treatment, and is capable of predicting the prognosis of cancer and is responsible for the proliferation, metastasis and survival of cancer cells. Since the expression can be suppressed, it may be widely used for the treatment of more effective cancer.

1 shows that MAEL isoforms are expressed in various somatic cancer cell lines. (A): It is schematic which shows the structure of MAEL isoform protein. (B): Western blot analysis showing expression of MAEL from human cancer cell lines. (C): siRNA and plasmid were simultaneously introduced into the HeLa cell line, and total protein was extracted 72 hours later. (D): MAEL isoform 3 was identified in HeLa cells and MDA-MB231 cells using RT-PCR analysis.
2 shows that MAEL isoforms are expressed at high levels in proliferating cells. (A): Intrinsic expression of MAEL mRNA in various cell lines was detected by RT-PCR. Various cell lines synthesized cDNA, and cDNA was synthesized using reverse transcription PCR. RT-PCR of GAPDH RNA was used as a control. (B): Western blot analysis of MAEL expression from various cancer cell lines (C): Western blot analysis using tissues derived from HCC showed pronounced expression of MAEL in human tissues.
3 shows that MAEL is essential for the survival of various cancer cells. (A) HeLa, MDA-MB-231, 293T, WI38, IMR90 and HUVEC cells are shown. The cells were transduced with siCon and siMAEL for 72 hours and observed under an optical microscope. (B) Cell numbers of HeLa, MDA-MB-231, 293T, WI38, IMR90 and HUVEC cells are shown. After 3 days after the introduction of siRNA, the cells were counted by the trypan blue exclusion method. (c) Cells were transduced with siCon and siMAEL and analyzed by Western blot.
4 shows that MAEL depletion reduces cancer cell survival signaling and induces apoptosis and senescence. (A) Western blot analysis. Cell extracts were obtained from HeLa cells and WI38 cells into which siRNA was introduced. Cell lysates were obtained and Western blot analysis detected the levels of MAEL, pERK, EGFR, pAKT and β-actin. (B) The present inventors used HeLa cell line. The cells were separated into single cells, washed with PBS, and stained according to the manufacturer's protocol using a FITC Annexin V apoptosis detection kit (BD). (C) Western blot analysis. SiRNA and plasmid were simultaneously introduced into HeLa cells.
5 is a graph showing the correlation between the expression level of MAEL and the prognosis after liver cancer treatment.
6 shows that MAEL is essential for genetic integrity in cancer cells. (A) HeLa, MDA-MB-231, 293T, WI38, IMR90 and HS68 cells were introduced with siCon or siMAEL for 72 hours (D3). After the cells were fixed and stained with PI, the amount of DNA was analyzed. (B) siRNAs were introduced into HeLa cells for 24, 36, 48 and 60 hours. Expression of MAEL, pChk1 (ser345), PARP, pH2AX and β-actin was analyzed by Western blot analysis. (C) 3 days after introduction of MAEL or control siRNA into HeLa cells, gamma-H2AX and 53BP1 were introduced and immunostained. Nuclei were visualized using DAPI (4 ', 6'-diamidino-2-phenylindole) staining.
7 shows that depletion of MAEL induces aging. (A) Aging-related β-galactosidase (SA-β-gal) activity assay results. SiCon and siMAEL were introduced into MRC5 cells. Cells were fixed and SA-β-gal stained (left). Stained cells exhibiting SA-b-gal activity were counted and expressed as percentage of total bird catch (right). (B) siRNA was introduced into MRC5 cells. Expression of MAEL and p21 was analyzed by Western blot analysis.
8 shows that inhibition of ATM / ATR signaling inhibits apoptosis mediated by MAEL depletion. (A) Proliferation was determined by crystal violet analysis. SiMAEL and siATM / siATR were introduced into HeLa cells and MDA-MB-231 cells. The cells were observed for 10 days and colonies stained with crystal violet. (B) FACS analysis results. SiRNA was introduced into HeLa cells for 72 hours (D3). After fixing the cells and staining with PI, the amount of DNA was analyzed. (C) Western blot analysis. SiLa, siMAEL and siATM were introduced into HeLa cells.
9 shows that MAEL is essential for Myc / Ras induced transformation. (A) Colony transformation results of MEF transformants. After 14 days of inoculation of cells (8 × 10 ⁵ ), colonies were stained with crystal violet and photographed. (B) Colony transformation assay results. Two weeks later colony counts were counted. Experiments were performed in duplicate.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: materials and methods

Example  1-1: Cell Culture

Human cervical cancer HeLa cell line, human embryonic kidney cell 293T cell line, human fibropod cell HT1080 cell line, lung fibroblast WI38 cell line, human embryonic fibroblast IMR90 cell line and human foreskin fibroblast HS68 Cell lines were cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin. In addition, human breast cancer cells MDAMB-231 cell line was cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin / streptomycin. Each of the cells was incubated at 37 ℃ and 5% C0 ₂ culture conditions.

Example  1-2: with plasmid siRNA Transduction of

Myc expression vectors for MAEL were constructed by cloning of corresponding PCR products.

First, the MAEL sequence was amplified using pfu DNA polymerase, forward primer 5'-ATGCCGAACCGTAAGGCCAGCCG-3 '(SEQ ID NO: 1) and reverse primer 5'-AGGGAAGTTGGGCCTGTTACT-3' (SEQ ID NO: 2). The fragment was cloned into the pIRES-puro-Myc vector using BamHI and NotI loci.

To delete the MAEL, shRNA was expressed in pSuper-puro. The pSuper-puro-MAEL plasmid was constructed by inserting sense and antisense mouse sequences using loop sequences between the BglII and HindIII multicloning loci. The insert was constructed with the following two annealing primers.

5'-gatcccccagcaatagtgtgacacccaattcaagagattgggtgtcacactattgctgtttttggaaa-3 '(SEQ ID NO: 3)

5'-agcttttccaaaacagcaatagtgtgacacccaatctcttgaattgggtgtcacactattgctgggg-3 '(SEQ ID NO: 4)

MAEL siRNA (1: 5'-CCAGCCGGAATGCTTACTA-3 '(SEQ ID NO: 5), 2: 5'-GGAACTGGCCACCTATCTA-3') using lipofectamine RNAiMAX (13778-150, Invitrogen) to achieve a final concentration of 20 nM in cells. SEQ ID NO: 6) 3: 5'-CAGCAATAGTGTGACACCCAA-3 '(SEQ ID NO: 7)) and a negative control (siCon) were introduced.

Example  1-3: Immunofluorescence analysis

Cells were seeded on chamber slides containing DMEM medium containing 10% FBS and 1% penicillin / streptomycin. The cells were fixed with 100% methanol in ice and 4 ° C. freezer and washed three times with cold PBS. Then, the antibody was treated with a blocking solution (1% BSA in PBS) for 1 hour at room temperature, and the anti-phospho-H2AX (Ser139, millipore, # 05-636) antibody was treated with the blocking solution for 2 hours. And then washed three times with PBS. The reaction was treated and observed with a secondary antibody (Invitrogen) bound to Alexa Fluor 488 or Alexa Fluor 594, wherein the nuclei were stained with a contained mounting solution (DAPI), and the stained cells were confocal laser scanning microscope. Was observed.

Example  1-4: Flow cytometry

Cells were harvested and suspended in 300 μl PBS. The cells were fixed in cold 100% ethanol at 4 ° C. for one day. The sample was washed twice with PBS. The cells were stained with propidium iodide (40 μl / ml) and RNase (50 μg / ml) for 15-20 minutes. After staining, cells were analyzed by FACSCalibour (Bectorn Dickeinson).

Example  1-5: Quantitative Reverse transcription - PCR  reaction

RNA was secured for the synthesis of cDNA to be used in the reaction. RNA of the cancer cell line was obtained using the RNeasy mini kit (QIAGEN, # 74106) according to the manufacturer's method. The concentration and amount of total RNA were calculated by measuring absorbance at 260/280 nm using a Nano Drop spectrophotometer & Bioanalyzer (Agilent 2100 Bioanalyzer). Reverse transcription was performed using 2 μg total RNA and iScript reverse transcriptase (Bio-Rad, # 170-8890). PCR was used for each primer, template cDNA and Maxime primer premix (intron). For proper amplification, 25 cycles of GAPDH and 31 cycles of MAEL were performed. CDNA was synthesized according to the manufacturer's protocol. The sequences of the MAEL and GAPDH primers used are shown in Table 1.

Example  1-6: Real time Reverse transcription - PCR  reaction

A total of 100 HBV-positive hepatocellular carcinoma tissues were randomly selected from the hepatocellular carcinoma tissues secured by the Atomic Energy Medical Center. Tissues were ground using a homogenizer on liquid nitrogen. RNA was obtained from the RNeasy mini kit (QIAGEN, # 74106) according to the manufacturer's method. The concentration and amount of total RNA were calculated by measuring absorbance at 260/280 nm using a Nano Drop spectrophotometer & Bioanalyzer (Agilent 2100 Bioanalyzer). Reverse transcription was performed using 2 μg total RNA and iScript reverse transcriptase (Bio-Rad, # 170-8890), and cDNA was synthesized according to the manufacturer's protocol. Quantitative PCR analysis was performed using the SYBR green method (Bio-Rad, iQ SYBR Green supermix # 170-8880). The sequences of the MAEL and 18S primers used are shown in Table 1. These reactions were performed three times. Relative expression levels of MAEL target genes were normalized to the mean value of 18S rRNA as a reference gene. We analyzed using the comparative threshold cycle (2-ΔΔC (t)) method.

PCR primer designation Sequence (5 '-> 3') SEQ ID NO: qRT-PCR
LINE1-5'UTR-s
LINE1-5'UTR-as
LINE1-ORF1-s
LINE1-ORF1-as
LINE1-ORF2-s
LINE1-ORF2-as
HER-V-LTR-s
HER-V-LTR-as
HER-V-ORF1-s
HER-V-ORF1-as
HER-V-ORF2-s
HER-V-ORF2-as
AAGGGGTGACGGTCGCACCTGGAA
TGCTGTGCTAGCAATCAGCGAGA
TCCTCGAGAAGAGCAACTCCA
GGGTTTCTGCCGAGAGATCC
ATGGCCATACTGCCCAAGGT
TGGCTTAGGATTGACTTGGCA
CAGATGCTTGAAGGCAGCAT
ACGTTGGACAATACCTGGCT
TGGGCAACCATTGTCGGGAAAC
GGCTTATTCCCTGAAACACTTGGGA
GGCTTATTCCCTGAAACACTTGGGA
TCATCAAGGCTGCAAGCAGCATAC
8
9
10
11
12
13
14
15
16
17
18
19 RT-PCR
MAEL-s
MAEL-as
GAPDH-s
GAPDH-as
3'UTRMAEL-s
3'UTRMAEL-as
18S-s
18S-as
TGGCCACTCTCTTTGGAATC
GCATTTCCAATTCTTCCAGC
GTCAGTGGTGGACCTGACCT
TGATGTAGCCAAATTCGTTG
cagtaacaggcccaacttcc
ggaagcagaacaatccctcaa
AAACGGCTACCACATCCAAG
CGCTCCCAAGATCCAACTAC
20
21
22
23
24
25
26
27 si-RNA
si-MAEL # 1
si-MAEL # 2
si-MAEL # 3
si-MAEL # 4
si-MAEL # 5
si-m-MAEL # 1
si-m-MAEL # 2
si-m-MAEL # 3
CCAGCCGGAATGCTTACTA
GGAACTGGCCACCTATCTA
CAGCAATAGTGTGACACCCAA
TGAACGTGGGCATAACCAA
GATAGAACCAGAGTCAACT
GCTTCCTCCCTTGTGAAAT
GGAACTGGCCACCTATTTA
CAGCAACAGTGTGACACCCAA
28
29
30
31
32
33
34
35 Sh-RNA
sh-m-MAEL # 1
sh-m-MAEL # 2
sh-m-MAEL # 3
CCAGCCGCAATGCCTACTA
GGAACTGGCCACCTATTTA
CAGCAACAGTGTGACACCCAA
36
37
38

Example  1-7: Reverse transcription - PCR  reaction

Total RNA was isolated from cells using the RNeasy Mini kit (QIAGEN, # 74106) according to the manufacturer's protocol. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad, # 170-8890). Reverse transcription-PCR reactions were performed using Maxime PCR PreMix (i-StarTaq) (intron, # 25167). Oligonucleotide primer sequences are as follows:

MAEL forward: 5'-TGGCCACTCTCTTTGGAATC-3 '(SEQ ID NO: 20)

MAEL reverse: 5'-GCATTTCCAATTCTTCCAGC-3 '(SEQ ID NO: 21)

GAPDH forward: 5'-GTCAGTGGTGGACCTGACCT-3 '(SEQ ID NO: 22)

GAPDH reverse: 5'-TGATGTAGCCAAATTCGTTG-3 '(SEQ ID NO: 23)

Real time PCR reaction was performed using Biorad iQ Supermic (# 170-8860). The oligonucleotide primer sequence corresponds to 3'UTR of MAEL and is as follows:

3'UTRMAEL forward: 5'-CAGTAACAGGCCCAACTTCC-3 '(SEQ ID NO: 24)

3'UTRMAEL reverse: 5'-GGAAGCAGAACAATCCCTCAA-3 '(SEQ ID NO: 25)

18s forward: 5'-AAACGGCTACCACATCCAAG-3 '(SEQ ID NO 26)

18s reverse: 5'-CGCTCCCAAGATCCAACTAC-3 '(SEQ ID NO: 27)

Example  1-8: Western Blot  analysis

Cells were washed with PBS and TNN buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40 and protease inhibitor cocktail tablet (Roche, Indianapolis, IN, http: // www. Roche-applied-science.com)], and the concentration of the protein was determined using a Bio-Rad protein assay (Bio-Rad, Hercules, CA, http://www.bio-rad.com). A portion of the total protein (30-50 μg protein / lane) was applied to SDS-PAGE and transferred to the NC membrane for 2 hours at 100 V. The membrane was TBS-T buffer containing 4% skim milk powder (20 mmol). / L Tris-HCl, pH 7.6, 137 mmol / L NaCl and 0.01% Tween 20) was blocked at room temperature for 1 hour and reacted by treating the primary antibody at room temperature. MAEL (Abcam, # ab28661), gamma-H2AX (ser139) (Millipore # 05-636), PARP (Cell Signaling, # 9542), phospho-ATM (Ser1981) (Cell Signaling, # 2853s), β-actin (Santa) cruz, # sc-47778) After washing the membrane with TBS-T, Horseradish peroxidase I was washed for four hours a fused secondary antibody was treated at room temperature for 1 hour. In TBS-T was carried out, and then color development reaction using the film chemiluminescence (Santa cruz).

Example  1-9: Aging-related β- Galactosidase (β- gal ) analysis

MRC5 cells were washed with PBS and fixed with 2% formaldehyde-0.2% glutaraldehyde. The cells were washed and stained with SA-β-gal staining solution (1 mg / ml 5-bromo-4-chloro-indolyl-β-D-galactopyranoside, 1X PBS (pH 6.0), 2 mM MgCl ₂ , 5 mM potassium ferricyanide, 5 mM) potassium ferrocyanied) overnight at 37 ° C. Cells stained blue on an optical microscope were determined to be aged cells and any total of 300 cells on the slides were counted to determine the proportion of SA-β-gal positive cells.

Example  1-10: Clonogenic  analysis

SiRNA was introduced into HeLa cell line and MDA-MB-231 cell line overnight. siRNA was prepared by targeting the 5'-cagcaatagtgtgacacccaa-3 'sequencing with siMAEL # 3. After 24 hours, cells were seeded in a 60 mm culture dish at a rate of 1000 cells per culture dish and incubated for 10 days. Colonies of cells were fixed with 10% formaldehyde for 10 minutes and stained with 0.1% crystal violet for 30 minutes.

Example  1-11: Colony  Formation analysis

MEF cell lines were cultured in a 100 mm culture dish (8 × 10 ⁵ cells / ml) and the medium was changed 3-4 hours before transduction. MEF cell lines are transduced to each respectively of 2㎍ expression vector (Myc, Ras) and expression of the vector 4㎍ (MAEL, sh-MAEL # 2 , sh-MAEL # 3, control vector), 0.2M CaCl 2 solution And 2X HBS solution were mixed and reacted for 10-12 minutes. Transduced cells were allowed to stand overnight. The next day, the medium was removed, washed once with warm PBS, then fresh medium was added and incubated again for 2 weeks. Cells were then stained with 10% formaldehyde and then stained with 0.1% crystal violet and the colonies observed were counted.

Example  2: Results

Example  2-1: Somatic cell  In cancer cells MAEL Isoform  Expression check of 3

As described above, MAEL was identified as one of the prognostic factors in microarray data of 143 HCC patient tissues. To confirm the expression of MAEL in cancer cells, including HCC cancer cell lines, Western blot analysis was performed using MAEL specific antibodies on proteins derived from HeLa-, 293T-, and MDA-MB-231 cell lines (FIG. 3B). . However, no expected 50 kDa protein band was detected. Instead, single bands of slightly larger size than 40 kDa were detected. The protein band was specifically reduced by treatment of siRNA targeting MAEL in humans (FIG. 3C).

We searched the reported human isoforms of MAEL on the NCBI website (http://www.ncbi.nlm.nih.gov) and found that they were either 44kDa (isoform 2) or 41kDa (isoform 3) sizes. Isoform of was confirmed. Isoform 2 is a form in which exon 2 is deleted and cleaved, and detoxification codon is initiated in the second ATG of the full length MAEL (FIG. 3A). In order to confirm whether MAEL expressed in various cancer cells is isoform 2 or isoform 3, RT PCR using a specially designed primer was performed. The primers can amplify two isoforms, but are specifically designed to change the size of each amplification product by specific deletion in isoform 2. As shown in FIG. 3D, the amplified product was isoform 3.

The results were confirmed using siRNA specific to each isoform (siRNA # 1) or siRNA specific to two isoforms (siRNA # 2, # 3). All siRNAs effectively reduced the expression of polynucleotides encoding exogenous overexpressed full-length MAELs. In addition, siRNA # 1, which could not reduce isoform 3, could not reduce intrinsic MAEL, while siRNA # 2 and # 3 decreased intrinsic MAEL.

Therefore, the results showed that the polynucleotide encoding MAEL is expressed in the form of isoform 3 in various cell lines.

Example  2-2: Cancer tissue  And in various cancer cells MAEL Overexpression of

First, since the cancer / testis associated gene MAEL is expressed in testes and cancer cell lines, the expression of MAEL was confirmed by RT-PCR analysis in various cancer cell lines and normal cell lines (FIG. 2A).

The cell lines used are as follows. Normal cells were immortalized BJ-T using IMR90, BJ, Wi38, and TERT genes, which are normal cell lines derived from foreskin cells (HDF), and cancer cell lines and transformation cell lines derived from various tissues and Available liver cancer cell lines were analyzed. HeLa, a cervical cancer cell line, 293T, a renal cell transformed with Large T antigen, HT1080 fibrosarcoma, MDA-MB-231, a breast cancer cell line, and H441, a lung cancer cell line were used. Heb3B, HepG2, Huh7, SK- Hep1, SNU182, SNU354, SNU368, SNU387, SNU423, SNU449, SNJ475, SNU709, SNU739, SNU878 were used. Each cell was incubated at 37 ° C. in a CO2 incubator using DMEM, RPMI, MEM medium with 10% FBS and 1% antibiotics (recommended media from Korea Cell Line Bank and US Cell Line Bank (ATCC)).

Next, polynucleotide expression patterns encoding these MAELs were confirmed once again in cancer cell lines by immunoblot analysis. That is, the MAEL isoform 3 was not expressed in normal cell lines but only in various cancer cell lines (FIG. 1B).

Finally, expression of polynucleotides encoding MAEL was confirmed in human HCC patient samples (FIG. 2C). As shown in FIG. 2C, polynucleotides encoding MAEL were expressed at high levels in most HCCs compared to their normal liver tissues.

In summary, it can be seen that MAEL, a CT gene (Cancer / Testis associated gene), is expressed in HCC and various cancer cell lines but is not detected in normal cell lines.

Example  2-3: in cancer cells MAEL Check the role of

Since we found that MAEL is expressed in cancer cells that proliferate to high levels, we studied the function of MAEL in cancer cells. Specifically, siRNA was introduced into three cell lines-HeLa, 293T, MDA-MB-231-and three normal foreskin cells-IMR90, Wi38, and HS68-, which had relatively high expression of MAEL. , Cells were observed (FIG. 1). As a result, it was found that siMAEL effectively inhibited the growth of cancer cells and increased in cancer cells, whereas cell death showed relatively low efficiency in normal cells (FIGS. 3A and 3B).

From the above results, it was found that cancer cells proliferating at high levels react more sensitively to inhibition of MAEL than normal cells.

Example  2-4: Cancer Cell Survival On signal path MAEL Check the role of

First, in order to identify what physiological changes occur in cells in which MAEL expression is suppressed, the present inventors identified a major signaling pathway essential for cancer cell survival. As a result, the activity of ERK and AKT and the expression level of EGFR were severely lowered (Fig. 4A).

Next, apoptosis analysis was performed using FACS using PI and annexin V staining. As a result, the overlapping positive dots were significantly increased in HeLa cells treated with siMAEL, and the expression of truncated PARP, an apoptosis marker protein, was also increased (FIG. 4B).

Finally, the effect of apoptosis was mitigated by MAEL overexpression. As a result, MAEL overexpression inhibited apoptosis and cleavage of PARP (FIG. 4C).

From the above results, it was confirmed that the effect of siMAEL on cancer cells was caused by specifically inhibiting the polynucleotide encoding MAEL, not by other genes or other additional effects.

Example  2-5: In liver cancer MAEL Prognosis by expression of

MAEL was found to increase expression in cancer tissues and cells, and furthermore, in order to investigate whether expression has an effect on prognosis, 100 cases of liver cancer patients from Korea Atomic Medical Center were obtained and the prognosis according to MAEL expression was analyzed. . In other words, MAD and 18S expression on cDNA synthesized from extracted RNA were obtained by real time PCR to obtain CT value based on 18S value, and then the expression level of MAEL was classified into high and low group by Kaplan-Meier. The prognosis of 7 years was analyzed using the assay (FIG. 5). 5 is a graph showing the correlation between the expression level of MAEL and the prognosis after liver cancer treatment. As shown in FIG. 5, the prognosis is significantly lower in the patient group having a high expression level of MAEL (red line), and the prognosis is relatively low, whereas the prognosis is relatively high in the patient group having a low expression level of MAEL (black line). It was confirmed that it represents.

From the above results, it was found that prognostic prediction after liver cancer treatment was possible by confirming the expression level of MAEL.

Example  2-6: natural DNA  Injury and induction of aging MAEL Check the role of

First, cell cycle analysis was performed by PI staining using flow cytometry (FIG. 6A). As a result, the G2 / M peak was increased in the cancer cells treated with siMAEL, it was confirmed that no effect in the normal cells. These results indicate that there is a possibility that siMAEL may cause DNA damage caused by DNA damage that induces checkpoint activation where the cell cycle is stopped at G2 phase.

In addition, Western blot analysis was performed using antibodies related to DNA damage signaling. As a result, it was found that activation of Chk1, a major DNA damage checkpoint protein, and gamma-H2AX, a representative DNA damage marker protein, was increased in cells depleted of MAEL (FIG. 6B).

In addition, as a result of immunostaining using confocal microscopy, activation signals of p53BP1 and gamma-H2AX increased in HeLa cells depleted in MAEL, and nucleus size increased in cells depleted in MAEL, resulting in severe DNA damage. It was confirmed that this occurred (Fig. 6C).

On the other hand, since DNA damage may induce apoptosis as well as aging, siMAEL was introduced into transformed MRC5 cells and β-gal analysis was performed. MRC5 cells treated with siMAEL increased β-gal signal (FIG. 7A) and DNA damage signal also increased.

In addition, since the reaction protein that induces aging in response to DNA damage is known as p21, the expression level of p21 was tested in MRC5 cells depleted in MAEL. P21 increased in cells depleted of MAEL (FIG. 7B).

On the other hand, siMAEL was shown to induce DNA damage and apoptosis. To investigate whether cell death by siMAEL is mediated by DNA damage that induces cell death, the inventors have used MAEL with ATM or ATR to play a role in activating DNA damage checkpoints in HeLa and MDA-MB-231 cells. Depleted and surviving cells were analyzed by clonality assay (FIG. 8A). Cell death induced by siMAEL was reversed by siATM and siATR, which means that cell death induced by siMAEL is mediated by ATM and ATR leading to DNA damage checkpoints. To further confirm these results, flow cytometry (FACS) and western blot analysis were performed (FIGS. 8B and 8C).

Example  2-7: Myc / Ras On transformation induced by MAEL Check the role of

Since MAEL acted on cancer cells proliferating to high levels, it was assumed that MAEL would play a role during normal cell transformation into immortalized cancer cells.

Transformed foci formation assay was performed using cancer cell Myc, Ras plasmid and MEF (p53-/-) cells of passage 4. Colony formation assay showed that MAEL overexpressed cells were more efficient in colony formation using pIRESpuro-Myc-MAEL plasmid. However, MAEL depletion with shMAEL plasmid dramatically reduced Myc / Ras induced foci formation (FIG. 9A). Quantitative analysis of colony numbers is shown in FIG. 9.

<110> KOREA INSTITUTE OF RADIOLOGICAL & MEDICAL SCIENCES <120> Use of MAEL for the Predicting Prognosis and Treatment of Cancer <130> PA120224 / KR <160> 38 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 atgccgaacc gtaaggccag ccg 23 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agggaagttg ggcctgttac t 21 <210> 3 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gatcccccag caatagtgtg acacccaatt caagagattg ggtgtcacac tattgctgtt 60 tttggaaa 68 <210> 4 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 agcttttcca aaacagcaat agtgtgacac ccaatctctt gaattgggtg tcacactatt 60 gctgggg 67 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> MAEL siRNA 1 <400> 5 ccagccggaa tgcttacta 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> MAEL siRNA 2 <400> 6 ggaactggcc acctatcta 19 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MAEL siRNA 3 <400> 7 cagcaatagt gtgacaccca a 21 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer LINE1-5'UTR-s <400> 8 aaggggtgac ggtcgcacct ggaa 24 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer LINE1-5'UTR-as <400> 9 tgctgtgcta gcaatcagcg aga 23 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer LINE1-ORF1-s <400> 10 tcctcgagaa gagcaactcc a 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer LINE1-ORF1-as <400> 11 gggtttctgc cgagagatcc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer LINE1-ORF2-s <400> 12 atggccatac tgcccaaggt 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer LINE1-ORF2-as <400> 13 tggcttagga ttgacttggc a 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer HER-V-LTR-s <400> 14 cagatgcttg aaggcagcat 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer HER-V-LTR-as <400> 15 acgttggaca atacctggct 20 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer HER-V-ORF1-s <400> 16 tgggcaacca ttgtcgggaa ac 22 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer HER-V-ORF1-as <400> 17 ggcttattcc ctgaaacact tggga 25 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer HER-V-ORF2-s <400> 18 ggcttattcc ctgaaacact tggga 25 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer HER-V-ORF2-as <400> 19 tcatcaaggc tgcaagcagc atac 24 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer MAEL-s <400> 20 tggccactct ctttggaatc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer MAEL-as <400> 21 gcatttccaa ttcttccagc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer GAPDH-s <400> 22 gtcagtggtg gacctgacct 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer GAPDH-as <400> 23 tgatgtagcc aaattcgttg 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 3'UTRMAEL-s <400> 24 cagtaacagg cccaacttcc 20 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer 3'UTRMAEL-as <400> 25 ggaagcagaa caatccctca a 21 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 18S-s <400> 26 aaacggctac cacatccaag 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 18S-as <400> 27 cgctcccaag atccaactac 20 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> si-MAEL # 1 <400> 28 ccagccggaa tgcttacta 19 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> si-MAEL # 2 <400> 29 ggaactggcc acctatcta 19 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> si-MAEL # 3 <400> 30 cagcaatagt gtgacaccca a 21 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> si-MAEL # 4 <400> 31 tgaacgtggg cataaccaa 19 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> si-MAEL # 5 <400> 32 gatagaacca gagtcaact 19 <210> 33 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> si-m-MAEL # 1 <400> 33 gcttcctccc ttgtgaaat 19 <210> 34 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> si-m-MAEL # 2 <400> 34 ggaactggcc acctattta 19 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> si-m-MAEL # 3 <400> 35 cagcaacagt gtgacaccca a 21 <210> 36 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> sh-m-MAEL # 1 <400> 36 ccagccgcaa tgcctacta 19 <210> 37 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> sh-m-MAEL # 2 <400> 37 ggaactggcc acctattta 19 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sh-m-MAEL # 3 <400> 38 cagcaacagt gtgacaccca a 21

Claims (13)

A composition for predicting prognosis after cancer, comprising an agent for measuring the level of mRNA or protein derived from a polynucleotide encoding MAEL (MAELstrom).
The method of claim 1,
The agent for measuring the level of mRNA comprises a primer pair, a probe (probe) or anti-sense nucleotides that specifically bind to the MAEL gene.
In claim 1,
The agent for measuring the expression level of the protein comprises an antibody specific for the protein.
The method of claim 1,
Wherein said cancer is selected from the group consisting of cervical cancer, breast cancer, and liver cancer.
A cancer prognosis kit comprising a composition according to any one of claims 1 to 4.
6. The method of claim 5,
Wherein said kit is an RT-PCR kit, a competitive RT-PCR kit, a real time RT-PCR kit, a real time RT-PCR kit, a DNA chip kit or a protein chip kit.
(i) measuring the mRNA of the MAEL gene or the protein encoded by the gene from a biological sample isolated from the individual for whom the treatment of cancer has been performed; And
(ii) determining that the cancer has been treated or improved when the level of mRNA of the measured gene or the protein encoded by the gene is reduced than that of a sample prior to cancer treatment. How to Provide Information.
8. The method of claim 7,
The method for measuring mRNA level is reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time quantitative RT-PCR, RNase protection assay (RNase protection method), Northern blotting or DNA chip technology.
8. The method of claim 7,
The method of measuring the level of the protein is Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunoasiffusion (radial immunodiffusion), Ouchterlony immunodiffusion (Ouchterlony immunodiffusion) ), Rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography , FACS analysis (fluorescenceactivated cell sorter analysis) or protein chip technology (protein chip technology).
A composition for treating or preventing cancer, comprising a substance that inhibits the expression or activity of a MAEL protein.
11. The method of claim 10,
The substance is an antisense oligonucleotide, aptamer, siRNA or shRNA for the MAEL gene.
11. The method of claim 10,
The substance is an antibody or antigen-binding fragment thereof that inhibits the activity of the MAEL protein.
Measuring the level of mRNA of a MAEL gene or a protein encoded by the gene after administration of a substance expected to be able to treat cancer.

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