KR101802473B1 - COMPOSITION FOR DIAGNOSING LEUKEMIA USING TMEM57 OR NudC - Google Patents

Abstract

The present invention relates to a composition for the diagnosis of leukemia, a kit, a method for providing information for diagnosis, and a screening method for a preventive or therapeutic agent. When the composition for the diagnosis of leukemia according to the present invention, the kit, the method for providing information for diagnosis, and the screening method for a prophylactic or therapeutic agent are used, the expression of the gene encoding TMEM57 or NudC is expressed in acute lymphocytic leukemia or acute myelogenous leukemia , It is possible to diagnose leukemia at an early stage by comparison with normal cells and can be useful for diagnosis of acute lymphocytic leukemia or acute myelogenous leukemia.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for diagnosing leukemia using TMEM57 or NudC gene,

The present invention relates to a composition for diagnosing leukemia using a gene encoding TMEM57 or NudC.

Leukemia is a term collectively referred to as a disease or disease in which white blood cells proliferate positively.

The types of leukemia can be divided into myeloid leukemia and lymphocytic leukemia according to the leukemia originating from leukemia, and classified into acute leukemia and chronic leukemia according to the progression rate.

On the other hand, clinical manifestations of leukemia vary according to the type of disease and the nature of the affected cells. Lymphocytic leukemia is caused by lymphoid blood cells, myeloid leukemia is caused by bone marrow blood cells, and chronic myelogenous leukemia is caused by malignant clones of mature cells, that is, pluripotent hematopoietic stem cells, and acute myelogenous leukemia Leukemia is caused by disorders of myeloid cells that begin to differentiate during the relatively early stage of hematopoiesis.

In order to diagnose such leukemia, in the past, the number of leukocyte in the peripheral blood of a patient was measured. When the measured value exceeded the normal value, a method of suspicion of leukemia was used. However, in diseases other than leukemia such as cold, Since the number of leukocytes is increased by the increase of the immune response, there is a possibility that a positive error may be caused only by the measurement of the white blood cell count.

Therefore, there has been a continuing effort to develop a diagnostic method for leukemia that can diagnose only a specific disease of more reliable leukemia.

Recently, molecular genetics has been developed, and specific leukemia-related chromosomes and gene abnormalities have been elucidated in more than 80-90% of malignant hematologic diseases, and molecular biologic diagnosis methods based on chromosomal abnormalities such as high-affinity method, fluorescence in situ hybridization, Enzyme chain reaction, etc. have been proposed, and the use of this diagnostic method improves the success rate of leukemia diagnosis. In particular, in the case of acute lymphoblastic leukemia, a genetic mutation affecting tumor suppressor genes, oncogenes and some chromosomes is known to be the main cause of the disease, A method of judging whether or not the disease has occurred is being used.

Although genes such as CYP, GST, NAT, MTHFR, NQO1, XRCC1, MDR1, cyclin D1, Trk A and CCND1 have been known as genes to be used for diagnosis of such genetic mutations (see Patent Document 1) Since the number of patients who can reliably detect the onset of leukemia using the above-mentioned gene as a marker is still small, there is a need for more diverse gene markers for diagnosis of leukemia.

Patent Document 1: Korean Patent Publication No. 10-2015-0018276

The present inventors have developed a novel marker marker for leukemia diagnosis in order to solve the above-mentioned problems of the prior art. As a result of these efforts, the inventors of the present invention have found that a leukemia cell line, particularly, an acute lymphoblastic leukemia (ALL) The expression level of NudC was decreased in the cell line of patients with acute myeloid leukemia (AML), and the expression of TMEM57 was reduced in patients with acute myeloid leukemia (AML). Thus, the gene was identified as a gene marker for leukemia diagnosis The present invention has been completed.

The present invention provides a composition for diagnosing leukemia comprising an agent for measuring the mRNA level of a gene encoding TMEM57 or NudC or the level of a protein expressed therefrom.

The present invention also provides a kit for the diagnosis of leukemia comprising a composition for diagnosing leukemia comprising an agent for measuring the mRNA level of a gene encoding TMEM57 or NudC or the level of a protein expressed therefrom.

(A) measuring the level of mRNA of the gene encoding TMEM57 or NudC or the level of the protein expressed therefrom, from a biological sample of a patient suspected of having leukemia; And (b) comparing the measured mRNA level or the level of the protein expressed therefrom with a level measured from a sample of a normal control, and determining that the leukemia has occurred if a significant difference is found, Lt; RTI ID = 0.0 > information. ≪ / RTI >

(A) measuring the level of expression of a gene encoding TMEM57 or NudC or the level of a protein encoded by the gene from a sample of an experimental animal in which leukemia has occurred as a control; (b) administering to said experimental animal a candidate agent expected to be able to treat leukemia; (c) measuring the level of expression of the gene encoding TMEM57 or NudC or the level of the protein encoded by the gene from a sample of an experimental animal to which the candidate substance is administered; And (d) comparing the level of expression of the gene measured in the control group or the level of the protein encoded by the gene with the level measured in the test group compared to the level measured in the control group, And determining that the substance can be used as a prophylactic agent for preventing the onset of leukemia or a therapeutic agent for treating leukemia. The present invention also provides a screening method for a prophylactic or therapeutic agent for leukemia.

As one embodiment for achieving the above object, the present invention provides a composition for diagnosing leukemia comprising an agent for measuring the mRNA level of a gene encoding TMEM57 or NudC or the level of a protein expressed therefrom.

The present inventors have carried out various studies to develop a novel marker for leukemia diagnosis, and found that the TMEM57 or NudC gene expression is different from that of normal individuals in leukemia patients. By confirming, the gene was noted. To confirm whether the TMEM57 or NudC-encoding gene can be used for the diagnosis of leukemia.

In the case of the gene coding for NudC, gene expression was decreased in patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) among the leukemia types than in normal individuals.

In addition, the gene encoding TMEM57 was specifically expressed in ALL patients, lower than normal individuals.

As used herein, the term "TMEM57" refers to Transmembrane protein 57, and the specific nucleotide sequence and protein information of TMEM57 is known from NCBI (GenBank number AAH32427.1).

Also, the term "NudC" as used herein is a nucleus distribution protein, and the specific nucleotide sequence and protein information of the NudC is known from NCBI (GenBank number NM_006600.3).

In addition, the leukemia stage referred to herein is the same as that according to the French-American-British (FAB) classification.

More specifically, "MO stage (non-differentiated acute myelogenous leukemia state) of leukemia cells in AML patients" means a stage in which bone marrow cells do not show signs of differentiation, accounting for about 5% of adult AML patients, It is known as bad leukemia. In addition, "M1 level of leukemia cells in AML patients (leukemia status of bone marrow constitution)" means a stage in which bone marrow cells show differentiation into granulocytic leukocytes, accounting for about 15% of adult AML patients . "M2 phase (leukemia state of bone marrow constitution) of leukemia cells of AML patient" means that bone marrow cells are out of granular leukocyte state and is said to account for about 25% of adult AML patients. Particularly, in the step M2, granule leukocyte maturation can be observed, and among the AML species, the prognosis is good. In the M3 stage of leukemia cells of AML patients, most abnormal cells are granular leukocytes between myeloblasts and myelocytes, and most cells are small particles and nuclei with various sizes and shapes . In particular, there is a specificity that can be treated using ATRA (All Trans-Retinoic Acid) compared to other subtypes, and the prognosis of AML is known to be the best stage. In addition, the M4 stage of leukemia cells in AML patients means that about 30% or more of cells having nuclear nucleus as acute myeloid leukemia are composed of bone marrow and mononuclear leukemia. The M5 stage of leukemia cells in patients with AML is mononuclear leukemia, which means that about 80% of the nucleated cells are mononuclear. The M6 stage of leukemic cells in patients with AML indicates that at least 50% of the cells with nuclear leukemia consist of abnormal hair follicles. The M7 stage of leukemia cells in patients with AML means leukemia with germ cell leukemia.

As used herein, the term "diagnosis " means identifying the presence or characteristic of a pathological condition. In the present invention, the "diagnosis" can be interpreted as observing and confirming whether or not leukemia develops.

The term "agent for measuring the mRNA level of a gene " used herein means a preparation used for measuring the level of mRNA transcribed from the target gene in order to confirm the expression of the target gene contained in the sample (RT-PCR), competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), northern blotting, , A DNA chip analysis method, and the like, but is not particularly limited to such a probe or a primer capable of specifically binding to a target gene.

As used herein, the term "primer " refers to a nucleic acid sequence having the same meaning as a conventional primer, for example, a short free 3 'hydroxyl group, and includes a complementary template and a base pair quot; refers to a short nucleic acid sequence that can form a base pair and serves as a starting point for template strand duplication. Such a primer can initiate DNA synthesis in the presence of a reagent for polymerization (i. E., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at a suitable buffer solution and temperature.

In the present invention, the primer may be a primer that can be used for amplification of a gene encoding TMEM57 or NudC, and as long as it is capable of complementarily binding with the gene to amplify the gene, the nucleotide sequence of the primer When amplifying a gene encoding NudC, but not limited thereto, the nucleotide sequence of SEQ ID NOs: 1 and 2 attached to the sequence listing, the sequence of SEQ ID NO: 3 attached to the sequence listing when amplifying the gene encoding TMEM57, 4 < / RTI > nucleotide sequence.

As used herein, the term "probe" may have the same meaning as a probe in the conventional sense, for example, RNA or DNA corresponding to a few bases or hundreds of bases that can achieve specific binding with a gene or mRNA And may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, May be labeled for detection.

In the present invention, the probe may be a probe capable of complementarily binding to a gene encoding TMEM57 or NudC, and the nucleotide sequence of the probe, as long as it can be complementary to the TMEM57 or NudC gene, It is not limited.

As used herein, the term "agent for measuring the level of protein" means a preparation used for measuring the level of a target protein contained in a sample, preferably western blotting, enzyme linked immunosorbent assay, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement An antibody used in a method such as Complement Fixation Assay, FACS and protein chip assay.

The term "antibody" in the present specification means a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. The antibody can be obtained by cloning each gene into an expression vector according to a conventional method, A protein encoded by the marker gene can be obtained and can be produced from the obtained protein by a conventional method. The form of the antibody is not particularly limited, and any polyclonal antibody, monoclonal antibody or antigen-binding antibody thereof may be included in the antibody of the present invention, and any immunoglobulin antibody may be included, Specific antibodies of the invention. In addition, the antibody comprises a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains.

Refers to a fragment having at least an antigen-binding function, and may be, for example, Fab, F (ab ') 2, F (ab') 2 and Fv.

In the present invention, the antibody may be an antibody capable of specifically binding to TMEM57 or NudC protein, preferably a polyclonal antibody capable of specifically binding to the protein, a monoclonal antibody or a It can be a part.

According to one embodiment of the present invention, NudC expression levels in patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were compared with those of normal bone marrow cells, (See FIG. 1). In addition, the level of TMEM57 expression was decreased in patients with acute myeloid leukemia (AML) compared with normal subjects.

Therefore, the TMEM57 or NudC can be used as a marker for the diagnosis of leukemia, and the agent for measuring the mRNA level of the gene encoding TMEM57 or NudC or the level of the protein expressed therefrom can effectively diagnose leukemia I could see the point.

The present invention also provides a kit for the diagnosis of leukemia comprising the composition for diagnosing leukemia.

The kit of the present invention can be used for diagnosing leukemia by detecting the level of mRNA of a gene encoding TMEM57 or NudC or the level of protein expressed therefrom from a sample of a patient suspected of having leukemia, , But are not limited to, primers, probes or antibodies for measuring the mRNA level of the gene or the level of the protein expressed therefrom, as well as one or more other component compositions, solutions or devices suitable for the assay It is possible.

As used herein, the term "sample " means a direct subject that is isolated from a patient with leukemia and measures the level of expression of TMEM57 or NudC, for example, but not limited to, blood, bone marrow, .

As a specific example, the kit for measuring the level of mRNA expression of the TMEM57 or NudC gene of the present invention may be a kit containing necessary elements necessary for conducting RT-PCR.

The RT-PCR kit can be used in combination with a test tube or other appropriate container, a reaction buffer (pH and magnesium concentration varies), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase , DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. In addition, it may contain a primer pair specific to a gene used as a quantitative control.

As another example, the kit of the present invention may contain the necessary elements necessary for performing DNA chip analysis. The kit for DNA chip analysis may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, a preparation, and an enzyme for producing a fluorescent-labeled probe. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.

As another example, the kit of the present invention may be a kit for analyzing a protein chip for measuring the level of a protein expressed from TMEM57 or a NudC gene. The kit may be used for immunological detection of an antibody, A substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, and the like.

The substrate is not particularly limited, but a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, and a glass slide glass can be used, and the coloring enzyme is not particularly limited The fluorescent substance may be FITC, RITC or the like, and the coloring substrate liquid is not particularly limited, but ABTS (2, 3, 4, 5, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) or OPD (o-phenylenediamine), TMB (tetramethylbenzidine).

The present invention also provides a method for providing information for the diagnosis of leukemia using said diagnostic composition or kit.

The method of providing information for the diagnosis of leukemia comprises the steps of: (a) measuring the mRNA level of a gene encoding TMEM57 or NudC or the level of a protein expressed therefrom from a biological sample of a patient suspected of having leukemia; And (b) comparing the measured mRNA level or the level of the protein expressed therefrom with a level measured from a sample of a normal control, and determining that the leukemia has occurred if the difference is significant.

The leukemia is not particularly limited, but may be, for example, acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML).

When the method of providing information for the diagnosis of leukemia is used, when the mRNA level of the gene encoding TMEM57 or the level of the protein expressed therefrom is significantly decreased, it is determined that AML has been obtained, and the gene encoding NudC If the mRNA level or the level of the protein expressed therefrom is significantly decreased, it can be judged that AML or ALL is developed. In addition, when it is determined that ALL has been developed, the ALL can be judged to have developed one of B-cell ALL and Pro-B ALL.

Meanwhile, the method of measuring the mRNA level of the gene or the level of the protein expressed therefrom is the same as described above.

The present invention also provides a method for screening an agent for the prevention or treatment of leukemia.

More specifically, a method for screening an agent for the prevention or treatment of leukemia comprises administering to a test animal a candidate substance which is expected to prevent or treat the onset of leukemia, and administering TMEM57 or NudC And a method for screening an agent for the prevention or treatment of leukemia comprising the step of measuring the mRNA level of the gene or the level of the protein expressed therefrom.

Measuring the level of expression of the gene of the present invention in the cell or the level of the protein encoded by the gene in the absence of a candidate substance capable of preventing or treating leukemia and measuring the level of the gene of the present invention in the presence of the candidate substance The expression level or the level of the protein encoded by the gene is measured and compared with each other. Subsequently, the substance that changes the expression level of the gene of the present invention or the level of the protein encoded by the gene when the candidate substance is present, As a prophylactic or therapeutic agent.

Specifically, the present invention provides a method of screening for an agent for the prevention or treatment of leukemia, comprising the steps of: (a) determining the level of expression of a gene encoding TMEM57 or NudC from a sample of an experimental animal in which a control leukemia has occurred, Measuring a level; (b) administering to said experimental animal a candidate agent expected to be able to treat leukemia; (c) measuring the level of expression of the gene encoding TMEM57 or NudC or the level of the protein encoded by the gene from a sample of an experimental animal to which the candidate substance is administered; And (d) comparing the level of expression of the gene measured in the control group or the level of the protein encoded by the gene with the level measured in the test group compared to the level measured in the control group, Determining that the substance can be used as a prophylactic agent for preventing the onset of leukemia or a therapeutic agent for treating leukemia.

In this case, when the leukemia is ALL or AML, when the level measured in the test group shows a higher level than the level measured in the control group, it can be judged that the leukemia can be used as a therapeutic agent.

When the composition for the diagnosis of leukemia according to the present invention, the kit, the method for providing information for diagnosis, and the screening method for a prophylactic or therapeutic agent are used, the expression of the gene encoding TMEM57 or NudC is expressed in acute lymphocytic leukemia or acute myelogenous leukemia , It is possible to diagnose leukemia at an early stage by comparison with normal cells and can be useful for diagnosis of acute lymphocytic leukemia or acute myelogenous leukemia.

The accompanying drawings are included to provide a further understanding of the invention to those skilled in the art, and the technical spirit of the invention is not limited thereto.
1 is a graph showing that NudC expression is decreased in leukemic cells of patients with acute lymphoblastic leukemia (ALL) and patients with acute myeloid leukemia (AML).
FIG. 2 is a graph showing a decrease in the expression of TMEM57 in leukemic cells of patients with acute myeloid leukemia (AML).
FIG. 3 is a graph showing the expression levels of TMEM57, NOTCH and HES1 at different stages of leukemic cell differentiation in patients with acute myeloid leukemia (AML).

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] Confirmation of correlation between NudC and leukemia

In order to confirm the correlation between NudC gene and leukemia, the following experiment was conducted.

First, the diagnosis of leukemia was made according to the criteria of the World Health Organization in 2008. monocyte was subjected to concentration gradient centrifugation from the bone marrow of ALL or AML patients using ficoll hypaque (Sigma-aldrich, St. Louis, USA). Thereafter, the cells were washed twice with a phosphate-buffered saline. Total RNA of mononuclear cells was extracted using AccuPrepGenomic DNA extraction kit and ExiPrep cell total RNA kit (Bioneer, Daejeon, Korea) and cDNA was generated using Superscript III Reverse Transcriptase (Applied Biosystems). At this time, the mRNAs expressed using the primer sets of SEQ ID NOS: 1 and 2 and SYBR Premix Ex Taq (TaKaRa) were quantitatively analyzed and β-actin mRNA was used as a control for standardization. Real-time quantitative PCR was performed using Rotor-Gene Q (QIAGEN), and changes in gene expression were calculated using the Com Ct method. FIG. 1 shows the results of confirming the degree of NudC expression of monocyte cells of AML or ALL patients.

As can be seen in FIG. 1, the expression of NudC in monocytes of AML or ALL patients was significantly reduced compared with that in normal mononuclear cells.

Therefore, it was confirmed that NudC can be used as a marker for diagnosing AML or ALL.

[Example 2] Confirmation of correlation between TMEM57 and leukemia

In order to confirm the correlation between TMEM57 gene and leukemia, the following experiment was conducted.

First, the diagnosis of leukemia was made according to the criteria of the World Health Organization in 2008. density gradient centrifugation of mononuclear cells from bone marrow of ALL or AML patients was performed using ficoll hypaque (Sigma-aldrich, St. Louis, USA). Thereafter, the cells were washed twice with a phosphate-buffered saline. Total RNA of mononuclear cells was extracted using AccuPrep genomic DNA extraction kit and ExiPrep cell total RNA kit (Bioneer, Daejeon, Korea) and cDNA was generated using Superscript III Reverse Transcriptase (Applied Biosystems). At this time, the mRNAs expressed using the primer set of SEQ ID NOS: 3 and 4 and SYBR Premix Ex Taq (TaKaRa) were quantitatively analyzed, and β-actin mRNA was used as a control for standardization. Real-time quantitative PCR was performed using Rotor-Gene Q (QIAGEN), and changes in gene expression were calculated using the Com Ct method. In addition, the expression of NOTCH and HES1 genes was confirmed in the same manner.

FIG. 2 shows the results of confirming the degree of TMEM57 expression in monocytes of the AML patients. In addition, the expression pattern of TMEM57, NOTCH and HES1 according to the differentiation time of the cells is shown in Fig.

As can be seen from Figs. 2 and 3, it was confirmed that expression of TMEM57 specifically in only monocytes of AML patients was markedly reduced compared with expression in bone marrow. In addition, TMEM57 showed a decrease in the expression of AML patients from M0 to M2.

Therefore, it was confirmed that TMEM57 can be used as a marker for diagnosing AML patients.

<110> SOOKMYUNG WOMEN'S UNIVERSITY INDUSTRY ACADEMIC COOOPERATION FOUNDATION <120> COMPOSITION FOR DIAGNOSING LEUKEMIA USING TMEM57 OR NudC <130> P-1 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Unknown <220> <223> Forword primer for NudC <400> 1 ccagatcaag gagctaactg 20 <210> 2 <211> 20 <212> DNA <213> Unknown <220> <223> Reverse primer for NudC <400> 2 ctcatgattc tctgcatcct 20 <210> 3 <211> 20 <212> DNA <213> Unknown <220> <223> Forward primer for TMEM57 <400> 3 cttcacctcg aagtcatagc 20 <210> 4 <211> 20 <212> DNA <213> Unknown <220> <223> Reverse primer for TMEM57 <400> 4 gctagtgcat ttctgcttct 20

Claims (16)

A composition for diagnosing leukemia comprising an agent that measures the mRNA level of a gene encoding TMEM57 or the level of a protein expressed therefrom,
Wherein the leukemia is an acute myelogenous leukemia (AML) disease. The composition for diagnosing leukemia according to claim 1, wherein the agent for measuring the level of the gene mRNA comprises a primer or a probe specifically binding to the gene. delete 3. The composition for diagnosing leukemia according to claim 2, wherein the primers are primer pairs having the nucleotide sequences of SEQ ID NOS: 3 and 4. The composition for diagnosing leukemia according to claim 1, wherein the agent for measuring the level of the protein comprises an antibody specific to the protein. delete A kit for the diagnosis of acute myelogenous leukemia (AML) comprising the composition according to claim 1. 8. The kit for diagnosing acute myeloid leukemia (AML) according to claim 7, wherein the kit is an RT-PCR kit, a DNA chip kit or a protein chip kit. (a) measuring the mRNA level of a gene encoding TMEM57 or the level of a protein expressed therefrom from a biological sample of a patient suspected of having leukemia; And
(b) comparing the measured mRNA level or the level of the protein expressed therefrom with a level measured from a sample of a normal control, and determining that leukemia has occurred if there is a significant difference, In a method of providing information,
Wherein said patient is a patient suffering from an acute myelogenous leukemia (AML) disease. delete 10. The method of claim 9, wherein the mRNA level of the gene is selected from the group consisting of RT-PCR, competitive RT-PCR, real-time quantitative RT-PCR, , RNase protection method, Northern blotting, or DNA chip technology assay. The method according to any one of claims 1 to 5, 10. The method of claim 9, wherein the level of the protein is selected from the group consisting of western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Immunohistochemical staining, immunoprecipitation assays, complement fixation assays, immunofluorescence, immunochromatography (immunohistochemical staining), immunohistochemical staining, immunohistochemical staining, immunohistochemical staining, wherein the assay is performed using any one selected from the group consisting of immunochromatography, fluorescence activated cell sorter analysis and protein chip technology assay. 10. The method according to claim 9, wherein when the mRNA level of the gene encoding TMEM57 or the level of the protein expressed therefrom decreases, it is determined that acute myelogenous leukemia (AML) has been developed. How to. delete delete delete

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