JP2019158753A - Biomarkers for lymphangioleiomyomatosis - Google Patents

Abstract

To provide biomarkers for lymphangioleiomyomatosis and a method of using the same.SOLUTION: Disclosed herein is at least one biomarker selected from a group consisting of a group of proteins (A) and a group of miRNAs (B) in extracellular vesicles in a bodily fluid collected from a subject.SELECTED DRAWING: None

Description

  The present invention relates to a biomarker for lymphangioleiomyomatosis, use thereof, and the like.

  Lymphangioleiomyonatosis (LAM) is a chronic, progressive, systemic rare or intractable disease in which smooth muscle cell-like LAM cells proliferate in the lymph nodes of the body, metastasize to the lung, and form multiple cysts. It is. The pathological condition is a low-grade neoplastic disease caused by LAM cells (tumors) caused by abnormalities in the TSC genes (Tscl and Tsc2), which are tumor suppressor genes, metastasize mainly to the lungs through lymphatic vessels. The prevalence rate in Japan is about 1.9 to 4.5 per million population, and mainly affects young women. When lung tissue destruction presents and progresses to chronic respiratory failure, oxygen therapy and even lung transplantation are required. The underlying diseases are classified into TSC-LAM that occurs with tuberous sclerosis complex (TSC) and sporadic LAM that occurs independently (Sporadic-LAM). As a biomarker (BM) for LAM, serum VEGF-D reflects diagnostic and therapeutic effects and is an item of diagnostic criteria, but that alone is not sufficient. Therefore, there is an urgent need to develop additional biomarkers (BM) useful for differentiation and treatment.

  Serum is considered the most ideal sample that can be repeatedly collected non-invasively, but more than 99% is useful from the viewpoint of proteomics because it is a large amount of protein (such as albumin) that is considered as a contaminant from the viewpoint of BM. Is not allowed. For example, disease-specific proteins have not been detected even in the recent LAM patient blood prodeomics. The BM found for analysis using serum containing a large amount of contaminants is only serum contaminants, which is far from LAM-specific BM (Non-patent Document 1).

Banville, PLoS One. 2014 Aug 18; 9 (8): e105365.

  An object of the present invention is to provide a biomarker for lymphangioleiomyomatosis and a method for using the same.

  As a result of diligent research in view of the above problems, the present inventor has found that a specific protein group in extracellular vesicles of a body fluid collected from a subject is useful as a biomarker for lymphangioleiomyomatosis. As a result of further research based on this finding, the present invention was completed. That is, the present invention includes the following aspects.

Item 1.
A method for examining lymphangioleiomyomatosis, comprising:
(1) Protein group (A) and miRNA group (B) in extracellular vesicles of body fluid collected from the subject:
(A) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, CD81 antigen, and protein group consisting of CD63 antigen, and (B) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p , MiR-519d-5p, miR-1915-3p, miR-4707-5p, miR-3663-3p, miR-4755-3p, miR-3654, miR-3184-5p, mi R-5705, miR-4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR-4482-3p, miR-4481, miR-4515, miR-6127, miR- MiRNA group consisting of 3614-5p, miR-6873-5p, miR-4532, miR-6831-5p, miR-451a, miR-16-5p, and miR-1224-3p,
A test method comprising a step of detecting at least one biomarker selected from the group consisting of:

Item 2.
The detected biomarkers are protein group (A1) and miRNA group (B1):
(A1) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, and protein group consisting of CD81 antigen, and (B1) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p, miR- 519d-5p, miR-1915-3p, miR-4707-5p, miR-3663-3p, miR-4755-3p, miR-3654, miR-3184-5p, miR-5705, miR- 4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR-4482-3p, miR-4481, miR-4515, miR-6127, miR-3614-5p, miR- MiRNA group consisting of 6873-5p, miR-4532, and miR-6831-5p,
If it is at least one biomarker selected from the group consisting of:
And (2a) including a step of determining that the subject suffers from lymphangioleiomyomatosis when the amount or concentration of the biomarker detected in the step (1) is equal to or higher than a cutoff value. Item 2. The inspection method according to Item 1.

Item 3.
The detected biomarkers are protein group (A2) and miRNA group (B2):
(A2) a protein group consisting of CD63 antigen, and (B2) a miRNA group consisting of miR-451a, miR-16-5p, and miR-1224-3p,
If it is at least one biomarker selected from the group consisting of:
Further, (2b) includes a step of determining that the subject is suffering from lymphangioleiomyomatosis when the amount or concentration of the biomarker detected in the step (1) is not more than a cutoff value. Item 2. The inspection method according to Item 1.

Item 4.
Item 4. The examination method according to any one of Items 1 to 3, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.

Item 5.
Item 5. The examination method according to any one of Items 1 to 4, wherein the subject is a human.

Item 6.
Protein group (A):
(A) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, CD81 antigen, and protein group consisting of CD63 antigen, and
miRNA group (B):
(B) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p, miR-519d-5p, miR-1915-3p, miR-4707-5p, miR-3663 -3p, miR-4755-3p, miR-3654, miR-3184-5p, miR-5705, miR-4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR -4482-3p, miR-4481, miR-4515, miR-6127, miR-3614-5p, miR-6873-5p, miR-4532, miR-6831-5p, miR-451a, miR-16-5p, and miRNA group consisting of miR-1224-3p,
A test agent for lymphangioleiomyomatosis comprising a detection agent for at least one biomarker selected from the group consisting of:

Item 7.
Protein group (A):
(A) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, CD81 antigen, and protein group consisting of CD63 antigen, and
miRNA group (B):
(B) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p, miR-519d-5p, miR-1915-3p, miR-4707-5p, miR-3663 -3p, miR-4755-3p, miR-3654, miR-3184-5p, miR-5705, miR-4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR -4482-3p, miR-4481, miR-4515, miR-6127, miR-3614-5p, miR-6873-5p, miR-4532, miR-6831-5p, miR-451a, miR-16-5p, and miRNA group consisting of miR-1224-3p,
An agent for preventing or treating lymphangioleiomyomatosis comprising an inhibitor of at least one biomarker selected from the group consisting of:

Item 8.
In the extracellular vesicles of body fluids collected from animals treated with the test substance,
Protein group (A):
(A) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, CD81 antigen, and protein group consisting of CD63 antigen, and
miRNA group (B):
(B) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p, miR-519d-5p, miR-1915-3p, miR-4707-5p, miR-3663 -3p, miR-4755-3p, miR-3654, miR-3184-5p, miR-5705, miR-4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR -4482-3p, miR-4481, miR-4515, miR-6127, miR-3614-5p, miR-6873-5p, miR-4532, miR-6831-5p, miR-451a, miR-16-5p, and miRNA group consisting of miR-1224-3p,
A method for screening an active ingredient of a preventive or therapeutic agent for lymphangioleiomyomatosis, using as an index the amount or concentration of at least one biomarker selected from the group consisting of:

Item 9.
In the extracellular vesicles of body fluids collected from animals treated with the test substance,
Protein group (A):
(A) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, CD81 antigen, and protein group consisting of CD63 antigen, and
miRNA group (B):
(B) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p, miR-519d-5p, miR-1915-3p, miR-4707-5p, miR-3663 -3p, miR-4755-3p, miR-3654, miR-3184-5p, miR-5705, miR-4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR -4482-3p, miR-4481, miR-4515, miR-6127, miR-3614-5p, miR-6873-5p, miR-4532, miR-6831-5p, miR-451a, miR-16-5p, and miRNA group consisting of miR-1224-3p,
An evaluation method for inducing or increasing malignancy of lymphangioleiomyomatosis using as an index the amount or concentration of at least one biomarker selected from the group consisting of:

  According to the present invention, a biomarker for lymphangioleiomyomatosis can be provided. By using the biomarker, examination of lymphangioleiomyomatosis and the like can be performed more simply, more efficiently, and at a lower cost. Furthermore, by using the biomarker, it may be possible to prevent or treat lymphangiomyomatosis, to screen for an active ingredient of a preventive or therapeutic agent for lymphangiomyomatosis, and the like.

The number of particles and the particle size of the extracellular vesicle fraction are shown (Test Example 1). Healthy volunteers show the results of extracellular vesicle fractions obtained from healthy subjects, and Sporadic-LAM and TSC-LAM are the fractions of extracellular vesicles obtained from subjects suffering from the disease. Results are shown. An electron microscope observation image of an extracellular vesicle fraction obtained from a subject suffering from Sporadic-LAM is shown (Test Example 1).

  In this specification, the expressions “containing” and “including” include the concepts of “containing”, “including”, “consisting essentially of”, and “consisting only of”.

1． In one aspect, the present invention is a method for examining lymphangiomyomatosis, comprising:
(1) A step of detecting at least one biomarker selected from the group consisting of a protein group (A) and a miRNA group (B) in an extracellular vesicle of a body fluid collected from a subject (step 1) (In the present specification, it may be referred to as “the method for examining lymphangioleiomyomatosis of the present invention”). This will be described below.

1-1. Process (1)
“Lymphangiomyomatosis (LAM)” to be examined is not particularly limited, and may be Sporadic-LAM (S-LAM) or TSC-LAM. The inspection object is preferably S-LAM.

  The subject is a target organism of the test method of the present invention, and the species is not particularly limited. Examples of the biological species of the subject include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, and preferably humans.

  The state of the subject is not particularly limited. Examples of specimens include specimens that are unclear whether or not they have lymphangioleiomyomatosis, specimens that have already been determined to have lymphangiomyomatosis by another method, or that suffers from lymphangiomyomatosis Samples that have already been determined by other methods if not, samples that are being treated for lymphangioleiomyomatosis, and the like.

  The body fluid is not particularly limited. Examples of the body fluid include whole blood, serum, plasma, spinal fluid, saliva, joint fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, and preferably whole blood, serum. , Plasma and cerebrospinal fluid, and more preferably whole blood, serum and plasma. Body fluids may be used alone or in combination of two or more.

  Body fluids can be collected from a subject by methods known to those skilled in the art. For example, whole blood can be collected by blood collection using a syringe or the like. Serum is a portion obtained by removing blood cells and a specific blood coagulation factor from whole blood, and can be obtained, for example, as a supernatant after coagulating whole blood. Plasma is a part obtained by removing blood cells from whole blood, and can be obtained, for example, as a supernatant when subjected to centrifugation under conditions that do not coagulate whole blood.

  The extracellular vesicle is not particularly limited as long as it is a membrane vesicle secreted or released from the cell. Extracellular vesicles are usually defined as membrane vesicles that carry information between cells locally and throughout the body by transporting intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of the cell. The Examples of extracellular vesicles include exosomes, microvesicles, apoptotic bodies, ectosomes, microparticles, and secretory microvesicles.

  Extracellular vesicles can be purified, separated, concentrated, etc. from body fluids according to or according to known methods. Examples of methods for purifying, separating and concentrating extracellular vesicles include, for example, ultracentrifugation (eg, pellet down method, sucrose cushion method, density gradient centrifugation method, etc.), a method using an immunoaffinity carrier, gel filtration, Examples thereof include a flow fractionation method and a FACS method. Further, purification, separation, concentration and the like of extracellular vesicles can be performed using a commercially available kit. These methods may be employed singly or in combination of two or more.

  The detection target in the step (1) is at least one biomarker selected from the group consisting of the protein group (A) and the miRNA group (B) (in the present specification, these are collectively referred to as “target biomarker”). It may be shown.)

  The protein group (A) is a lymphangiomyomatosis-specific biomarker, and lymphangiomyomatosis can be differentiated by using this as an index.

  Protein group (A) consists of (A) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD ( P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteinoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6 , Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, CD81 antigen, and CD63 antigen.

  Protein group (A), protein group (A1): Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1 -antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein The protein group consisting of S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, and CD81 antigen is a protein group whose amount in the lymphangiomyomatosis sample is higher than that in the healthy sample.

  Protein group (A2): Protein group (A2): The protein group consisting of CD63 antigen is a protein group whose amount in a lymphangiomyomatosis sample is lower than that in a normal sample.

  Among the protein group (A), from the viewpoint that the quantitative ratio to the normal sample is higher, preferably fibrinogen beta chain, fibrinogen gamma chain, putative alpha-1-antitrypsin-related protein, protein S100-A8, Versican core protein, etc. Is mentioned.

  In the case of humans, the protein group (A) is a protein identified by the UniProtKB accession number shown in Table 1-2 in the Examples described later. In the case of other species, it is the ortholog of the protein specified by the UniProtKB accession number shown in Table 1.

  The miRNA group (B) is a lymphangiomyomatosis-specific biomarker, and lymphangiomyomatosis can be differentiated by using this as an index.

  The miRNA group (B) consists of (B) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p, miR-519d-5p, miR-1915-3p, miR- 4707-5p, miR-3663-3p, miR-4755-3p, miR-3654, miR-3184-5p, miR-5705, miR-4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR-4482-3p, miR-4481, miR-4515, miR-6127, miR-3614-5p, miR-6873-5p, miR-4532, miR-6831-5p, miR-451a, It is a miRNA group consisting of miR-16-5p and miR-1224-3p.

  In miRNA group (B), miRNA group (B1): miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p, miR-519d-5p, miR-1915-3p , MiR-4707-5p, miR-3663-3p, miR-4755-3p, miR-3654, miR-3184-5p, miR-5705, miR-4450, miR-4439, miR-6872-5p, miR-3194 From -3p, miR-7851-3p, miR-4482-3p, miR-4481, miR-4515, miR-6127, miR-3614-5p, miR-6873-5p, miR-4532, and miR-6831-5p The miRNA group is a miRNA group in which the amount in the lymphangiomyomatosis sample is higher than that in the healthy sample.

  In miRNA group (B), miRNA group (B2): miRNA quality group consisting of miR-451a, miR-16-5p, and miR-1224-3p is more in the amount in lymphangiomyomatosis samples than in normal samples Is also a low miRNA group.

  Among the miRNA groups (B), miR-4730, miR-6132, miR-744-5p, miR-4633-3p, and the like are preferable from the viewpoint of a higher quantitative ratio with respect to a normal sample.

  The miRNAs of the miRNA group (B) are miRNAs identified by IDs shown in Table 3 in Examples described later in the case of humans. In the case of other species, the miRNA corresponding to the miRNA specified by the ID shown in Table 3.

  The number of target biomarkers in step (1) may be only one type, or may be a combination of two or more types. By combining more detection targets (for example, 2, 5, 10, 20, and 30 or more types), it becomes possible to perform examination of lymphangioleiomyomatosis more accurately.

  Detection is usually performed by measuring the amount or concentration of the target biomarker. The “density” is not limited to the absolute density, but may be a relative density, a weight per unit volume, or raw data measured to know the absolute density.

  The method for detecting the target biomarker is not particularly limited as long as it is a method capable of specifically detecting a part or all of the target biomarker. Specific detection methods include, for example, mass spectrometry that detects peptides constituting the target biomarker, immunological measurement using an antibody that specifically recognizes the target biomarker, Northern blotting, RT -PCR method, DNA chip analysis method, etc. can be mentioned. The amino acid sequence information of the target biomarker can be obtained by searching the EBI (http://www.ebi.ac.uk/IPI/IPIhelp.html) database based on the UniProtKB accession number. .

  Preferred examples of the immunological measurement method include immunohistochemical staining, ELISA, sandwich ELISA, EIA, RIA, and Western blotting.

In mass spectrometry, a peptide sample is turned into gaseous ions using an ion source (ionization), and the peptide sample ionized by moving it in a vacuum and using electromagnetic force or by a time-of-flight difference in the analysis section is mass-charged. This is a measurement method using a mass spectrometer that can be separated and detected according to the ratio. Ionization using an ion source includes the EI method, CI method, FD method, FAB method, MALDI method, and ESI method. The method of separating ionized peptide samples in the analysis section can be selected as appropriate. Magnetic field deflection type, quadrupole type, ion trap type, time of flight (TOF) type, Fourier transform A separation method such as an ion cyclotron resonance type can be appropriately selected. In addition, tandem mass spectrometry (MS / MS) or triple quadrupole mass spectrometry combining two or more mass spectrometry methods can be used. When the sample contains a phosphorylated peptide, the sample can be concentrated using iron ion-immobilized affinity chromatography (Fe-IMAC) before introducing the sample into the mass spectrometer. Moreover, the peptide which comprises a target biomarker can be isolate | separated and refine | purified by liquid chromatograph (LC) or HPLC, and it can be set as a sample. Moreover, a detection part and a data processing method can also be selected suitably. When mass spectrometry is used to detect and quantify a peptide that constitutes a target biomarker, a peptide labeled with a stable isotope of known amino acid sequence and known concentration is used as an internal standard. It can be. As such a stable isotope-labeled peptide, one or more of amino acids in a peptide constituting a target biomarker to be detected contains one or more of ¹⁵ N, ¹³ C, ¹⁸ O, and ² H. Thus, the type, position, number, etc. of amino acids can be appropriately selected, and such stable isotope-labeled peptides can be obtained using the F-moc method (Amblard., Et al. Methods Mol Biol. 298: 3-24 (2005)) can be chemically synthesized, but iTRAQ (registered trademark) reagent, ICAT (registered trademark) reagent, ICPL (registered trademark) reagent, NBS ( It can also be prepared using a labeling reagent such as a registered trademark reagent.

  When using the Northern blot method, specifically, the above-mentioned expression system in which the probe is labeled with a radioisotope (32P, 33P, etc .: RI) or a fluorescent substance, and then transferred to a nylon membrane or the like according to a conventional method. After hybridization with the derived mRNA, a signal derived from the label of the probe (RI or a labeling substance such as a fluorescent substance) is generated from the diagnostic agent and the mRNA derived from the subject's sample. Examples thereof include a method of detecting and measuring with a radiation detector BAS-1800II (manufactured by Fuji Film) or a fluorescence detector. In addition, using the AlkPhos Direct Labeling and Detection System (manufactured by Amersham Pharmacia Biotech), the probe is labeled according to the protocol, hybridized with the mRNA derived from the above expression system, and then the signal derived from the probe labeled product is multibiotic. A method of detecting and measuring with Major STORM860 (manufactured by Amersham Pharmacia Biotech) can also be used.

  When using the RT-PCR method, specifically, a cDNA is prepared from RNA derived from the above expression system according to a conventional method, and a target region can be amplified using this as a template so that a target region can be amplified (the above cDNA ( A method of detecting the amplified double-stranded DNA obtained by hybridizing the positive strand that binds to the -strand) and the reverse strand that binds to the + strand and performing the PCR method according to a conventional method. it can. The detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance. For example, a method in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method and hybridized with a labeled probe to detect it can be used. The produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems). In addition, prepare an RT-PCR reaction solution according to the protocol using SYBR Green RT-PCR Reagents (Applied Biosystems) and react with ABI PRISM 7700 Sequence Detection System (Applied Biosystems) to detect the reaction product. You can also.

  When using DNA chip analysis, prepare a DNA chip to which a DNA probe (single-stranded or double-stranded) is attached, and then hybridize it with cRNA prepared by conventional methods from RNA derived from the above expression system. In addition, a method of detecting the formed double strand of DNA and cRNA by binding to a labeled probe can be mentioned.

  Western blotting uses a primary antibody followed by a labeled antibody (an antibody that binds to the primary antibody) labeled with a radioactive isotope such as 125I, a fluorescent substance, or an enzyme such as horseradish peroxidase (HRP) as a secondary antibody. Illustrated is a method for detecting and measuring signals derived from labeling substances such as radioisotopes and fluorescent substances of the obtained labeling compounds with a radiation measuring instrument BAS-1800II (manufactured by Fuji Film Co., Ltd.), a fluorescence detector, etc. The Moreover, after using a primary antibody, it can detect according to the said protocol using ECL Plus Western Blotting Detection System (made by Amersham Pharmacia Biotech), and can also measure with multibioimager STORM860 (made by Amersham Pharmacia Biotech).

  According to the test method of the present invention including the step (1), the amount and / or concentration of the target biomarker that is a detection index of lymphangioleiomyomatosis can be provided, thereby detecting lymphangiomyomatosis. Etc. can be assisted.

  Test results of the test method of the present invention including step (1) are as follows: therapeutic effect determination, elucidation of pathophysiology of lymphangioleiomyomatosis, prognosis of lymphangiomyomatosis, patient stratification, selection of treatment method (individualization) Medical, therapeutic response), refractory in lymphangiomyomatosis, evaluation of remodeling, differentiation of lymphangiomyomatosis histology and phenotype, etc.

1-2. Process (2)
In one embodiment of the test method of the present invention, when the detected biomarker is at least one biomarker selected from the group consisting of the protein group (A2) and the miRNA group (B2), (2a It is preferable that the method includes a step of determining that the subject suffers from lymphangioleiomyomatosis when the amount or concentration of the biomarker detected in the step (1) is equal to or higher than a cutoff value. According to the test method of the present invention including the step 2a, it becomes possible to determine lymphangioleiomyomatosis.

  In one embodiment of the test method of the present invention, when the detected biomarker is at least one biomarker selected from the group consisting of the protein group (A1) and the miRNA group (B1), (2b It is preferable that the method includes the step of determining that the subject is suffering from lymphangioleiomyomatosis when the amount or concentration of the biomarker detected in the step (1) is not more than a cutoff value. According to the test method of the present invention including the step 2b, it becomes possible to determine lymphangioleiomyomatosis.

  The cut-off value can be appropriately set by those skilled in the art from the viewpoint of sensitivity, specificity, positive predictive value, negative predictive value, etc., for example, collected from a subject not suffering from lymphangioleiomyomatosis. Based on the amount and / or concentration of the target biomarker in the extracellular vesicles of the collected body fluid, a predetermined value or a predetermined value can be obtained. The cutoff value is, for example, the amount and / or concentration of the target biomarker in the extracellular vesicles of body fluid collected from a subject not suffering from lymphangioleiomyomatosis (in the case of multiple subjects, the average value) , Median, etc.), for example, a value of 1 to 1.5 times.

  In a preferred embodiment of step (2), when the subject is a specimen being treated for lymphangiomyomatosis, the cutoff value is determined, for example, the amount of the target biomarker in a past sample for the same specimen and / or By setting the value based on the concentration, the therapeutic effect can be determined.

2． When the test method of the present invention including the diagnostic step (2) with higher accuracy of lymphangioleiomyomatosis determines that the subject suffers from lymphangioleiomyomatosis, the test method of the present invention Furthermore, by combining the process of applying a diagnosis by a doctor of lymphangioleiomyomatosis, the lymphangiomyomatosis can be diagnosed with higher accuracy. In addition, since the test method of the present invention can detect lymphangioleiomyomatosis more accurately, combining the above steps with the test method of the present invention makes it possible to more efficiently and more accurately “be affected by lymphangioleiomyomatosis”. Can be diagnosed.

3． When it is determined that the subject suffers from lymphangioleiomyomatosis according to the examination method of the present invention including the treatment step (2) of lymphangioleiomyomatosis, or in addition to the examination method of the present invention or the above-mentioned When it is diagnosed that the patient has lymphangioleiomyomatosis as described in “2. Diagnosis of lymphangioleiomyomatosis with higher accuracy”, the method of applying the examination method of the present invention and diagnosis by a doctor And (3) subjecting the subject to be diagnosed or diagnosed as having lymphangioleiomyomatosis, by treating the subject with the disease, Can be treated. In addition, since the inspection method of the present invention can detect lymphangioleiomyomatosis more accurately, the inspection method of the present invention or the combination of the inspection method of the present invention and the process of applying a diagnosis by a doctor. By combining step 3, a subject suffering from lymphangioleiomyomatosis can be treated more efficiently and more reliably.

  A method for treating lymphangioleiomyomatosis is not particularly limited, but typically includes medication. The drug used for the drug treatment is not particularly limited, and examples thereof include sirolimus, everolimus, gonadotropin-releasing hormone derivatives, medroxyprogesterone and the like. A pharmaceutical can be used 1 type, 2 types, or 3 or more types in combination.

Four. In one aspect, the present invention relates to a lymphatic vessel comprising a detection agent for at least one biomarker selected from the group consisting of a protein group (A) and a miRNA group (B). The present invention relates to a test agent for ductal myomatosis (sometimes referred to as “the test agent of the present invention” in this specification). This will be described below.

  The protein group (A), miRNA group (B), lymphangioleiomyomatosis and the like are the same as defined in “1. Method for examining lymphangioleiomyomatosis” above.

  The detection agent is not particularly limited as long as it can specifically detect the target biomarker. Examples of the detection agent include an antibody against the target biomarker, a primer for the target biomarker, a probe, and the like.

  The detection agent may be modified as long as its function is not significantly impaired. Examples of the modification include addition of a label such as a fluorescent dye, an enzyme, a protein, a radioisotope, a chemiluminescent substance, biotin and the like.

  As the fluorescent dye used in the present invention, those generally labeled with nucleotides and used for detection and quantification of nucleic acids can be suitably used. For example, HEX (4, 7, 2 ′, 4 ′, 5 ′, 7 '-hexachloro-6-carboxylfluorescein (green fluorescent dye), fluorescein, NED (trade name, Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, Applied Biosystems, yellow) Green fluorescent dye), rhodamine or a derivative thereof (for example, tetramethylrhodamine (TMR)), but is not limited thereto. As a method for labeling nucleotides with a fluorescent dye, an appropriate one of known labeling methods can be used [see Nature Biotechnology, 14, 303-308 (1996)]. Commercially available fluorescent labeling kits can also be used (for example, oligonucleotide ECL 3'-oligo labeling system manufactured by Amersham Pharmacia).

  The detection agent can be used by being immobilized on an arbitrary solid phase. For this reason, the test agent of the present invention can be provided in the form of a substrate on which a detection agent is immobilized (for example, a microarray chip on which a probe is immobilized, another example is an ELISA plate on which an antibody is immobilized).

  The solid phase used for immobilization is not particularly limited as long as it can immobilize antibodies, polynucleotides, etc. For example, a glass plate, nylon membrane, microbead, silicon chip, capillary or other substrate is used. Can be mentioned. The immobilization of the detection agent on the solid phase is not particularly limited. The immobilization method is well known in the art depending on the type of immobilization probe, for example, using a commercially available spotter (such as manufactured by Amersham) in the case of a microarray [for example, photolithographic technique (Affymetrix), In situ synthesis of oligonucleotides using inkjet technology (Rosetta Inpharmatics).

  The antibody is not particularly limited as long as it selectively recognizes (specifically) the target biomarker. Here, “selectively (specifically) recognize” means that the target biomarker can be specifically detected in, for example, Western blotting or ELISA, but is not limited thereto, and is not limited thereto. However, what is necessary is just to be able to judge that the detected object is derived from the target biomarker.

  The antibody includes a part of the antibody having antigen-binding properties such as a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or a Fab fragment or a fragment generated by a Fab expression library. The antibody of the present invention also includes an antibody having an antigen binding property to a polypeptide consisting of at least continuous amino acids of the target biomarker, usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids.

  Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology, Chapter 11.12 to 11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, an oligopeptide having a partial amino acid sequence of the target biomarker using a target biomarker expressed and purified in Escherichia coli or the like according to a conventional method or according to a conventional method Can be synthesized to immunize non-human animals such as rabbits, and obtained from the sera of the immunized animals according to a conventional method. On the other hand, in the case of a monoclonal antibody, spleen obtained by immunizing a non-human animal such as a mouse with a target biomarker expressed and purified in Escherichia coli according to a conventional method, or an oligopeptide having a partial amino acid sequence of the target biomarker It can be obtained from hybridoma cells prepared by cell fusion of cells and myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).

  Biomarkers of interest used as immunizing antigens for the production of antibodies are based on known gene sequence information, DNA cloning, construction of each plasmid, transfection into a host, culture of transformants and protein from the culture. It can be obtained by a recovery operation. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). Can be done.

  Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding the target biomarker in a desired host cell is prepared, introduced into the host cell, transformed, and the transformant is cultured. By collecting the target protein from the obtained culture, a protein as an immunizing antigen for producing the antibody of the present invention can be obtained. The partial peptide of the target biomarker can also be produced by a general chemical synthesis method (peptide synthesis) according to known gene sequence information.

  The antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of the target biomarker. The oligo (poly) peptide used for the production of such an antibody does not need to have a functional biological activity, but desirably has the same immunogenic properties as the target biomarker. An oligo (poly) peptide preferably having this immunogenic property and consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of the subject biomarker can be exemplified.

  Production of antibodies against such oligo (poly) peptides can also be carried out by enhancing the immunological reaction using various adjuvants depending on the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Active substances, human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are included.

  The primer, probe, etc. are not particularly limited as long as they selectively recognize (specifically) the target biomarker and nucleic acid derived therefrom. Here, “selectively (specifically)” means that the target biomarker can be specifically detected, for example, in the Northern blot method, and the target biomarker or a nucleic acid derived therefrom in the RT-PCR method. (CDNA, etc.) means that it is specifically amplified, but is not limited to this, as long as those skilled in the art can determine that the detected product or amplified product is derived from the target biomarker Good.

Specific examples of the primer and probe include the polynucleotide described in (a) below and the polynucleotide described in (b) below:
(A) a polynucleotide having at least 15 consecutive bases in the base sequence of the target biomarker and / or a polynucleotide complementary to the polynucleotide; and (b) a base sequence of the target biomarker or a base sequence complementary thereto. And at least one selected from the group consisting of polynucleotides having at least 15 bases that hybridize under stringent conditions.

  Complementary polynucleotide or complementary base sequence (complementary strand, reverse strand) is a full-length sequence of a polynucleotide consisting of the base sequence of the target biomarker, or a base sequence of at least 15 bases in length in the base sequence A polynucleotide or base that is in a base-complementary relationship based on a base-pair relationship such as A: T and G: C with respect to the partial sequence that it has (for convenience, these are also referred to as “positive strands”) It means an array. However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows it to hybridize with the target positive strand under stringent conditions. You may have. Note that stringent conditions here bind the complex or probe as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, the conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” can be mentioned. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although there is no particular limitation, the more stringent hybridization conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.”, and the more severe hybridization conditions are “0.1 × SSC, 0.1% SDS, 65 ° C.”. Can do. Specifically, as such a complementary strand, a strand consisting of a base sequence that is completely complementary to the base sequence of the target positive strand, and at least 90%, preferably 95%, more preferably Examples thereof include a chain composed of a base sequence having 98% or more identity, more preferably 99% or more identity.

  Primers, probes, and the like can be designed using, for example, the vector NTI (manufactured by Infomax) based on the base sequence of the target biomarker. Specifically, a primer or probe candidate sequence obtained by applying the base sequence of the target biomarker to the software of Vector NTI, or a sequence partially including at least the sequence can be used as a primer or a probe.

  The base length of the primer, probe, etc. is not particularly limited as long as it has a length of at least 15 consecutive bases as described above, and can be appropriately set depending on the application. Examples of the base length include 15 to 35 bases when used as a primer, and 15 to 35 bases when used as a probe, for example.

  The test agent of the present invention may be in the form of a composition. The composition may contain other components as necessary. Examples of other components include a base, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, a moisturizer, a colorant, and a fragrance. And chelating agents.

  The test agent of the present invention may be in the form of a kit. In addition to the above-mentioned detection agent or the above-mentioned composition containing the same, the kit may contain a substance that can be used for detection of a target biomarker in an extracellular vesicle of a body fluid of a subject. Specific examples of such materials include various reagents (for example, secondary antibodies, buffers, etc.), instruments (for example, instruments for purification, separation, and concentration of extracellular vesicles (for example, columns)).

Five. Prophylactic or therapeutic agent for lymphangioleiomyomatosis In one aspect thereof, the present invention contains an inhibitor of at least one biomarker selected from the group consisting of protein group (A) and miRNA group (B). And an agent for the prevention or treatment of lymphangioleiomyomatosis (sometimes referred to herein as “the agent of the present invention”). This will be described below.

  The protein group (A), miRNA group (B) and the like are the same as defined in “1. Test method for lymphangioleiomyomatosis” above.

  As an inhibitor, Preferably the expression inhibitor of a target biomarker is mentioned.

  The target biomarker expression inhibitor is not particularly limited as long as it can suppress the expression level of the target biomarker, its mRNA, its precursor, etc. For example, gene-specific small interfering RNA (siRNA) of the target biomarker ), Target biomarker gene-specific microRNA (miRNA), target biomarker gene-specific antisense nucleic acid, expression vector thereof; target biomarker gene-specific ribozyme; target biomarker gene gene by CRISPR / Cas system Editors etc. are mentioned.

  Expression suppression refers to the expression level of the target biomarker, its mRNA, etc., for example, 1/2, 1/3, 1/5, 1/10, 1/20, 1/30, 1/50, 1 / It means that it is suppressed to 100, 1/200, 1/300, 1/500, 1/1000, 1/10000 or less, and the expression level of these is set to 0.

  The gene siRNA of the target biomarker is not particularly limited as long as it is a double-stranded RNA molecule that specifically suppresses the expression of the gene encoding the target biomarker. In one embodiment, the siRNA is preferably 18 or more bases, 19 or more bases, 20 or more bases, or 21 or more bases in length, for example. Moreover, it is preferable that siRNA is 25 bases or less, 24 bases or less, 23 bases or less, or 22 bases or less, for example. It is assumed that the upper limit value and the lower limit value of the siRNA length described here are arbitrarily combined. For example, the lower limit is 18 bases and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases; the lower limit is 19 bases, and the upper limit is 25 bases, 24 bases, 23 bases, or 22 bases A certain length; a lower limit of 20 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 bases; a lower limit of 21 bases and an upper limit of 25 bases, 24 bases, 23 bases, or 22 Combinations of lengths that are bases are envisioned.

  The siRNA may be shRNA (small hairpin RNA). shRNA can be designed so that a part thereof forms a stem-loop structure. For example, if the sequence of a certain region is a sequence a and the complementary strand to the sequence a is a sequence b, these sequences are present in a single RNA strand in the order of sequence a, spacer, and sequence b. And can be designed to have a total length of 45-60 bases. The sequence a is a partial sequence of a base sequence encoding a target biomarker to be targeted, and the target region is not particularly limited, and any region can be a candidate. The length of the sequence a is 19 to 25 bases, preferably 19 to 21 bases.

  The gene-specific siRNA of the subject biomarker may have an additional base at the 5 'or 3' end. The length of the additional base is usually about 2 to 4 bases. The additional base may be DNA or RNA, but the use of DNA may improve the stability of the nucleic acid. Examples of such additional base sequences include ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', ttttt-3 Examples include, but are not limited to, ', uuuuu-3'.

  The siRNA may have a protruding portion sequence (overhang) at the 3 ′ end, and specifically includes those to which dTdT (dT represents deoxythymidine) is added. Further, it may be a blunt end (blunt end) without end addition. The siRNA may have a different number of bases in the sense strand and the antisense strand. For example, the antisense strand has “asymmetrical interfering RNA (overhang) at the 3 ′ end and the 5 ′ end. aiRNA) ". A typical aiRNA has an antisense strand consisting of 21 bases, a sense strand consisting of 15 bases, and has an overhang structure of 3 bases at each end of the antisense strand.

  Although the position of the target sequence of the gene-specific siRNA of the target biomarker is not particularly limited, in one embodiment, from the 5′-UTR and the start codon to about 50 bases, and from a region other than the 3′-UTR It is desirable to select a target sequence. For the selected target sequence candidate group, whether or not there is homology in the 16-17 base sequence in the non-target mRNA is determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ It is preferable to check using a homology search software such as) to confirm the specificity of the selected target sequence. About the target sequence whose specificity was confirmed, a sense strand having a 3 'terminal overhang of TT or UU at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 bases and TT or A double-stranded RNA consisting of an antisense strand having a 3 ′ end overhang of UU may be designed as an siRNA. In addition, for the shRNA that is a precursor of siRNA, an arbitrary linker sequence (for example, about 5-25 bases) that can form a loop structure is appropriately selected, and the sense strand and the antisense strand are connected via the linker sequence. It can be designed by connecting.

The sequence of siRNA and / or shRNA can be searched using search software provided free of charge on various websites. Examples of such sites include the following.
SiRNA Target Finder provided by Ambion (http://www.ambion.com/techlib/misc/siRNA_finder.html) Insert design tool for pSilencer® Expression Vector (http://www.ambion.com/ jp / techlib / misc / psilencer_converter.html) GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi).

  siRNA is synthesized by each of the sense strand and antisense strand of the target sequence on the mRNA with a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. It can also be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving it with an RNA-cleaving protein dicer.

  The gene-specific miRNA of the target biomarker is optional as long as it inhibits translation of the gene encoding the target biomarker. For example, an miRNA may pair with the 3 'untranslated region (UTR) of the target and inhibit its translation, rather than cleaving the target mRNA like an siRNA. The miRNA may be any of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA), and mature miRNA. The length of miRNA is not particularly limited, the length of pri-miRNA is usually several hundred to several thousand bases, the length of pre-miRNA is usually 50 to 80 bases, and the length of mature miRNA is usually 18 ~ 30 bases. In one embodiment, the gene specific miRNA of the subject biomarker is preferably a pre-miRNA or a mature miRNA, more preferably a mature miRNA. Such gene-specific miRNA of the target biomarker may be synthesized by a known method or purchased from a company that provides synthetic RNA.

  The gene-specific antisense nucleic acid of the target biomarker is a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of mRNA of the gene encoding the target biomarker, or a part thereof. And a nucleic acid having a function of suppressing the synthesis of the target biomarker by forming a specific and stable duplex. The antisense nucleic acid may be any of DNA, RNA, and DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA: DNA hybrid formed by the target RNA and the antisense DNA is recognized by endogenous ribonuclease H (RNase H) and causes selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the gene of the target biomarker. The intron sequence can be determined by comparing the genomic sequence and the cDNA base sequence of the target biomarker gene using a homology search program such as BLAST or FASTA.

  The length of the target region of the gene-specific antisense nucleic acid of the target biomarker is not limited as long as the antisense nucleic acid hybridizes and consequently translation into the target biomarker is inhibited. The gene-specific antisense nucleic acid of the target biomarker may be the entire sequence or a partial sequence of mRNA encoding the target biomarker. In view of easiness of synthesis, antigenicity, intracellular migration, and the like, an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferred, but is not limited thereto. More specifically, 5 ′ end hairpin loop, 5 ′ end untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3 ′ end untranslated region, 3 ′ end palindrome of the target biomarker gene A region or a 3 ′ end hairpin loop or the like may be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.

  The gene-specific antisense nucleic acid of the target biomarker not only hybridizes with the mRNA and initial transcription product of the target biomarker gene to inhibit translation into proteins, but also binds to these genes that are double-stranded DNA Thus, it may be one that forms a triplex and can inhibit transcription to RNA (antigene).

The nucleotide molecules constituting the gene-specific siRNA of the target biomarker, the gene-specific miRNA of the target biomarker, and the gene-specific antisense nucleic acid of the target biomarker are stable (chemical and / or anti-enzyme) In order to improve specific activity (affinity with RNA), various chemical modifications may be included. For example, in order to prevent degradation by a hydrolase such as nuclease, a phosphate residue (phosphate) of each nucleotide constituting an antisense nucleic acid is selected from, for example, phosphorothioate (PS), methylphosphonate, phosphorodithio. It can be substituted with a chemically modified phosphate residue such as a phosphorodithioate. In addition, the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R═CH ₃ (2′-O-Me), CH ₂ CH ₂ OCH ₃ (2′-O-MOE), CH ₂ CH ₂ NHC (NH) NH ₂ , CH ₂ CONHCH ₃ , or CH ₂ CH ₂ CN) may be substituted. Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. May be applied. Moreover, a part of nucleotide molecules constituting siRNA or miRNA may be replaced with natural DNA.

  The target biomarker gene-specific siRNA, the target biomarker gene-specific miRNA, and the target biomarker gene-specific antisense nucleic acid are mRNA or initial based on the cDNA sequence or genomic DNA sequence of the target biomarker gene. It can be prepared by determining the target sequence of the transcript and synthesizing a complementary sequence using a commercially available DNA / RNA automatic synthesizer. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a known method.

  For the target biomarker gene-specific siRNA, target biomarker gene-specific miRNA, or target biomarker gene-specific antisense nucleic acid expression vector, target biomarker gene-specific siRNA, target biomarker gene-specific The target miRNA or the gene-specific antisense nucleic acid of the target biomarker is not particularly limited as long as it is incorporated in an expressible state. Typically, the expression vector comprises a promoter sequence and a gene-specific siRNA of a subject biomarker, a gene-specific miRNA of a subject biomarker, or a coding sequence of a gene-specific antisense nucleic acid of a subject biomarker (if necessary In addition, a polynucleotide containing a transcription termination signal sequence), and optionally other sequences. The promoter is not particularly limited, and for example, a CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β actin promoter, CAG promoter and other RNA polymerase II (polII) promoters; mouse and human U6-snRNA promoters, Examples include RNA polymerase III (polIII) promoters such as human H1-RNase P RNA promoter and human valine-tRNA promoter, and among these, polIII promoters can be used to accurately transcribe short RNAs. preferable. Other sequences are not particularly limited, and various known sequences that can be included in the expression vector can be employed. Examples of such sequences include the origin of replication and drug resistance genes. Examples of the drug resistance gene and the vector include those described above.

  Another example of the target biomarker gene expression inhibitor is a gene-specific ribozyme of the target biomarker. “Ribozyme” in a narrow sense means RNA having an enzyme activity for cleaving nucleic acid, but in the present application, it includes DNA as long as it has sequence-specific nucleic acid cleaving activity. The most versatile ribozyme nucleic acid is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends (about 10 bases in total) adjacent to the part having the hammerhead structure are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has an advantage that it does not attack genomic DNA because it uses only RNA as a substrate. If the mRNA of the target biomarker gene has a double-stranded structure by itself, a single target sequence can be obtained by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. Can be chained [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when ribozymes are used in the form of expression vectors containing the DNA that encodes them, they should be hybrid ribozymes in which tRNA-modified sequences are further linked in order to promote the transfer of transcripts to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

  The application target of the agent of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats and deer. .

  The form of the agent of this invention is not specifically limited, According to the use of the agent of this invention, the form normally used in each use can be taken.

  As the form, when the use is a medicine, health enhancer, nutritional supplement (eg supplement), etc., for example, tablets (inner disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, etc.) ), Pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jelly preparations suitable for oral intake Forms (oral dosage forms), nasal drops, inhalants, rectal suppositories, inserts, enemas, jellies, injections, patches, lotions, creams, etc. Oral dosage form).

  As a form, when the use is a food composition, a liquid, gel or solid food such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, infant formula , Cake mix, powdered or liquid dairy products, bread, cookies and the like.

  As a form, when the use is an oral composition, for example, liquid (solution, emulsion, suspension, etc.), semi-solid (gel, cream, paste, etc.), solid (tablet, particulate agent, capsule, Film, kneaded material, molten solid, waxy solid, elastic solid, etc.), more specifically, dentifrice (toothpaste, liquid toothpaste, liquid toothpaste, powder toothpaste etc.), mouthwash, Examples thereof include a coating agent, a patch, a mouth freshener, and a food (for example, chewing gum, tablet candy, candy, gummi, film, troche, etc.).

  The agent of the present invention may further contain other components as necessary. Other components are not particularly limited as long as they are components that can be blended in, for example, pharmaceuticals, food compositions, oral compositions, health enhancers, nutritional supplements (such as supplements), etc. , Carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, and the like.

  The content of the inhibitor of the target biomarker of the agent of the present invention depends on the type of inhibitor, application, use mode, application target, application target state, and the like, and is not limited. It can be 100% by weight, preferably 0.001 to 50% by weight.

  The amount of application (eg, administration, ingestion, inoculation, etc.) of the composition of the present invention is not particularly limited as long as it is an effective amount that exhibits a medicinal effect, and is generally 0.1 to 1000 per day as the weight of the active ingredient. mg / kg body weight. The above dose is preferably administered once a day or divided into 2 to 3 times a day, and can be appropriately increased or decreased depending on age, disease state, and symptoms.

6． The screening method of the active ingredient of the preventive or therapeutic agent of lymphangioleiomyomatosis The present invention, in one aspect thereof, is a protein group (A) in an extracellular vesicle of a body fluid collected from an animal treated with a test substance. And a method for screening an active ingredient of a preventive or therapeutic agent for lymphangioleiomyomatosis, using as an index the amount or concentration of at least one biomarker selected from the group consisting of miRNA group (B) (in the present specification, , Sometimes referred to as “the active ingredient screening method of the present invention”). This will be described below.

  For body fluid, extracellular vesicle, protein group (A), miRNA group (B), lymphangiomyomatosis, measurement of the amount or concentration of the target biomarker, etc. Is the same as the definition in "."

  The species of animal is not particularly limited. Examples of animal biological species include various mammalian animals such as humans, monkeys, mice, rats, dogs, cats, rabbits, and preferably humans.

  The test substance can be widely used regardless of a naturally occurring compound or an artificially produced compound. Moreover, the composition which mixed not only the refined compound but various compounds, and the extract of animals and plants can also be used. The compounds include not only low molecular compounds but also high molecular compounds such as proteins, nucleic acids, and polysaccharides.

  In the active ingredient screening method of the present invention, more specifically, when the biomarker used as an index is at least one biomarker selected from the group consisting of a protein group (A1) and a miRNA group (B1) When the value of the index is lower than the amount or concentration (control value) of the corresponding biomarker in the extracellular vesicles of a body fluid collected from an animal not treated with the test substance, the test substance is A step of selecting as an active ingredient of a prophylactic or therapeutic agent for vascular myomasis (or a candidate substance for an active ingredient of a prophylactic or therapeutic agent for lymphangiomyomatosis).

  In another specific example of the active ingredient screening method of the present invention, the biomarker as an index is at least one biomarker selected from the group consisting of a protein group (A2) and a miRNA group (B2) When the value of the index is higher than the amount or concentration (control value) of the corresponding biomarker in the extracellular vesicles of a body fluid collected from an animal that has not been treated with the test substance, A step of selecting as an active ingredient of a prophylactic or therapeutic agent for vascular myomasis (or a candidate substance for an active ingredient of a prophylactic or therapeutic agent for lymphangiomyomatosis).

  The corresponding biomarker means the same protein as the target biomarker used as an index.

  “High” means, for example, that the value of the index is 2 times, 5 times, 10 times, 20 times, 50 times, or 100 times the control value.

  “Low” means, for example, that the value of the index is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.

7． In one aspect of the present invention, the present invention relates to a protein group (A) in an extracellular vesicle of a body fluid collected from an animal treated with a test substance, and A method for evaluating the induction or malignancy of lymphangioleiomyomatosis using the amount or concentration of at least one biomarker selected from the group consisting of miRNA group (B) as an index (herein, “the present invention It may be indicated as “toxicity assessment method”. This will be described below.

  For body fluid, extracellular vesicle, protein group (A), miRNA group (B), lymphangiomyomatosis, measurement of the amount or concentration of the target biomarker, animal species, test substance, etc. This is the same as the definition in “. Method for examining lymphangioleiomyomatosis” and “6. Screening method for active ingredient of prophylactic or therapeutic agent for lymphangiomyomatosis”.

  More specifically, in the toxicity evaluation method of the present invention, the biomarker as an index is at least one biomarker selected from the group consisting of the protein group (A1) and the miRNA group (B1). When the value of the index is higher than the amount or concentration (control value) of the corresponding biomarker in the extracellular vesicles of a body fluid collected from an animal that has not been treated with the test substance, A step of determining that there is induction or malignancy of the tube myomatosis.

  The toxicity evaluation method of the present invention, as another specific example, when the biomarker as an index is at least one biomarker selected from the group consisting of the protein group (A2) and the miRNA group (B2), When the value of the index is lower than the amount or concentration (control value) of the corresponding biomarker in the extracellular vesicles of a body fluid collected from an animal that has not been treated with the test substance, A step of determining that there is induction or malignancy of the tube myomatosis.

  The corresponding biomarker means the same protein as the target biomarker used as an index.

  “High” means, for example, that the value of the index is 2 times, 5 times, 10 times, 20 times, 50 times, or 100 times the control value.

  “Low” means, for example, that the value of the index is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Test Example 1. Preparation of extracellular vesicular fraction Each human subject diagnosed with lymphangioleiomyomatosis (LAM) (8: Sporadic-LAM (S-LAM, 3) + TSC-LAM (5)) Extracellular vesicle fractions were prepared from sera, sera of healthy human subjects (4: female), and human subjects (10) diagnosed with chronic obstructive pulmonary disease (COPD). The extracellular vesicle fraction was prepared by stepwise ultracentrifugation. Specifically, it was performed as follows. Serum was diluted with PBS and centrifuged (2000 g, 4 ° C., 30 minutes). The supernatant was passed through a 0.22 μm filter and ultracentrifuged (100,000 g, 4 ° C., 90 minutes). The pellet was washed with PBS and then ultracentrifuged under the same conditions. This washing-ultracentrifugation step was repeated twice in total. The finally obtained pellet was suspended in PBS to obtain an extracellular vesicle fraction.

  Subsequently, the number and particle size of the extracellular vesicles were measured for the obtained extracellular vesicle fraction. Specifically, it was measured using a nanosite (Nippon Quantum Design Co., Ltd., Nanoparticle Tracking Analysis (NTA) Version 2.3 Build 0025). This is an analysis based on the difference in Brownian motion speed for each particle size, tracking (tracking) each individual scattered light reflected on the screen, and using each moving speed (diffusion coefficient) in the liquid. The particle diameter (hydrodynamic diameter) can be calculated. The results are shown in FIG.

  In addition, extracellular vesicles were observed by immunoelectron microscopy. Specifically, it was performed as follows. 5 to 8 μL of the fixed extracellular vesicle solution was placed on the grid and allowed to stand for 15 minutes to fix the extracellular vesicles to the form bar of the grid. After washing 3 times with PBS, blocking reaction (1% BSA / PBS, 10 minutes) followed by primary antibody reaction (Invitrogen AHS0902 Mouse (monoclonal) Anti-Human Leukemia and Platelet Associated Antigen CD9 Clone: MM2 / 57, (100-fold dilution, 2.5 hours, room temperature). After washing 6 times with PBS, secondary antibody reaction (CRL EMGMHL10 Anti-IgG (H + L), Mouse, Goat-Poly, Gold 10 nm, EM, 200-fold dilution, 1 hour, room temperature) was performed. After washing 6 times with PBS, it was treated with 1% GA / PBS for 15 minutes and further washed 3 times with PBS. Washed with water, treated with 0.5% UA (uranyl acetate), dried and observed with an electron microscope. A representative example of the results obtained is shown in FIG.

  As shown in FIGS. 1 and 2, exosomes are present in the obtained fraction, and it is found that the number of particles and the particle diameter are almost the same between healthy subjects and LAM subjects. It was.

Test Example 2. Proteomics analysis (TMT label, LC-MS / MS)
TMT label-LC-MS / MS proteomics analysis was performed according to a previously reported document (Journal of Proteome Research., 2017, 16 (2), pp 1077-1086.). The outline is as follows. The peptide sample obtained by trypsin digesting the protein in the extracellular vesicle fraction obtained in Test Example 1 was labeled with TMT 10-plex reagent. The labeled sample was fractionated with an SCX column and subjected to LC-MS / MS analysis using a mass spectrometer (LTQ-Orbitrap XL, manufactured by Thermo Fisher Scientific). The obtained raw data is analyzed using an analysis program (Proteome Discoverer ver.1.3 (Thermo Fisher Scientific) equipped with Mascot v2.3.1 (Matrix Science) for UniProt / SwissProt) to quantify various proteins. I got the data.

  The amount of the S-LAM specimen in the extracellular vesicle fraction (S-LAM group) is significantly higher than the amount of the healthy subject in the extracellular vesicle fraction (control group) (p value = less than 0.05) A protein that is high (1.25 times or more) and has no significant difference from the control group in the amount of COPD specimen in the extracellular vesicle fraction (COPD group) is determined to have a certain degree of effectiveness as a LAM biomarker. Selected (Group 1).

  In addition, proteins in the extracellular vesicle fraction of the S-LAM group and TSC-LAM specimens are higher than the control group (1.25 times or more), and the COPD group has no significant difference from the control group. And selected as a LAM biomarker (group 2).

  Furthermore, S-LAM group is higher than control group (1.25 times or more), and proteins judged to be functionally related to LAM are also determined to have a certain degree of effectiveness as LAM biomarkers. (Group 3).

  Table 1 shows the amount ratio of the selected protein and the control group.

Test Example 3. Proteomics analysis 2 (SRM / MRM)
SRM proteomics analysis was performed according to a previously reported document (Molecular & Cellular Proteomics 13: 10.1074 / mcp. M113.037093, 1471-1484, 2014.). The outline is as follows. One or two peptides (trypsin digested fragments) that are specifically detected by the SRM method are selected based on the amino acid sequence information of the protein, and a stable isotope-labeled peptide (SI peptide consisting of the same amino acid sequence as the corresponding peptide) ) Was used as an internal standard peptide. The protein of the extracellular vesicle fraction obtained in Test Example 1 is trypsin-digested, mixed with an internal standard peptide, and subjected to relative quantitative analysis by the SRM method using a mass spectrometer (TSQ Vantage, manufactured by Thermo Fisher Scientific) did. Based on the obtained quantitative data, the S-LAM group is higher than the control group, and the protein judged to be functionally related to LAM is determined to have a certain degree of effectiveness as a LAM biomarker, Proteins to be LAM biomarkers were selected.

  Table 2 shows the amount ratio of the selected protein and the control group.

Test Example 4. A microarray analysis was performed on the basis of miRNA (microRNA) in the extracellular vesicle fraction obtained in miRNA analysis test example 1 (Toray Industries, Inc.) (3D-Gene (registered trademark) miRNA Oligo chip).

  The S-LAM group is higher than the control group (more than 2 times), or the S-LAM group is lower than the control group (less than 0.5 times), the protein is determined to have a certain degree of effectiveness as a LAM biomarker, Selected.

  Table 3 shows the amount ratio of the selected protein and the control group. In Table 3, 1-3 represent the data of each subject of the control group, 4-6 represent the data of the subject of each S-LAM group, I represents the average of 1-3, II represents 4-4 Represents the average of 6, ratio represents II / I.

  Furthermore, among the above miRNAs, miR-4730, miR-6132, miR-744-5p, miR-1915-3p, miR-3663-3p, according to the standard that it can be detected in 2/3 specimens in the disease group, miR-4707-5p was selected as a more promising biomarker.

Claims (9)

A method for examining lymphangioleiomyomatosis, comprising:
(1) Protein group (A) and miRNA group (B) in extracellular vesicles of body fluid collected from the subject:
(A) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, CD81 antigen, and protein group consisting of CD63 antigen, and (B) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p , MiR-519d-5p, miR-1915-3p, miR-4707-5p, miR-3663-3p, miR-4755-3p, miR-3654, miR-3184-5p, mi R-5705, miR-4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR-4482-3p, miR-4481, miR-4515, miR-6127, miR- MiRNA group consisting of 3614-5p, miR-6873-5p, miR-4532, miR-6831-5p, miR-451a, miR-16-5p, and miR-1224-3p,
A test method comprising a step of detecting at least one biomarker selected from the group consisting of: The detected biomarkers are protein group (A1) and miRNA group (B1):
(A1) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, and protein group consisting of CD81 antigen, and (B1) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p, miR- 519d-5p, miR-1915-3p, miR-4707-5p, miR-3663-3p, miR-4755-3p, miR-3654, miR-3184-5p, miR-5705, miR- 4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR-4482-3p, miR-4481, miR-4515, miR-6127, miR-3614-5p, miR- MiRNA group consisting of 6873-5p, miR-4532, and miR-6831-5p,
If it is at least one biomarker selected from the group consisting of:
And (2a) including a step of determining that the subject suffers from lymphangioleiomyomatosis when the amount or concentration of the biomarker detected in the step (1) is equal to or higher than a cutoff value. The inspection method according to claim 1. The detected biomarkers are protein group (A2) and miRNA group (B2):
(A2) a protein group consisting of CD63 antigen, and (B2) a miRNA group consisting of miR-451a, miR-16-5p, and miR-1224-3p,
If it is at least one biomarker selected from the group consisting of:
Further, (2b) includes a step of determining that the subject is suffering from lymphangioleiomyomatosis when the amount or concentration of the biomarker detected in the step (1) is not more than a cutoff value. The inspection method according to claim 1. The examination method according to claim 1, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum. The test method according to claim 1, wherein the subject is a human. Protein group (A):
(A) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, CD81 antigen, and protein group consisting of CD63 antigen, and
miRNA group (B):
(B) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p, miR-519d-5p, miR-1915-3p, miR-4707-5p, miR-3663 -3p, miR-4755-3p, miR-3654, miR-3184-5p, miR-5705, miR-4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR -4482-3p, miR-4481, miR-4515, miR-6127, miR-3614-5p, miR-6873-5p, miR-4532, miR-6831-5p, miR-451a, miR-16-5p, and miRNA group consisting of miR-1224-3p,
A test agent for lymphangioleiomyomatosis comprising a detection agent for at least one biomarker selected from the group consisting of: Protein group (A):
(A) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, CD81 antigen, and protein group consisting of CD63 antigen, and
miRNA group (B):
(B) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p, miR-519d-5p, miR-1915-3p, miR-4707-5p, miR-3663 -3p, miR-4755-3p, miR-3654, miR-3184-5p, miR-5705, miR-4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR -4482-3p, miR-4481, miR-4515, miR-6127, miR-3614-5p, miR-6873-5p, miR-4532, miR-6831-5p, miR-451a, miR-16-5p, and miRNA group consisting of miR-1224-3p,
An agent for preventing or treating lymphangioleiomyomatosis comprising an inhibitor of at least one biomarker selected from the group consisting of: In the extracellular vesicles of body fluids collected from animals treated with the test substance,
Protein group (A):
(A) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, CD81 antigen, and protein group consisting of CD63 antigen, and
miRNA group (B):
(B) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p, miR-519d-5p, miR-1915-3p, miR-4707-5p, miR-3663 -3p, miR-4755-3p, miR-3654, miR-3184-5p, miR-5705, miR-4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR -4482-3p, miR-4481, miR-4515, miR-6127, miR-3614-5p, miR-6873-5p, miR-4532, miR-6831-5p, miR-451a, miR-16-5p, and miRNA group consisting of miR-1224-3p,
A method for screening an active ingredient of a preventive or therapeutic agent for lymphangioleiomyomatosis, using as an index the amount or concentration of at least one biomarker selected from the group consisting of: In the extracellular vesicles of body fluids collected from animals treated with the test substance,
Protein group (A):
(A) Fibrinogen beta chain, Fibrinogen gamma chain, Bleomycin hydrolase, Transferrin receptor protein 1, Fibrinogen alpha chain, Protein S100-A12, Properdin, Aminopeptidase N, tRNA-dihydrouridine (20) synthase [NAD (P) +]-like, G-protein coupled receptor family C group 5 member C, Receptor-type tyrosine-protein phosphatase C, C4b-binding protein alpha chain, Vitamin K-dependent protein S, Suprabasin, Alpha-1-antichymotrypsin, Complement C1q subcomponent subunit C, Proteoglycan 4, Lactadherin, Protein S100-A9, Alpha-1-antitrypsin, Collagen alpha-3 (VI) chain, Putative alpha-1-antitrypsin-related protein, Protein S100-A8, Protein S100-A6, Angiopoietin-1, Carbonic anhydrase 1, Glutathione peroxidase 3, Versican core protein, CD81 antigen, and protein group consisting of CD63 antigen, and
miRNA group (B):
(B) miR-4730, miR-6132, miR-744-5p, miR-4633-3p, miR-6858-3p, miR-519d-5p, miR-1915-3p, miR-4707-5p, miR-3663 -3p, miR-4755-3p, miR-3654, miR-3184-5p, miR-5705, miR-4450, miR-4439, miR-6872-5p, miR-3194-3p, miR-7851-3p, miR -4482-3p, miR-4481, miR-4515, miR-6127, miR-3614-5p, miR-6873-5p, miR-4532, miR-6831-5p, miR-451a, miR-16-5p, and miRNA group consisting of miR-1224-3p,
An evaluation method for inducing or increasing malignancy of lymphangioleiomyomatosis using as an index the amount or concentration of at least one biomarker selected from the group consisting of: