WO2021034091A1

WO2021034091A1 - Method for diagnosing glioblastoma

Info

Publication number: WO2021034091A1
Application number: PCT/KR2020/011011
Authority: WO
Inventors: 이승주; 김경곤; 김정훈; 이은엽; 전한울
Original assignee: 재단법인 아산사회복지재단; 울산대학교 산학협력단
Priority date: 2019-08-19
Filing date: 2020-08-19
Publication date: 2021-02-25

Abstract

The present invention relates to a method for diagnosing or predicting the prognosis of glioblastoma. The present inventors have confirmed that there is a close correlation between the expression of a large number of specific proteins in the tissue of patients with glioblastoma, which is a malignant glioma, and predictions of the onset and prognosis of glioblastoma, and it is expected that confirming the expression level of said proteins or mRNA thereof can provide useful information for treating and predicting the prognosis of glioblastoma.

Description

How to diagnose glioblastoma

The present invention relates to a method for diagnosing glioblastoma or predicting prognosis.

This application claims priority based on Korean Patent Application No. 10-2019-0101010 filed on August 19, 2019, and all contents disclosed in the specification and drawings of the application are incorporated in this application.

Glioma (glioma) is a tumor that accounts for 60% of primary brain tumors, and it is a malignant tumor that does not have any special treatment other than radiation therapy. Among them, glioblastoma (GBM), which is classified as the most malignant, has very high resistance to radiation and anticancer treatment compared to other cancers, so once diagnosed, the expected survival period is only 1 year. In addition, in the case of such a brain tumor, it is difficult to deliver drugs for treatment due to the cerebral vascular barrier. In particular, due to a relatively lack of understanding of cranial nerve biology, the development of therapeutic agents for it has been slow. Moreover, glioblastoma exhibits an aggressive variant when compared to other brain tumors, which can lead to lethal outcomes within weeks if not treated as soon as possible.

To determine the prognosis of existing brain tumor patients, it is common to synthesize the progress of clinical treatment, radiation therapy, and chemotherapy, and determine the survival prognosis including the possibility of recurrence by considering the malignancy, location, and age of the brain tumor. Although it was a method, it cannot be said to be an accurate prognostic method because there are various differences for each type of patient and results for clinical treatment are also different. Therefore, the necessity of developing a new diagnostic and therapeutic target capable of diagnosing malignant brain tumors and predicting prognosis is emerging.

On the other hand, glioblastoma is a representative refractory cancer that is difficult to treat, and the primary treatment for glioblastoma is anticancer-radiation therapy after surgery. In the case of recurrence after surgery or chemo-radiation treatment, there are few treatment options, and anti-angiogenic therapy (bevacizumab is used) is used. In addition to the aforementioned recurrent glioblastoma, bevacizumab treatment is used in cases where surgical treatment is difficult.

The problem with bevacizumab treatment is that it may work in some patients, but not at all in certain patients, so the response to treatment is inconsistent. Since this is not known in advance, high-priced drugs (800 to 10 million won/month) are arbitrarily used, but the anti-tumor effect is excellent in patients with anti-vascular treatment effects. Therefore, for efficient anti-vascular treatment, it is necessary to develop biomarkers capable of predicting anti-vascular treatment response.

An object of the present invention is to provide a method for providing information for diagnosing glioblastoma, comprising the following steps.

Obtaining a biological sample; And

STING (Stimulator of interferon genes), CALU (Calumenin), SEPT5 (Septin5), CD99, CIT (Citron Rho-interacting kinase), PXMP4 (Peroxisomal membrane protein 4), Nectin-2, ALG3 (Dol- P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase), DLL3 (Delta-like protein 3), Noelin-2, TNS1 (Tensin-1), CAV1 (Caveolin-1) ), FCN2(Ficolin-2), KIFC1(Kinesin Family Member C1), HIDE1(Highly expressed in immature dendritic cell transcript 1), CLEC2B(C-type lectin domatin family 2 member B), CD207(C-type Lectin Domain Family) 4 Member K) and CLEC12A (C-type lectin domain family 12 member A) measuring the expression level of one or more selected proteins or mRNAs thereof from the group consisting of.

Another object of the present invention is to provide a method of providing information for predicting the prognosis of glioblastoma patients, including the following steps.

Obtaining a biological sample of a patient with glioblastoma; And

In the obtained sample, at least one from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4, Nectin-2, ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A Measuring the expression level of the selected protein or mRNA thereof.

Another object of the present invention is from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4, Nectin-2, ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A. It is to provide a composition for diagnosing glioblastoma or predicting prognosis, comprising an agent for measuring the expression level of one or more selected proteins or mRNA thereof.

Another object of the present invention is to provide a kit for diagnosing glioblastoma or predicting prognosis comprising the composition and instructions.

Another object of the present invention is to provide a method for screening for glioblastoma prevention or treatment, comprising the following steps.

Contacting the cell with the test substance;

STING, CALU, SEPT5, CD99, CIT, PXMP4, Nectin-2, ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and one or more selected protein or mRNA thereof from the group consisting of CLEC12A Measuring the expression level of; And

Selecting a test substance having a difference in the expression level of the selected protein or its mRNA compared to a control group not contacted with the test substance.

Another object of the present invention is to provide an animal model of glioblastoma in which the STING (Stimulator of interferon genes) gene is deleted.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a method of providing information for diagnosing glioblastoma, comprising the following steps.

Obtaining a biological sample;

In the obtained sample, at least one from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4, Nectin-2, ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A Measuring the expression level of the selected protein or mRNA thereof; And

A) determining that the expression of one or more proteins selected from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4, and Nectin-2, or mRNA thereof, is low compared to the control group, and that it has glioblastoma or is affected; or

B) If the expression level of one or more proteins selected from the group consisting of ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A is high compared to the control group, glioblastoma has occurred. Or determining that it will take.

In one embodiment of the present invention, the protein expression level measurement is Western blot, ELISA, radioimmunoassay, radiation immunodiffusion method, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, immunostaining, It can be measured by one or more methods selected from the group consisting of immunoprecipitation assay, complement fixation assay, mass spectrometry, FACS, and protein chip.

In another embodiment of the present invention, the mRNA expression level measurement is one or more from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization, and DNA chip. It can be measured in a selected way.

In another embodiment of the present invention, the biological sample is from the group consisting of tissue, cells, blood, plasma, serum, saliva, nasal fluid, sputum, joint capsule fluid, amniotic fluid, ascites, cervical secretion, vaginal secretion, urine and cerebrospinal fluid. It may be one or more selected, preferably may be a tissue.

In another embodiment of the present invention, the tissue may be a formalin-fixed paraffin-embedded (FFPE) sample.

In another embodiment of the present invention, the control group may be a normal person who does not suffer from glioblastoma, but is not limited thereto.

In addition, it provides a method for diagnosing glioblastoma, comprising the following steps.

Obtaining a biological sample;

In addition, the present invention provides a method for providing information for predicting the prognosis of glioblastoma patients, including the following steps.

Obtaining a biological sample of a patient with glioblastoma; And

In biological samples, one or more proteins selected from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4, Nectin-2, ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A Or measuring the expression level of its mRNA.

In an embodiment of the present invention, the prognosis prediction may be a prognosis prediction for an angiogenesis inhibitor treatment, but is not limited thereto.

In another embodiment of the present invention, the angiogenesis inhibitor may be bevacizumab, but is not limited thereto.

In another embodiment of the present invention, the method is when the expression level of one or more proteins selected from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4 and Nectin-2 or mRNA thereof is high compared to the first control, It can be determined that the prognosis is good, but it is not limited thereto. Here, the first control group may be a normal person or a treatment non-responsive group.

In another embodiment of the present invention, the method controls the expression level of one or more proteins selected from the group consisting of ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A or their mRNA. 2 If it is high compared to the control group, it may be determined that the prognosis is poor, but is not limited thereto. Here, the second control group may be a normal person or a treatment responder.

In addition, the present invention is one or more from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4, Nectin-2, ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A It provides a composition for diagnosing glioblastoma or predicting prognosis, comprising an agent for measuring the expression level of the selected protein or mRNA thereof.

In addition, the present invention provides a kit for diagnosing glioblastoma or predicting prognosis, including the composition and instructions.

In addition, the present invention is one or more from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4, Nectin-2, ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A It provides the use of an agent for measuring the expression level of a selected protein or mRNA thereof for glioblastoma diagnosis or prognosis.

In addition, the present invention provides a screening method for a glioblastoma prevention or therapeutic agent comprising the following steps.

Contacting the test substance with the cell or animal model;

In one embodiment of the present invention, the step of selecting the test substance does not contact the test substance with the expression level of one or more proteins selected from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4, and Nectin-2 or its mRNA. If it is higher than that of the control group, it may be determined as a treatment candidate, but is not limited thereto.

In another embodiment of the present invention, the step of selecting the test substance comprises at least one selected protein or mRNA thereof from the group consisting of ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A. When the expression level of is low compared to the control group not contacted with the test substance, it may be determined as a treatment candidate, but is not limited thereto.

In addition, the present invention provides a glioblastoma animal model in which the STING (Stimulator of interferon genes) gene is deleted.

In one embodiment of the present invention, the animal model may be used for screening for glioblastoma therapeutic agents, but is not limited thereto.

The present inventors have confirmed that there is a close correlation between the expression of a number of specific proteins in the tissues of patients with glioblastoma, which is a malignant glioma, and predicting the onset and prognosis of glioblastoma.By confirming the level of expression of the protein or its mRNA, glioblastoma It can provide useful information for predicting the treatment and prognosis of patients.

1 is a result showing the increased protein and decreased protein expressed in glioblastoma patients administered with an angiogenesis inhibitor.

2 shows a process of extracting a protein from a paraffin section.

3 shows the results of measuring the expression level of STING in the response group of glioblastoma patients administered with an angiogenesis inhibitor.

4 shows the results of measuring the expression level of Calumenin (CALU) in the response group of glioblastoma patients administered with an angiogenesis inhibitor.

5 shows the results of measuring the expression level of Septin-5 (SEPT5) in the response group of glioblastoma patients administered with an angiogenesis inhibitor.

6 shows the results of measuring the expression level of CD99 in the response group of glioblastoma patients administered with an angiogenesis inhibitor.

7 shows the results of measuring the expression level of Citron Rho-interacting kinase (CIT) in the response group of glioblastoma patients administered with an angiogenesis inhibitor.

8 shows the results of measuring the expression level of Peroxisomal membrane protein 4 (PXMP4) in the response group of glioblastoma patients administered with an angiogenesis inhibitor.

9 shows the results of measuring the expression level of Nectin-2 in the response group of glioblastoma patients administered with an angiogenesis inhibitor.

Figure 10 is a measurement of the expression level of ALG3 (Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase) in the non-responder group of glioblastoma patients administered with an angiogenesis inhibitor. It shows the results.

Figure 11 shows the results of measuring the expression level of Delta-like protein 3 (DLL3) in the non-responder group of glioblastoma patients administered with an angiogenesis inhibitor.

12 shows the results of measuring the expression level of Noelin-2 in the non-responder group of glioblastoma patients administered with an angiogenesis inhibitor.

13 shows the results of measuring the expression level of Tensin-1 (TNS1) in the non-responder group of glioblastoma patients administered with an angiogenesis inhibitor.

14 shows the results of measuring the expression level of Caveolin-1 (CAV1) in the non-responder group of glioblastoma patients administered with an angiogenesis inhibitor.

15 shows the results of measuring the expression level of Ficolin-2 (FCN2) in the non-responder group of glioblastoma patients administered with an angiogenesis inhibitor.

16 shows the results of measuring the expression level of KIFC1 in the non-responder group of glioblastoma patients administered with an angiogenesis inhibitor.

17 shows the results of measuring the expression level of HIDE1 in the non-responder group of glioblastoma patients administered with an angiogenesis inhibitor.

18 shows the results of measuring the expression level of CLEC2B (C-type lectin domatin family 2 member B) according to the prognosis of glioblastoma patients.

19 shows the results of measuring the expression level of CD207 (C-typle lectin domain family) according to the prognosis of glioblastoma patients.

20 shows the results of measuring the expression level of CLEC12A (C-type lectin domain family 12 member A) according to the prognosis of glioblastoma patients.

21 shows the results of immunostaining the FFPE tissues of glioblastoma patients, and confirming the expression position and expression pattern of the STING protein.

22 shows the results of immunostaining FFPE tissues of patients with glioblastoma, and confirming the expression position and expression pattern of ALG3 protein.

23 shows the overall survival rate and progression free survival according to the level of STING and ALG3 expression of glioblastoma patients.

Figure 24 shows the results of confirming the tumor growth rate in the mouse deficient in the STING gene.

Hereinafter, the present invention will be described in detail.

In the present specification,'mRNA (messenger RNA or messenger RNA)' is an RNA that serves as a blueprint for polypeptide synthesis (protein translation, translation) by transferring the genetic information of the nucleotide sequence of a specific gene to the ribosome during protein synthesis. . Using the gene as a template, single-stranded mRNA is synthesized through a transcription process.

In the present specification,'protein' is used interchangeably with'polypeptide' or'peptide', and, for example, refers to a polymer of amino acid residues as commonly found in proteins in a natural state.

One letter (three letters) of amino acids used herein means the following amino acids according to the standard abbreviation regulations in the field of biochemistry: A(Ala): alanine; C(Cys): cysteine; D(Asp): aspartic acid; E(Glu): glutamic acid; F(Phe): phenylalanine; G(Gly): glycine; H(His): histidine; I(IIe): isoleucine; K(Lys): lysine; L(Leu): leucine; M(Met): methionine; N(Asn): asparagine; O(Ply): pyrrolysine; P(Pro): proline; Q(Gin): glutamine; R(Arg): arginine; S(Ser): serine; T(Thr): threonine; U(Sec): selenocysteine; V(Val): valine; W(Trp): tryptophan; Y(Tyr): Tyrosine.

In the present specification, "complementary" refers to a targeting moiety in a nucleic acid molecule under a predetermined hybridization or annealing condition, specifically, a physiological condition (intracellular) to a target (eg, the SNP of the present invention) selectively. It means that it is sufficiently complementary enough to hybridize, and can have one or more mismatched nucleotide sequences, and has a meaning to encompass both substantially complementary and perfectly complementary. , More specifically, it means completely complementary.

In the present specification, the term “detection” refers to both measuring and confirming the presence (expression) of a target substance, or measuring and confirming a change in the abundance level (expression level) of a target substance. In the same context, measuring the expression level of the protein or its mRNA in the present invention is to measure the expression or not (i.e., to measure the presence or absence of expression), or to measure the level of qualitative or quantitative change of the protein or its mRNA Means to do. The measurement may be performed without limitation, including both a qualitative method (analysis) and a quantitative method. For measuring the presence or absence of the protein or its mRNA, the types of qualitative and quantitative methods are well known in the art, and the experimental methods described herein are included therein. Specific level comparison methods for each method are well known in the art.

In the present specification, the term “diagnosis” refers to determining the susceptibility of an object to a specific disease or disease, determining whether an object currently has a specific disease or disease, or having a specific disease or disorder. It is a concept that includes both determining the prognosis of an object, or therametrics (eg, monitoring the condition of an object to provide information on treatment efficacy). In the present specification, the diagnosis may preferably mean diagnosis, onset prediction, or prognosis, but is not limited thereto.

In the present specification, "expression" means that a protein or nucleic acid is produced in a cell.

First, the present invention relates to a method for providing information for diagnosing glioblastoma or a method for providing information for predicting prognosis of a patient with glioblastoma, including the following steps.

Obtaining a biological sample; And

STING (Stimulator of interferon genes), CALU (Calumenin), SEPT5 (Septin5), CD99, CIT (Citron Rho-interacting kinase), PXMP4 (Peroxisomal membrane protein 4), Nectin-2, Dol-P- Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase, DLL3 (Delta-like protein 3), Noelin-2, TNS1 (Tensin-1), CAV1 (Caveolin-1), FCN2 (Ficolin-2), KIFC1(Kinesin Family Member C1), HIDE1(Highly expressed in immature dendritic cell transcript 1), CLEC2B(C-type lectin domatin family 2 member B), CD207(C-type Lectin Domain Family 4 Member K ) And CLEC12A (C-type lectin domain family 12 member A) measuring the expression level of one or more selected proteins or mRNAs thereof from the group consisting of.

In the present invention, STING may be derived from mammals, preferably derived from humans. In addition, STING in the present invention may include or consist of an amino acid sequence of human STING (UniProtKB/Swiss-Prot: Q86WV6.1) represented by SEQ ID NO: 1, but is not limited thereto.

In the present invention, CALU may be derived from a mammal, preferably derived from a human. In addition, the CALU in the present invention may include or consist of the amino acid sequence of human CALU (UniProtKB/Swiss-Prot: O43852.2) represented by SEQ ID NO: 2, but is not limited thereto.

In the present invention, SEPT5 may be derived from mammals, preferably derived from humans. In addition, SEPT5 in the present invention may include or consist of the amino acid sequence of human SEPT5 (UniProtKB/Swiss-Prot: Q99719.1) represented by SEQ ID NO: 3, but is not limited thereto.

In the present invention, CD99 may be derived from mammals, preferably derived from humans. In addition, CD99 in the present invention may include or consist of the amino acid sequence of human CD99 (UniProtKB/Swiss-Prot: P14209.1) represented by SEQ ID NO: 4, but is not limited thereto.

In the present invention, the CIT may be derived from a mammal, preferably derived from a human. In addition, the CIT in the present invention may include or consist of a human CIT (UniProtKB/Swiss-Prot: O14578.2) amino acid sequence represented by SEQ ID NO: 5, but is not limited thereto.

In the present invention, PXMP4 may be derived from a mammal, preferably derived from a human. In addition, PXMP4 in the present invention may include or consist of the amino acid sequence of human PXMP4 (UniProtKB/Swiss-Prot: Q9Y6I8.3) represented by SEQ ID NO: 6, but is not limited thereto.

In the present invention, Nectin-2 may be derived from mammals, preferably derived from humans. In addition, Nectin-2 in the present invention may include or consist of the amino acid sequence of human Nectin-2 (UniProtKB/Swiss-Prot: Q92692.1) represented by SEQ ID NO: 7, but is not limited thereto.

In the present invention, ALG3 may be derived from mammals, preferably derived from humans. In addition, ALG3 in the present invention may include or consist of the amino acid sequence of human ALG3 (UniProtKB/Swiss-Prot: Q92685.1) represented by SEQ ID NO: 8, but is not limited thereto.

In the present invention, DLL3 may be derived from mammals, preferably derived from humans. In addition, DLL3 in the present invention may include or consist of the amino acid sequence of human DLL3 (UniProtKB/Swiss-Prot: Q9NYJ7.1) represented by SEQ ID NO: 9, but is not limited thereto.

In the present invention, Noelin-2 may be derived from mammals, preferably derived from humans. In addition, Noelin-2 in the present invention may include or consist of the amino acid sequence of human Noelin-2 (UniProtKB/Swiss-Prot: O95897.2) represented by SEQ ID NO: 10, but is not limited thereto.

In the present invention, TNS1 may be derived from mammals, preferably derived from humans. In addition, TNS1 in the present invention may include or consist of the amino acid sequence of human TNS1 (UniProtKB/Swiss-Prot: Q9HBL0.2) represented by SEQ ID NO: 11, but is not limited thereto.

In the present invention, CAV1 may be derived from mammals, preferably derived from humans. In addition, CAV1 in the present invention may include or consist of the amino acid sequence of human CAV1 (UniProtKB/Swiss-Prot: Q03135.4) represented by SEQ ID NO: 12, but is not limited thereto.

In the present invention, FCN2 may be derived from mammals, preferably derived from humans. In addition, FCN2 in the present invention may include or consist of the amino acid sequence of human FCN2 (UniProtKB/Swiss-Prot: Q15485.2) represented by SEQ ID NO: 13, but is not limited thereto.

In the present invention, KIFC1 may be derived from a mammal, preferably derived from a human. In addition, KIFC1 in the present invention may include or consist of the amino acid sequence of human KIFC1 (UniProtKB/Swiss-Prot: Q9BW19.2) represented by SEQ ID NO: 14, but is not limited thereto.

In the present invention, HIDE1 may be derived from mammals, and preferably derived from humans. In addition, HIDE1 in the present invention may include or consist of the amino acid sequence of human HIDE1 (UniProtKB/Swiss-Prot: A8MVS5.2) represented by SEQ ID NO: 15, but is not limited thereto.

In the present invention, CLEC2B may be derived from a mammal, preferably derived from a human. In addition, CLEC2B in the present invention may include or consist of the amino acid sequence of human CLEC2B (UniProtKB/Swiss-Prot: Q92478.2) represented by SEQ ID NO: 16, but is not limited thereto.

In the present invention, CD207 may be derived from mammals, preferably derived from humans. In addition, CD207 in the present invention may include or consist of the amino acid sequence of human CD207 (UniProtKB/Swiss-Prot: Q9UJ71.2) represented by SEQ ID NO: 17, but is not limited thereto.

In the present invention, CLEC12A may be derived from mammals, and preferably derived from humans. In addition, CLEC12A in the present invention may include or consist of the amino acid sequence of human CLEC12A (UniProtKB/Swiss-Prot: Q5QGZ9.3) represented by SEQ ID NO: 18, but is not limited thereto.

The biological sample may be used without limitation as long as it is collected from a subject, and may be, for example, cells or tissues obtained by biopsy, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, various secretions, urine, feces, and the like. Preferably, it may be to use a biological liquid sample (body fluid sample), for example tissue, cells, blood, plasma, serum, saliva, nasal fluid, sputum, joint capsule fluid, amniotic fluid, ascites, cervical secretion, vaginal secretion, urine And it may be to use cerebrospinal fluid. The present invention is of great technical significance in that it is possible to diagnose through bodily fluid samples (eg, plasma) other than the direct brain lesion tissue in which glioblastoma has occurred, and thus the effectiveness, speed, convenience and safety of diagnosis are increased. In the present invention, the sample may most preferably be a tissue, particularly a brain tissue, and the tissue may be a formalin-fixed paraffin-embedded (FFPE) sample. The biological sample may be a gene sample, and the gene sample includes DNA, mRNA, or cDNA synthesized from mRNA.

The sample may be prepared to increase the detection sensitivity of a protein marker. For example, a serum sample obtained from a patient is anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, It can be pretreated using a method such as sequential extraction or gel electrophoresis.

The sample can be pretreated prior to use for detection or diagnosis. For example, it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.

The measurement of the protein expression level is not particularly limited as long as it is by a protein expression measurement method known in the art, but for example, Western blot, ELISA, radioimmunoassay, radiation immunodiffusion method, Ouchterlony immune diffusion method (Ouchterlony immunodiffusion), rocket immunoelectrophoresis, immunostaining, immunoprecipitation assay, complement fixation assay, mass spectrometry, FACS, and protein chip.

The gene expression level measurement is a group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization, and DNA chip according to a gene expression measurement method known in the art. It may be one or more selected from.

In the present specification, the method is considered to have glioblastoma when the expression level of one or more proteins selected from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4 and Nectin-2 or its mRNA is low compared to the control group. The determining step; may further include, but is not limited thereto. Here, the control group may be a normal person who does not suffer from glioblastoma.

In the present specification, the method is when the expression level of one or more proteins selected from the group consisting of ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A or its mRNA is high compared to the control group. It may further include, but is not limited to, the step of determining whether or not to have glioblastoma. Here, the control group may be a normal person who does not suffer from glioblastoma.

In an embodiment of the present invention, it was confirmed that the growth rate of glioblastoma was increased in the STING knockout mouse model, and it was confirmed that the survival rate of the High STING/Low ALG3 group was higher, when the STING protein or gene expression was reduced. And/or if the expression of the ALG3 protein or gene is increased, it may be determined that it has or will have glioblastoma.

In this specification, "prognosis" refers to a prospect for a future symptom or course determined by diagnosing a disease. In cancer patients, the prognosis usually refers to the recurrence of cancer or metastasis within a certain period of time after surgical procedure or survival period. Prediction of the prognosis (or diagnosis of the prognosis) is a very important clinical task as it provides clues on the direction of future glioblastoma treatment, including chemotherapy, especially for glioblastoma patients. The prediction of prognosis includes prediction of the patient's response to the disease treatment and the course of treatment.

In the present specification, the prognosis prediction may be a prognosis prediction or a treatment response prediction for an angiogenesis inhibitor treatment, but is not limited thereto.

In the present invention, the term "predicting the prognosis for angiogenesis inhibitor treatment" means to predict whether a patient with glioblastoma will respond favorably or unfavorably to angiogenesis inhibitor treatment To do, or to predict the risk of resistance or angiogenesis inhibitor resistance. The prediction method of the present invention can be used clinically to make a treatment decision by selecting the most appropriate treatment regimen for a patient with glioblastoma. The appropriate treatment method may include radiation therapy and/or immunotherapy, but is not limited thereto.

In the present invention, the angiogenesis inhibitor is not limited as long as it is an angiogenesis inhibitor used for the treatment of glioblastoma patients, for example, bevacizumab, 2'3'-cGAMP, fumagiline, Shikonin, Tranilast, ursolic acid; Suramin; Thalidomide, lenalidomide; Phthalargine, as a non-limiting example, 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine, 1-(4-methylanilino)-4-(4-pyridyl Methyl)phthalazine, 1-(3-chloroanilino)-4-(4-pyridylmethyl)phthalazine, 1-anilino-4-(4-pyridylmethyl)phthalazine, 1-benzyl Amino-4-(4-pyridylmethyl)phthalazine, 1-(4-methoxyanilino)-4-(4-pyridylmethyl)phthalazine, 1-(3-benzyloxyanilino)- 4-(4-pyridylmethyl)phthalazine, 1-(3-methoxyanilino)-4-(4-pyridylmethyl)phthalazine, 1-(2-methoxyanilino}-4- (4-pyridylmethyl)phthalazine, 1-(4-trifluoromethylanilino)-4-(4-pyridylmethyl)phthalazine, 1-(4-fluoroanilino)-4- (4-pyridylmethyl)phthalazine, 1-(3-hydroxyanilino)-4-(4-pyridylmethyl)phthalazine, 1-(4-hydroxyanilino)-4-(4 -Pyridylmethyl)phthalazine, 1-(3-aminoanilino)-4-(4-pyridylmethyl)phthalazine, 1-(3,4-dichloroanilino)-4-(4-pyridyl Dylmethyl)phthalazine, 1-(4-bromoanilino)-4-(4-pyridylmethyl)phthalazine, 1-(3-chloro-4-methoxyanilino)-4-(4 -Pyridylmethyl)phthalazine, 1-(4-cyanoanilino)-4-(4-pyridylmethyl)phthalazine, 1-(3-chloro-4-fluoroanilino)-4- (4-pyridylmethyl)phthalazine, 1-(3-methylanilino)-4-(4-pyridylmethyl)phthalazine and PCT Patent Application Publication No. WO 98/035958 (Bold et al., the present invention) PCT Patent Application Publication No. WO, including other phthalazines, 1-(3,5-dimethylanilino)-4-(pyridin-4-ylmethyl)-isoquinoline described in (the entire contents of which are incorporated herein by reference). Isoquinoline disclosed in 00/09495 (Altmann et al., the entire contents of which are incorporated herein by reference); Phthalar disclosed in PCT Patent Application Publication No.WO 00/59509 (Bold et al., the entire contents of which are incorporated herein by reference) As a gin, these include E-1-(3-methylanilino)-4-[(2-(pyridin-3-yl)vinyl]phthalazine, Z-1-(3-methylanilino)-4- [(2-(pyridin-3-yl)vinyl]phthalazine, 1-(3-methylanil) Lino)-4-[(2-(pyridin-3-yl)ethyl]phthalazine, 1-(3-methylanilino)-4-[ {2-(pyridin-4-yl)vinyl]phthalazine , 1-(4-chloro-3-trifluoromethylanilino)-4-[(2-(pyridin-3-yl)ethyl]phthalazine, 1-(4-chloroanilino)-4-[ (2-(pyridin-3-yl)ethyl]phthalazine, 1-(3-chlorobenzylamino)-4-[(2-(pyridin-3-yl)ethyl]phthalazine, 1-(4- Chloro-3-trifluoromethylanilino)-4-[3-(pyridin-3-yl)propyl]phthalazine, 1-(4-chloroanilino)-4-[3-(pyridin-3- Yl)propyl]phthalazine, 1-(3-chloro-5-trifluoromethylanilino)-4-[3-(pyridin-3-yl)propyl]phthalazine and 1-(4-tertiary -Butylanilino)-4-[3-(pyridin-3-yl)propyl]phthalazine is included; And a monoclonal antibody; may be.

In the present invention, the method comprises a protein selected from the group consisting of STING, Calumenin, SEPT5, CD99, CIT, Peroxisomal membrane protein 4 and Nectin-2, or when the expression level of mRNA thereof is high compared to the first control, the prognosis is It may further include determining that it is good. Here, the first control group may be a normal person or a treatment non-responsive group. The treatment non-responder group refers to a patient who responds unfavorably to the treatment of glioblastoma, or does not exhibit a response or therapeutic effect among glioblastoma patients.

In the present invention, the method is STING, Calumenin, SEPT5, CD99, CIT, Peroxisomal membrane protein 4 and Nectin-2, when the expression level of one or more proteins selected from the group consisting of or mRNA thereof is high compared to the first control, angiogenesis It may further comprise the step of determining that the therapeutic responsiveness of the inhibitor is high. Here, the first control group may be a normal person or an angiogenesis inhibitor non-responsive group. The angiogenesis inhibitor non-responder group refers to a patient who responds unfavorably to the angiogenesis inhibitor treatment, or does not show a response or therapeutic effect among glioblastoma patients.

In the present invention, the method includes the expression level of one or more proteins selected from the group consisting of ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A or their mRNA expression level compared to the second control group. If it is high, it may further include determining that the prognosis is poor. Here, the second control group may be a normal person or a treatment responder. The treatment response group refers to a patient who responds favorably to glioblastoma treatment or shows a therapeutic effect among glioblastoma patients.

In the present invention, the method includes the expression level of one or more proteins selected from the group consisting of ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A or their mRNA expression level compared to the second control group. If it is high, the step of determining that the therapeutic responsiveness of the angiogenesis inhibitor is high may be further included. Here, the second control group may be an angiogenesis inhibitor non-responsive group. The angiogenesis inhibitor non-responder group refers to a patient who responds unfavorably to the angiogenesis inhibitor treatment, or does not show a response or therapeutic effect among glioblastoma patients.

When the composition of the present invention is for measuring the expression level of mRNA, the agent for measuring the mRNA expression level may be a set of probes or primers that specifically bind to mRNA.

The composition of the present invention comprising the mRNA-specific probe or primer set of the protein may further include an agent required for a known method of detecting RNA. Using the present composition, a known method for detecting RNA can be used without limitation to measure the level of the mRNA of the markers in a subject.

In the present invention, "primer" refers to a short nucleic acid sequence capable of forming a base pair with a complementary template with a nucleic acid sequence having a short free three-terminal hydroxyl group and serving as a starting point for template strand copying. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffers and temperatures.

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases or hundreds of bases for a specific binding to an mRNA, and is labeled to determine the presence or absence of a specific mRNA. I can confirm. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. Selection of suitable probes and conditions for hybridization can be modified based on those known in the art.

In the present invention, the primer or probe may be chemically synthesized using a method for synthesizing a solid support of phosphoramidite or other well-known methods. In addition, primers or probes can be variously modified according to methods known in the art within a range that does not interfere with hybridization with mRNA. Examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkers (e.g. methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc. ) Or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.), and binding of a labeling material using fluorescence or enzymes.

Meanwhile, the agent for measuring the protein expression level in the present invention may be an antibody or aptamer that specifically binds to the protein.

In the present invention, "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes all of polyclonal antibodies, monoclonal antibodies and recombinant antibodies. In addition, as long as it has antigen-antibody binding, a part of the whole antibody is also included in the antibody of the present invention, and all kinds of immunoglobulin antibodies that specifically bind to the protein of the present invention are included. For example, antibodies in complete form with two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules, i.e., Fab, F(ab'), F(ab') with antigen-binding function 2 and Fv, and the like. Furthermore, the antibody of the present invention includes special antibodies such as humanized antibodies and chimeric antibodies, and recombinant antibodies as long as it can specifically bind to the protein of the present invention.

In the present invention, the term "aptamer" refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure as a substance capable of specifically binding to an analyte to be detected in a sample. , Specifically, the presence of the target protein in the sample can be confirmed. The aptamer is synthesized by determining the sequence of an oligonucleotide having a high binding ability and selective to the target protein to be identified according to a general aptamer manufacturing method, and then applying the 5'or 3'end of the oligonucleotide. In order to bind to the functional group of the timer chip, it may be _{made by modifying it with -SH, -COOH, -OH or NH 2} , but is not limited thereto.

The composition of the present invention containing the protein-specific antibody may additionally include an agent required for a method of detecting a known protein, and a subject ( In the present invention, it is possible to measure the expression level of a protein in a glioblastoma patient or a biological sample obtained from a patient).

Since the nucleic acid sequence of the gene of the present invention is registered in the gene bank, those skilled in the art can design antisense oligonucleotides, primer pairs, or probes that specifically amplify specific regions of these genes based on the above sequences.

The antisense oligonucleotides, primers, or probes of the present invention can be chemically synthesized using the phosphor amidite solid support method, or other well known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkers (e.g. methyl phosphonate, phosphotriester, phosphoroami Date, carbamate, etc.) or to a charged linker (e.g. phosphorothioate, phosphorodithioate, etc.).

The composition of the present invention may further include an excipient, a carrier, a labeling substance capable of generating a detectable signal, a solubilizing agent, a detergent, etc., but is not limited thereto. When the labeling substance is an enzyme, a substrate capable of measuring enzyme activity and a reaction terminator may be included together. Examples of the carrier include, but are not limited to, a soluble carrier such as a physiologically acceptable buffer known in the art, such as PBS; Insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymers such as magnetic fine particles plated with metal on latex, other paper, glass, metal , Agarose, and combinations thereof.

In addition, the present invention provides a kit for diagnosing glioblastoma or predicting prognosis comprising the composition and instructions.

The kit of the present invention may include an antibody that recognizes a target protein as a marker or a primer, a probe that recognizes an mRNA as a marker, as well as one or more other component compositions, solutions, or devices suitable for an analysis method.

The kit of the present invention may include instructions including at least one selected from the group consisting of a description of how to use the kit, a glioblastoma diagnostic index, and a glioblastoma therapy.

As a specific embodiment, the kit may be a diagnostic kit, characterized in that it contains essential elements necessary to perform a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit contains each primer pair specific for a marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. In addition, a primer specific to the nucleic acid sequence of the control gene may be included. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (various pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitors DEPC. -May include DEPC-water, sterilized water, etc.

In another aspect, it may be a diagnostic kit characterized in that it contains essential elements necessary to perform the DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescently labeled probe. In addition, the substrate may include a cDNA or oligonucleotide corresponding to a control gene or a fragment thereof.

In addition, the kit of the present invention may include a washing solution or an eluent capable of removing substrates for color development reaction with enzymes and unbound proteins, and retaining only bound protein markers.

Contacting the cell with the test substance;

The term "test substance" used while referring to the screening method of the present invention means an unknown substance used in screening to test whether it affects the expression of the protein or gene of the present invention. The test substances are siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, PNA (peptide nucleic acids), antisense oligonucleotides, antibodies, aptamers, natural extracts, or Including, but not limited to, chemicals.

In addition, the cells used in the screening method may be provided in the form of an experimental animal, in which case the screening method of the present invention further comprises the step of inducing glioblastoma to the experimental animal, and contact with the test substance Parenteral or oral administration, including, but not limited to, stereotactic injection, and those skilled in the art will be able to select an appropriate method for testing the test substance on an animal.

Treating the test substance means adding the test substance to the cell or tissue culture medium and then culturing the cells for a certain time. When the cells are provided in the form of an experimental animal, contact with the test substance is not limited thereto, including parenteral or oral administration, or stereotactic injection, and those skilled in the art can select an appropriate method for testing the test substance on the animal. will be.

In the screening method, the step of selecting one or more proteins selected from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4, and Nectin-2 or a substance having increased mRNA levels thereof as a glioblastoma therapeutic agent; may further include However, it is not limited thereto.

In the screening method, at least one protein selected from the group consisting of ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A or a substance having reduced mRNA level thereof is selected as a glioblastoma therapeutic agent. The step of; may further include, but is not limited thereto.

In the screening method, the cell may be a cell that internally expresses the protein or its mRNA, or is transformed with a recombinant expression vector comprising a polynucleotide encoding the protein or its mRNA to overexpress the protein or its mRNA. It may be a cell that does.

In the present invention, "knock-out" means partial, substantial, complete deletion or inactivation of a gene.

In the present invention, the animal model is not limited as long as it is an animal that can be reared in a cage except for humans, and includes rodents, rabbits and monkeys, but may be preferably mice and rats for experimentation.

Rats belonging to rodents are the most used in animal experiments, and in particular, mice for experiments include mice (mus musculus: mice) and rats (rattus norvegicus: rats), and are experimental animals in that they are easy to raise, reproduce quickly, and are inexpensive. It is advantageous as, and studies on behavior patterns corresponding to various symptoms of patients have been matured, making it more suitable as an animal model.

The terms used in the present invention have been selected from general terms that are currently widely used while considering functions in the present invention, but this may vary depending on the intention or precedent of a technician working in the field, the emergence of new technologies, and the like. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning of the terms will be described in detail in the description of the corresponding invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall contents of the present invention, not a simple name of the term.

Throughout the specification of the present invention, when a certain part "includes" a certain constituent element, it means that other constituent elements may be further included rather than excluding other constituent elements unless otherwise stated. The terms "about", "substantially", etc. of the degree used throughout the specification of the present invention are used at or close to the numerical value when manufacturing and material tolerances specific to the stated meaning are presented, and the present invention To aid in understanding of, accurate or absolute figures are used to prevent unreasonable use of the stated disclosure by unscrupulous infringers.

Throughout the specification of the present invention, the term "combination of these" included in the expression of the Makushi format refers to one or more mixtures or combinations selected from the group consisting of components described in the expression of the Makushi format, the It means to include one or more selected from the group consisting of components.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the examples.

실시예 1. 실험 방법Example 1. Experimental method

Differences in protein expression were analyzed in the tissues of glioblastoma patients (FFPE, formalin fixed paraffin embedding) (7 responders to anti-vascular treatment, 7 non-responders) obtained before anti-angiogenic treatment, proteomic analysis was performed using LC-MS. Was used to extract a protein with a statistically significant difference among 5,000 cancer-related proteins.

In addition, to determine whether there is a difference in the prognosis of glioblastoma patients according to the expression of STING and ALG3 regardless of the treatment method, the STING and ALG3 patients of 7 glioblastoma patients with good prognosis and 7 glioblastoma patients with poor prognosis. The amount of expression was confirmed by immunostaining in FFPE tissue.

Specifically, a method of extracting a protein from a paraffin section for performing the experimental process of FIG. 2 is as follows.

First, after dividing the patient group administered with an angiogenesis inhibitor (bevacizumab, Avastin ^® ) for malignant glioma into responders and non-responders, each patient's paraffin tissue was cut into 10 μm thickness and Qiagen's Qproteome FFPE tissue kit was used The experiment was carried out. After collecting the cut tissue in a 1.5ml tube, 0.5ml heptane was added, vortexed for 10 seconds, and then incubated at room temperature for 1 hour to dissolve paraffin. Thereafter, 25 μl of methanol was added, vortexed for 10 seconds, centrifuged at 9000 g for 2 minutes to obtain a pellet formed at the bottom of the tube, and dried for 5 minutes. Then, 100 μl of extraction buffer (beta-mercaptoethanol was added) was mixed, incubated on ice for 5 minutes, vortexed, and then incubated at 100°C for 20 minutes and 80°C for 2 hours at 750 rpm. At the end of this process, after being put on ice for 1 minute, the sealing clip of the collection tube was removed, followed by centrifugation at 14000g and 4°C for 15 minutes. Then, the supernatant from which the protein was extracted was transferred to a new 1.5ml tube and quantified through BCA to measure the protein concentration. Further, into 1ml of ethanol to the tube and mixed to remove the supernatant was centrifuged for 2 min at 9000g and, 25mM NH ₄ HCO ₃ in 1% RapiGest SF 10μl with 25mM NH ₄ HCO ₃ of 50mM DTT 10μl pellet Reduction was performed. After incubation at 950 rpm for 1 hour while maintaining the temperature at 37° C., 10 μl of 100 mM iodoacetamide in _{25 mM NH 4} HCO _{3 was mixed to perform alkylation.} Thereafter, incubation was performed at 950 rpm for 1 hour at a temperature of 37° C., and vortexing was performed every 10 minutes. Thereafter, 90μl of 25mM NH ₄ HCO ₃ was added, followed by 20 μl of 0.25ug/ul trypsin in _{25mM NH 4} HCO _3. At this time, the ratio of enzyme and protein was set to 1:5. After incubation at a temperature of 37° C. for 16 hours or longer, 20 μl of 5% TFA was added to stop digestion, followed by incubation at 37° C. for 1 hour at 950 rpm. Then, after centrifugation at 13000rpm for 30 minutes at a temperature of 25°C, the supernatant was transferred to a 0.5ml tube, dried using a speedvac evaporator vacumn, and then dissolved in 0.1% formic acid to perform the LC-MS analysis below.

The conditions for liquid chromatography using the Thermo Ultimate 3000 system are as follows.

-Column: 50cm length, 75um I.D, 360 um O.D fused silica C18

LC rum time: 200 min

Flow rate: 350 nl/min

Gradient: 5% SolB to 50% SolB during 150 min gradient

SolA: 0.1% formic acid with 5% DMSO

SolB: 80% acetonitrile, 0.1% formic acid with 5% DMSO

The conditions for mass spectrometry using Thermo Q Exactive Plus Mass spectrometry are as follows.

MS1 resolution: 70000

MS1 maximum fill time: 20 msec

Data dependent acquisition (DDA) methods (Top 20)

MS2 resolution: 17500

MS2 maximum fill time: 100 msec

Auto gain control (AGC): 1e6

The spectral files for the peptide and fragment ions obtained through mass spectrometry were identified using the SwissProt human proteome sequence with Proteome Discoverer (version 2.2, Thermo) based on the Sequest HT algorithm. In addition, protein relative quantification was performed using label-free quantitation using the intensity of the laser peptide and the unique peptide of the proteins within each group. Principal component analysis and hierarchical cluster analysis were performed using the protein quantification values of each group. In addition, volcano plot analysis was performed using the P value and ratio at the protein level. Venn diagram analysis was performed using jvenn (http://jvenn.toulouse.inra.fr/app/index.html) to analyze differentially expressed proteins.

실시예 2. 교모세포종 환자의 항혈관 치료 반응성에 대한 단백질 발현 확인Example 2. Confirmation of protein expression for anti-vascular reactivity in glioblastoma patients

According to the above results, there were a total of 200 proteins showing statistically significant differences, and proteins expressed only in a specific group were extracted. Some were expressed only in the group that had a good anti-vascular treatment response, and some were expressed only in the group that had a poor response.

The results of comparing protein expression are shown in Table 1 and FIGS. 3 to 17. The blue bars in FIGS. 3 to 17 represent the average value of protein expression.

그룹group	발현량이 높은 단백질High expression protein
항혈관 치료 반응이 좋았던 그룹 (good-responder group)Good-responder group	1. STING (Stimulator of interferon gene protein) 2. Calumenin (isoform 4) (CALU) 3. Septin-5 (isoform 2) (SEPT5) 4. CD99 antigen (CD99) 5. Citron Rho-interacting kinase (isoform 4) (CIT) 6. Peroxisomal membrane protein 4 (PXMP4) 7. Nectin-2 (NECTIN2) 1. STING (Stimulator of interferon gene protein) 2. Calumenin (isoform 4) (CALU) 3. Septin-5 (isoform 2) (SEPT5) 4. CD99 antigen (CD99) 5. Citron Rho-interacting kinase (isoform 4) (CIT) 6.Peroxisomal membrane protein 4 (PXMP4) 7.Nectin-2 (NECTIN2)
항혈관 치료 반응이 없었던 그룹 (bad-responder group)The group that did not respond to anti-vascular treatment (bad-responder group)	1. Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase (ALG3) 2. Delta-like protein 3 (DLL3) 3. Noelin-2 4. Tensin-1 (TNS1) 5. Caveolin-1 (CAV1) 6. Ficolin-2 (FCN2) 7. Kinesin-like protein KIFC1(Kinesin Family Member C1, KIFC1) 8. Protein HIDE1(Highly expressed in immature dendritic cell transcript 1, HIDE1) 1.Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase (ALG3) 2. Delta-like protein 3 (DLL3) 3. Noelin-2 4. Tensin-1 (TNS1) 5. Caveolin-1 (CAV1) 6. Ficolin-2 (FCN2) 7. Kinesin-like protein KIFC1 (Kinesin Family Member C1, KIFC1) 8. Protein HIDE1(Highly expressed in immature dendritic cell transcript 1, HIDE1)

실시예 3. 교모세포종 환자의 예후 예측을 위한 단백질 발현 확인Example 3. Confirmation of protein expression for predicting prognosis of patients with glioblastoma

Samples were obtained in the same manner as in Example 1, and protein expression levels were measured according to the prognosis of glioblastoma patients regardless of the treatment method.

As a result, as shown in FIGS. 18 to 20, C-type lectin domatin family 2 member B (CLEC2B), C-type Lectin Domain Family 4 Member K (CD207), C in patients with poor prognosis among glioblastoma patients. It was confirmed that -type lectin domain family 12 member A (CLEC12A) was highly expressed.

실시예 4. 교모세포종 환자의 FFPE 조직에서 STING 단백질의 발현 확인Example 4. Expression of STING protein in FFPE tissue of glioblastoma patient

By immunostaining the FFPE tissue analyzed in Example 1, the expression location and expression pattern of the STING protein were confirmed in patients with good and poor treatment prognosis.

First, a paraffin tissue section slide cut into 4 μm was put in a 60ºC dry oven to dissolve the paraffin, and then immersed in 100% xylene for 10 minutes to perform deparaffination three times. From 100% ethanol to 95%-90%-80%-70%-distilled water, it was hydrated by sequentially immersing for 5 minutes each. Then, it was rinsed with distilled water for 10 minutes. Thereafter, using an antigen retrieval solution of pH 6, the tissue was placed in a jar for 30 minutes and heated to perform an Antigen unmasking process. Thereafter, a permeation process was performed using PBS-T (containing 0.3% Triton x 100) buffer. Thereafter, 1% of BSA was dissolved in PBS-T, followed by a blocking process at room temperature for 30 minutes, and then the primary antibody was diluted 1:100 and incubated overnight at 4°C. The next day, the primary antibody was washed and the secondary antibody with fluorescence was incubated for 2 hours. After the washing process, DAPI staining was performed, and after mounting, the tissue was observed with a confocal microscope.

As a result, as shown in Fig. 21, it was confirmed that the STING protein expression level was significantly higher in the patient with a good treatment prognosis compared to the group with a poor treatment prognosis.

실시예 5. 교모세포종 환자의 FFPE 조직에서 ALG3 단백질의 발현 확인Example 5. Expression of ALG3 protein in FFPE tissue of glioblastoma patient

The analyzed FFPE tissue was immunostained, and the expression position and expression pattern of the ALG3 protein were confirmed in patients with good and poor treatment prognosis.

Specifically, for immunostaining, paraffin tissue section slides cut into 4 μm were placed in a 60ºC dry oven to dissolve paraffin, and then deparaffinization was performed 3 times by immersing in 100% xylene for 10 minutes. From 100% ethanol to 95%-90%-80%-70%-50%-distilled water, it was hydrated by sequentially immersing for 5 minutes each. The slide was immersed in an antigen retrieval solution and heated in a steamer for 15 minutes to recover the receptor protein. After washing with PBS, it was _{treated with 3% H 2} O ₂ /Methanol for 20 minutes to remove endogenous peroxidase. After washing with PBS and blocking with 2.5% normal horse serum for 1 hour, rabbit anti-ALG3 antibody was diluted 1:100 and reacted overnight at 4ºC. After washing with PBS, the biotinylated anti-rabbit secondary antibody was reacted for 30 minutes at room temperature. After washing with PBS, the peroxidase-streptavidin complex was reacted at room temperature for 15 minutes, and then washed with PBS. After reacting for 10 minutes using a DAB Substrate kit, color development was confirmed, and then washed with distilled water. After dehydration in the reverse order of hydration, the cover slide was closed with a mounting solution and observed under a microscope.

As a result, as shown in FIG. 22, it was confirmed that the expression level of ALG3 protein was remarkably high only in patients with poor treatment prognosis.

실시예 6. 교모세포종 환자의 생존율 확인Example 6. Confirmation of survival rate of glioblastoma patients

As confirmed in Examples 4 and 5, patients with good prognosis showed high expression of STING, while patients with poor prognosis showed little expression of STING. Conversely, the expression level of ALG3 was hardly detected in patients with a good prognosis, and it was found to be high in patients with a poor prognosis.

In addition, the survival rate of patients according to the expression levels of STING and ALG3 was analyzed.

As a result, as shown in FIG.23, it was confirmed that the overall survival rate and progression free survival of patients with high STING/Low ALG3 glioblastoma were all better compared to those of Low STING/High ALG3 patients. Could

Therefore, it was confirmed that STING and ALG3 can be used not only as a prediction of anti-vascular treatment but also as an overall prognostic marker for glioblastoma.

실시예 7. STING 유전자가 결손된 마우스 동물모델 제작Example 7. Production of mouse animal model with a lack of STING gene

An animal model of glioblastoma was constructed by deleting the STING gene.

Specifically, in order to produce mice in which the STING gene was knocked-out all over the body, EGFR viii/viii mice were crossed to obtain systemic STING-deficient mice. Within 1-2 days of birth, a spontaneous glioblastoma animal model was constructed by injecting CRISPR cas9 TP53/PTEN targeting cre plasmid into the subventricular zone (SVZ) of the brain. Tumor formation was confirmed 6-8 weeks after plasmid injection, and tumor formation was confirmed by taking a mouse brain T2 image on a coronal plane through small animal MRI. After selecting only the tumor-forming mouse, 3 weeks passed and follow-up was performed to confirm the change in tumor size.

As a result, as shown in Fig. 24, it was confirmed that the growth rate of the tumor was increased in the mouse in which the STING gene was deleted.

This is a result showing that STING KO mice can be used as an animal model of glioblastoma.

As described above, the expression level of the protein may be measured for diagnosing glioblastoma, predicting the prognosis of a patient, and in particular, predicting the therapeutic response of an anti-angiogenic agent to a patient. In addition, the kit provided by the present inventors can be used to diagnose glioblastoma from a patient sample, as well as to quickly review the prognosis including the reactivity of an antiangiogenic agent.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

The present inventors have confirmed that there is a close correlation between the expression of a number of specific proteins in the tissues of patients with glioblastoma, which is a malignant glioma, and predicting the onset and prognosis of glioblastoma.By confirming the level of expression of the protein or its mRNA, glioblastoma It has industrial applicability as it can provide useful information for the treatment and prognosis of patients.

Claims

A method of providing information for diagnosis of glioblastoma, comprising the following steps:

Obtaining a biological sample;

STING (Stimulator of interferon genes), CALU (Calumenin), SEPT5 (Septin5), CD99, CIT (Citron Rho-interacting kinase), PXMP4 (Peroxisomal membrane protein 4), Nectin-2, ALG3 (Dol- P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase), DLL3 (Delta-like protein 3), Noelin-2, TNS1 (Tensin-1), CAV1 (Caveolin-1) ), FCN2(Ficolin-2), KIFC1(Kinesin Family Member C1), HIDE1(Highly expressed in immature dendritic cell transcript 1), CLEC2B(C-type lectin domatin family 2 member B), CD207(C-type Lectin Domain Family) 4 Member K) and CLEC12A (C-type lectin domain family 12 member A) measuring the expression level of one or more selected proteins or mRNAs thereof from the group consisting of; And

A) determining that the expression of one or more proteins selected from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4, and Nectin-2, or mRNA thereof, is low compared to the control group, and that it has glioblastoma or is affected; or

B) If the expression level of one or more proteins selected from the group consisting of ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A is high compared to the control group, glioblastoma has occurred. Or determining that it will take.
The method of claim 1,

The biological sample is characterized in that one or more selected from the group consisting of tissue, cells, blood, plasma, serum, saliva, nasal fluid, sputum, joint capsule fluid, amniotic fluid, ascites, cervical secretion, vaginal secretion, urine, and cerebrospinal fluid. .
A method for diagnosing glioblastoma, comprising the following steps:

Obtaining a biological sample;

STING (Stimulator of interferon genes), CALU (Calumenin), SEPT5 (Septin5), CD99, CIT (Citron Rho-interacting kinase), PXMP4 (Peroxisomal membrane protein 4), Nectin-2, ALG3 (Dol- P-Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase), DLL3 (Delta-like protein 3), Noelin-2, TNS1 (Tensin-1), CAV1 (Caveolin-1) ), FCN2(Ficolin-2), KIFC1(Kinesin Family Member C1), HIDE1(Highly expressed in immature dendritic cell transcript 1), CLEC2B(C-type lectin domatin family 2 member B), CD207(C-type Lectin Domain Family) 4 Member K) and CLEC12A (C-type lectin domain family 12 member A) measuring the expression level of one or more selected proteins or mRNAs thereof from the group consisting of; And

A) determining that the expression of one or more proteins selected from the group consisting of STING, CALU, SEPT5, CD99, CIT, PXMP4, and Nectin-2, or mRNA thereof, is low compared to the control group, and that it has glioblastoma or is affected; or

B) If the expression level of one or more proteins selected from the group consisting of ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A is high compared to the control group, glioblastoma has occurred. Or determining that it will take.
Information providing method for predicting the prognosis of glioblastoma patient, comprising the following steps:

Obtaining a biological sample of a patient with glioblastoma;

In biological samples, STING (Stimulator of interferon genes), CALU (Calumenin), SEPT5 (Septin5), CD99, CIT (Citron Rho-interacting kinase), PXMP4 (Peroxisomal membrane protein 4), Nectin-2, ALG3 (Dol-P- Man:Man(5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase), DLL3 (Delta-like protein 3), Noelin-2, TNS1 (Tensin-1), CAV1 (Caveolin-1), FCN2(Ficolin-2), KIFC1(Kinesin Family Member C1), HIDE1(Highly expressed in immature dendritic cell transcript 1), CLEC2B(C-type lectin domatin family 2 member B), CD207(C-type Lectin Domain Family 4 Member) K) and CLEC12A (C-type lectin domain family 12 member A) measuring the expression level of one or more selected protein or mRNA thereof from the group consisting of; And

i) STING, CALU, SEPT5, CD99, CIT, PXMP4 and Nectin-2, if the expression level of one or more selected protein or mRNA thereof from the group consisting of a high compared to the first control, predicting that the prognosis will be good; or

ii) When the expression level of one or more proteins selected from the group consisting of ALG3, DLL3, Noelin-2, TNS1, CAV1, FCN2, KIFC1, HIDE1, CLEC2B, CD207 and CLEC12A is high compared to the second control, the prognosis is Predicting bad things.
The method of claim 4,

The prognosis prediction is characterized in that the prognosis prediction for angiogenesis inhibitor treatment, the method.
The method of claim 5,

The method of claim 1, wherein the angiogenesis inhibitor is bevacizumab.
STING(Stimulator of interferon genes), CALU(Calumenin), SEPT5(Septin5), CD99, CIT(Citron Rho-interacting kinase), PXMP4(Peroxisomal membrane protein 4), Nectin-2, ALG3(Dol-P-Man:Man) (5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase), DLL3 (Delta-like protein 3), Noelin-2, TNS1 (Tensin-1), CAV1 (Caveolin-1), FCN2 (Ficolin -2), KIFC1 (Kinesin Family Member C1), HIDE1 (Highly expressed in immature dendritic cell transcript 1), CLEC2B (C-type lectin domatin family 2 member B), CD207 (C-type Lectin Domain Family 4 Member K) and CLEC12A (C-type lectin domain family 12 member A) comprising an agent for measuring the expression level of one or more selected proteins or mRNAs from the group consisting of, glioblastoma diagnosis or prognosis prediction composition.
A kit for diagnosing glioblastoma or predicting prognosis, comprising the composition and instructions of claim 7.
STING(Stimulator of interferon genes), CALU(Calumenin), SEPT5(Septin5), CD99, CIT(Citron Rho-interacting kinase), PXMP4(Peroxisomal membrane protein 4), Nectin-2, ALG3(Dol-P-Man:Man) (5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase), DLL3 (Delta-like protein 3), Noelin-2, TNS1 (Tensin-1), CAV1 (Caveolin-1), FCN2 (Ficolin -2), KIFC1 (Kinesin Family Member C1), HIDE1 (Highly expressed in immature dendritic cell transcript 1), CLEC2B (C-type lectin domatin family 2 member B), CD207 (C-type Lectin Domain Family 4 Member K) and CLEC12A (C-type lectin domain family 12 member A), the use of an agent for measuring the expression level of one or more selected proteins or mRNAs from the group consisting of glioblastoma diagnosis or prognosis.
A method for screening for glioblastoma prevention or treatment, comprising the following steps:

Contacting the cell with the test substance;

STING(Stimulator of interferon genes), CALU(Calumenin), SEPT5(Septin5), CD99, CIT(Citron Rho-interacting kinase), PXMP4(Peroxisomal membrane protein 4), Nectin-2, ALG3(Dol-P-Man:Man) (5)GlcNAc(2)-PP-Dol alpha-1,3-mannosyltransferase), DLL3 (Delta-like protein 3), Noelin-2, TNS1 (Tensin-1), CAV1 (Caveolin-1), FCN2 (Ficolin -2), KIFC1 (Kinesin Family Member C1), HIDE1 (Highly expressed in immature dendritic cell transcript 1), CLEC2B (C-type lectin domatin family 2 member B), CD207 (C-type Lectin Domain Family 4 Member K) and Measuring the expression level of one or more selected proteins or mRNAs thereof from the group consisting of CLEC12A (C-type lectin domain family 12 member A); And

Selecting a test substance having a difference in the expression level of the selected protein or its mRNA compared to a control group not contacted with the test substance.
Glioblastoma animal model in which the STING (Stimulator of interferon genes) gene is deleted.
The animal model according to claim 11, wherein the animal model is for screening a glioblastoma therapeutic agent.