KR20210047538A - Compositiion for predicting or diagnosing inverted papilloma and method for predicting or diagnosing using the same - Google Patents

Abstract

The present invention relates to a composition for predicting or diagnosing inverted papilloma. A phosphatidylinositol 3-kinase delta (PI3K-δ) biomarker provided by the present invention can improve the patient's inverted papilloma diagnosis accuracy by more accurately determining the onset of inverted papilloma, and can be usefully used to determine an appropriate treatment method or to increase the survival rate after the onset of inverted papilloma by making it possible to better predict the condition of the lesion and the clinical outcome of the patient after treatment.

Description

Composition for predicting or diagnosing inverted papilloma and method for predicting or diagnosing using the same}

The present invention relates to a composition for predicting or diagnosing reverse papilloma, and more particularly, for use in predicting and diagnosing reverse papilloma by measuring the activity of PI3K-δ (Phosphatidyl inositol 3 kinase) enzyme in nasal mucosa. Can be used.

Reversible papilloma occurring in the nasal cavity and sinuses is a benign epithelial tumor, which is relatively rare in 0.5-4% of primary tumors in the nasal cavity. , It is one of the major diseases that require continuous observation due to its association with malignant tumors.

The primary sites of the semi-transparent papilloma are the middle nasal and middle nasal turbinates, which are the nasal side walls, and they rarely originate from the sinuses, and most of them spread from the nasal cavity to the ethmoid sinus and the maxillary sinus opening. Most of the malignant changes in reverse papilloma progress to invasive squamous cell carcinoma, and there are more simultaneous malignant changes than secondary.

In the case of reverse papilloma, there are external approaches as surgical resection, lateral nasal incision (non-external) approach, sub-oral facial center approach, and osteoplastic flap surgery. Diagnosis is the most important for proper treatment at the right time because it can cause and there is the possibility of malignant changes.

Diagnosis of reverse papilloma includes endoscopy, radiological examination, and biopsy. With the advancement of endoscopic surgery, computed tomography has become a basic method for diagnosing sinusitis, and a lot of research and development are being made for radiographic diagnosis of reverse papilloma, but it still has the disadvantage of being unable to distinguish other tumors in the nasal cavity. On the other hand, there is an MRI method that can closely check the existence and extent of reverse papilloma, but this also has the disadvantage of not being able to distinguish between other tumors and reverse papilloma, and is applied to all patients complaining of nasal congestion symptoms in terms of cost. It is somewhat unreasonable to do. Moreover, since only surgical treatment exists for reverse papilloma, it is very important to first develop a biomarker specific for reverse papilloma in order to develop a new drug for it.

Therefore, there is a growing demand for a method for predicting or diagnosing reverse papilloma and a biomarker for this.

Matters described as the above-described background art are only for enhancing an understanding of the background of the present invention, and should not be taken as acknowledging that they correspond to the prior art already known to those of ordinary skill in the art.

Non-Patent Documents 1. Kwon Sam-Hyun and 6 others. Treatment of reverse papilloma: Comparison between non-extraoperative and endoscopic intranasal surgery. Lee Inji Han, Vol. 40, No. 8 1997.

The present inventors have made great efforts to develop a novel marker for predicting or diagnosing reverse papilloma in the nasal cavity and a method for predicting or diagnosing reverse papilloma using the same. As a result, by finding out that there is a positive correlation between the level of the PI3K-δ (Phosphatidylinositol 3-kinase delta) protein and the reverse papilloma, the present invention was completed.

Accordingly, an object of the present invention is to provide a composition for predicting or diagnosing reverse papilloma.

Another object of the present invention is to provide a method of providing information for predicting or diagnosing a reverse papilloma.

Another object of the present invention is to provide a method for providing information on the expression level of PI3K-δ in a semi-reversible papilloma in order to determine whether or not to use a PI3K-δ inhibitor.

Another object of the present invention is to provide a method for screening candidates for prevention, treatment or improvement of reverse papilloma.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides a composition for predicting or diagnosing reverse papilloma comprising an agent measuring the expression level of PI3K-δ (Phosphatidylinositol 3-kinase delta).

As a result of trying to discover novel markers for predicting or diagnosing reverse papilloma, the present inventors confirmed that there is a positive correlation between the level of PI3K-δ (Phosphatidylinositol 3-kinase delta) protein and the reverse papilloma. In addition, it was confirmed that there was a positive correlation between VEGF (vascular endothelial growth factor) and HIF-1α (hypoxia induced factor-1α) durtl reverse papilloma, a related indicator.

In the present invention, "diagnosis" refers to confirming the presence or characteristics of a pathological condition, wherein the diagnosis is to confirm whether or not reverse papilloma has occurred, determine the prognosis of a subject suffering from reverse papilloma, or information on treatment efficacy By monitoring the condition of the subject in order to provide a, it is possible to determine whether the onset of reverse papilloma occurs early.

In the present invention, the term "prediction" is to predict the risk of onset, and the prediction can determine the likelihood or risk of developing a reverse papilloma.

In the present invention, the term "biomarker" or "marker" may also be used as a diagnostic marker or diagnostic marker, and is a material that can be diagnosed by discriminating whether or not reverse papilloma has occurred in a biological sample. It may be a PI3K-δ protein showing an increase in the level of expression in one patient or a nucleic acid (mRNA) encoding it. In addition, in addition to an increase in PI3K-δ protein, VEGF and/or HIF-1α protein or a nucleic acid (mRNA) encoding the same may be included, which shows an increase in the expression level in patients suffering from reverse papilloma compared to normal samples.

In the present invention, the "protein marker" may be a PI3K-δ protein, or a combination of PI3K-δ protein and any one or more selected from VEGF and HIF-1α.

PI3K is an enzyme that phosphorylates the 3-hydroxyl group of the phosphoinositide (PI) inositol ring, one of the lipid signaling kinases. PI3K is divided into three groups, class Ⅰ, Ⅱ, and Ⅲ according to molecular structure, function, and substrate specificity, and Class I PI3K is again classified into class IA and class IB. PI3Kα, PI3Kβ and PI3Kδ belong to class IA, and PI3Kγ belongs to class IB. Class I PI3K is a heterodimer consisting of a catalytic subunit and a regulatory subunit with a molecular weight of 110 kDa. There are three types of catalytic subunits, p110α, p110β, and p110δ, and one of them and one of the regulatory subunits p85α, p50α, p55α, p85β, and p55γ combine to form a PI3K heterodimer. Among them, PI3K-δ (Phosphatidylinositol 3-kinase delta) is also known as'p110δ', and PI3K-α is known to act on insulin hormone regulation, cancer growth and metabolic regulation mechanism, and PI3K-δ is cell growth, It is known to affect movement and survival.

According to a preferred embodiment of the present invention, the expression of PI3K-δ (Phosphatidylinositol 3-kinase delta) protein specifically increased in a biological sample isolated from a subject in order to predict or diagnose reverse papilloma, and its amino acid sequence and base As for the sequence, the sequence information can be confirmed in a known gene database. Specifically, PI3K-δ (Phosphatidylinositol 3-kinase delta) may be a protein derived from all eukaryotes having PI3K-δ, including mammals such as humans, cattle, goats, sheep, pigs, mice, rabbits, etc., preferably It may be derived from a biological sample isolated from, and the PI3K-δ (Gene ID: 5293) may be more preferably searched through, and NCBI accession no. NC_000001.11, NC_000001.10, NG_023434.1 It may be translated from the gene of RefSeqGene.

The biological sample includes samples such as tissues, cells, whole blood, plasma, serum, blood, saliva, sputum, lymph fluid, cerebrospinal fluid, intercellular fluid, or urine, but is not limited thereto, but preferably may be tissue and specifically It may be nasal or sinus tissue.

According to a preferred embodiment of the present invention, the process of separating a protein from a biological sample may be performed using a generally known process.

According to a preferred embodiment of the present invention, it was confirmed that the expression of VEGF and HIF-1α proteins together with PI3K-δ was increased in a biological sample isolated from a subject in order to predict or diagnose reverse papilloma.

Therefore, in order to increase the accuracy in predicting or diagnosing reverse papilloma, an agent for measuring one or more expression levels selected from the group consisting of vascular endothelial growth factor (VEGF) and HIF-1α (Hypoxia-Inducible Factor-1 alpha) is further used. Can include.

The vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis by cells as a'vascular endothelial growth factor'. That is, it exerts an effect by acting on endothelial cells produced by cells (paracrine) or by acting on the same endothelial cells (autocrine) produced by the endothelial cells themselves. VEGF is known to bind to receptors present on the surface of endothelial cells, such as VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), or neuropilin-1 (NRP-1). VEGF binds to VEGFR-2 on the surface of endothelial cells, and almost all known angiogenesis, such as proliferation, survival, adsorption, migration, and tube formation. Be involved in the stage.

The Hypoxia inducible factor (HIF) is an important transcription factor that acts on the expression of VEGF. HIF is expressed by hypoxia and is known to affect the expression of VEGF, and it plays an important role in the expression of other hypoxia inducible genes. HIF is a heterodimer composed of α-unit that is degraded under normal oxygen partial pressure and β-unit that is not affected by oxygen partial pressure. It is known that HIF-1α and HIF-2α act on the Hypoxia response element (HRE) for transcription, and HIF-3α acts as an antagonist to the HIF system.

The amino acid sequence and nucleotide sequence of VEGF and HIF-1α of the present invention can be identified in known gene databases. For example, the amino acid sequence of the VEGF protein (Gene ID: 7422) is NCBI accession no. It may be translated from the genes of NC_000006.12, NG 008732.1, and NC_000006.11. The amino acid sequence of HIF-1α protein (Gene ID: 3091) is NCBI accession no. It may be translated from the genes of NC_000014.9, NG 029606.1, and NC_000014.8.

According to a preferred embodiment of the present invention, the agent for measuring the expression level of PI3K-δ is an antibody capable of specifically binding to PI3K-δ protein or a fragment thereof; Alternatively, it may be a primer or probe that specifically binds to the gene encoding the PI3K-δ.

In addition, the agent for measuring the expression level of one or more selected from the group consisting of vascular endothelial growth factor (VEGF) and HIF-1α (Hypoxia-Inducible Factor-1 alpha) is applied to VEGF and/or HIF-1α protein or fragment thereof. Antibodies capable of specifically binding; Alternatively, it may be a primer or probe that specifically binds to the gene encoding the VEGF and/or HIF-1α protein.

The antibody includes all monoclonal antibodies, polyclonal antibodies and recombinant antibodies. In the present invention, the monoclonal antibody is a hybridoma method well known in the art (Kohler and Milstein (1976) European journal of Immunology 6:511-519), or a phage antibody library (Clarkson et al, Nature, 352:624- 628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991). Polyclonal antibodies can be prepared by conventional methods, including injecting the protein antigen into an animal and obtaining serum containing the antibody from the animal. Such polyclonal antibodies can be prepared from any animal including dogs, goats, sheep, rabbits, monkeys, horses, pigs, and cattle. Further, the antibodies of the present invention include chimeric antibodies, humanized antibodies, human antibodies, and the like. In addition, the antibody used in the present invention includes a full-form antibody having two full-length light chains and two full-length heavy chains and functional fragments thereof. The functional fragment of the antibody refers to a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv.

The primer is a short nucleic acid sequence having a short free 3'hydroxyl group, which can form a complementary template and a base pair, and functions as a starting point for template strand copying. Means sequence. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. In the present invention, PCR amplification may be performed using a gene encoding PI3K-δ, VEGF and/or HIF-1α protein, or a sense and antisense primer thereof, and the reverse papilloma can be diagnosed through whether or not a desired product is produced. The PCR conditions, the length of the sense and antisense primers can be modified based on those known in the art.

The probe refers to a nucleic acid fragment such as RNA or DNA corresponding to a short number of bases to hundreds of bases that can achieve specific binding with an mRNA, and is labeled so that the presence or absence of a specific mRNA can be confirmed. 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. In the present invention, hybridization can be performed using a probe complementary to a gene encoding PI3K-δ, VEGF and/or HIF-1α protein, and the hybridization can be used to diagnose a reverse papilloma. Selection of suitable probes and conditions for hybridization can be modified based on those known in the art.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite 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 (eg, methyl phosphonate, phosphotriester, Phosphoroamidate, carbamate, etc.) or to a charged linker (eg phosphorothioate, phosphorodithioate, etc.).

In addition, the present invention is a protein whose expression level is relatively increased in a biological sample of a patient with reverse papilloma compared to a biological sample isolated from a normal person, comprising an agent measuring the expression level of the PI3K protein, for predicting or diagnosing reverse papilloma Kits are provided.

The kit may further include an agent for measuring one or more expression levels selected from the group consisting of vascular endothelial growth factor (VEGF) and HIF-1α (Hypoxia-Inducible Factor-1 alpha).

The kit of the present invention can measure the expression level of PI3K-δ protein by analyzing the antigen against the antibody through antigen-antibody binding reaction. In addition, the kit may further include one or more compositions, solutions or devices suitable for analysis of the protein level. For example, the kit may include a substrate, a buffer solution, a secondary antibody labeled with a detection label, and a chromogenic substrate for immunological detection of the antibody.

The antigen-antibody binding reaction is a conventional enzyme-linked immunosorbent assay (ELISA), radioimmnoassay (RIA), sandwich assay, western blot on a polyacrylamide gel, immunoblot assay, and immunity. It is preferably selected from the group consisting of the histochemical staining method (immnohistochemical staining), but is not limited thereto.

The fixture for the antigen-antibody binding reaction is composed of a nitrocellulose membrane, a PVDF membrane, a well plate synthesized of polyvinyl resin or polystyrene resin, and a slide glass made of glass. Those selected from the group may be used, but are not limited thereto.

The secondary antibody is preferably labeled with a conventional coloring agent that performs a color reaction, and HRP (Horseradish peroxidase), alkaline phosphatase, colloid gold, FITC (Poly L-lysine-fluorescein isothiocyanate) ), RITC (Rhodamine-B-isothiocyanate), or any one of a label selected from the group consisting of a fluorescent substance (Fluorescein) and a dye (Dye) may be used. Substrates that induce color development are preferable to be used depending on the label that reacts to color development, and TMB(3,3',5,5'-tetramethyl bezidine), ABTS[2,2'-azino-bis(3-ethylbenzothiazoline) -6-sulfonic acid)] and OPD (ophenylenediamine) It is preferable to use any one selected from the group consisting of, but is not limited thereto.

According to a preferred embodiment of the present invention, the kit for diagnosing a reverse induced species may be a kit including necessary components to perform an ELISA method such as an ELISA kit or a sandwich ELISA. The ELISA kit includes an antibody specific for the protein marker, and can bind to a reagent capable of detecting a bound antibody, for example, a labeled secondary antibody, a chromopores, an enzyme, and a substrate or antibody thereof. It may include other materials that are present.

In addition, the present invention relates to a protein chip for diagnosing reverse papilloma comprising the composition for diagnosing reverse papilloma. The protein chip is composed of one or more antibodies against the protein marker of the present invention disposed on the chip at high density. In the present invention, the detection label may be selected from the group consisting of enzymes, phosphors, ligands, luminaries, microparticles, redox molecules, and radioisotopes, but is not limited thereto.

The method of providing information for predicting or diagnosing reverse papilloma of the present invention includes the following steps:

(a) obtaining a biological sample isolated from the subject outside the body;

(b) measuring the expression level of PI3K-δ protein from the biological sample; And

(c) determining whether the PI3K-δ protein expression level contained in the biological sample of the subject is higher than the PI3K-δ protein expression level contained in the biological sample of the normal group.

First, (a) a biological sample isolated from the subject outside the body is obtained.

The biological sample is a tissue, cell, whole blood, plasma, serum, blood, saliva, sputum, lymph, cerebrospinal fluid, intercellular fluid, or urine, in which the protein expression level or the expression level of the gene encoding it differs due to the onset of reverse papilloma. Includes a sample, but is not particularly limited thereto. Preferably, the biological sample may be tissue, specifically nasal or sinus tissue.

According to a preferred embodiment of the present invention, the process of separating the biological sample separated from the subject from the outside of the body may be performed using a generally known process.

Next, (b) measuring the expression level of the PI3K-δ protein or the gene encoding it from the biological sample, and (c) the PI3K-δ protein contained in the biological sample of the subject or the gene encoding the expression level in the normal group Whether it is higher than the expression level of the PI3K-δ protein or the gene encoding it contained in the biological sample of

According to a preferred embodiment of the present invention, the method of providing information for predicting or diagnosing reverse papilloma is a group consisting of VEGF and HIF-1α in (d) a biological sample obtained from step (a) in order to increase the precision of diagnosis. Measuring the expression level of one or more selected from; And (e) additionally confirming whether the expression level of at least one selected from the group consisting of VEGF and HIF-1α contained in the biological sample of the subject is higher than the expression level contained in the biological sample of the normal group. Can include.

The expression level of the protein marker of the present invention can be measured and compared using an antibody that specifically binds to the corresponding protein. The antibody and the corresponding protein in the biological sample are allowed to form an antigen-antibody complex, and a method of detecting this is used.

The antigen-antibody complex refers to a combination of a corresponding protein antigen contained in a biological sample and an antibody that recognizes it. The detection of the antigen-antibody complex can be detected using methods known in the art, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, absorptive, chemical and other methods.

Methods for measuring or comparing the expression level of the protein marker include protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/ Ionization Time of Flight Mass Spectrometry analysis, radioimmunoassay (RIA), radioimmunodiffusion, Ouchteroni immune diffusion method, rocket immunoelectrophoresis, immunohistochemistry analysis, immunocytochemistry ) Analysis, immunoprecipitation, complement fixation method, two-dimensional electrophoresis analysis, liquid chromatography-Mass Spectrometry (LCMS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry) ), Western blot, and ELISA (enzyme linked immunosorbentassay), but are not limited thereto.

In the present invention, immunohistochemistry, immunoprecipitation, Western Blotting and ELISA (enzyme linked immunosorbentassay) were used to measure and compare the protein marker expression level itself.

In addition, the expression level of the gene encoding the protein marker may be measured or compared with the mRNA expression level using a primer or probe that specifically binds thereto. Measurement or comparison of the mRNA expression level is a reverse transcriptase polymerase chain reaction (RT-PCR), a competitive reverse transcriptase polymerase reaction, a real time-polymerase chain reaction, real-time PCR, and RNase. Protection assays, Northern blotting, next-generation sequencing (NGS) or DNA chips may be used, but are not particularly limited thereto.

When the expression level of the protein marker contained in the biological sample of the subject or the gene encoding the same is higher than the expression level of the protein marker contained in the biological sample of the normal group, the subject has a high risk of developing a reverse papilloma or develops a reverse papilloma. Information to determine that the disease has occurred can be provided.

According to a preferred embodiment of the present invention, the disease caused by overexpression of the protein marker is a reverse papilloma.

The method for providing information on the level of PI3K-δ expression in a semi-reversible papilloma to determine whether to use the PI3K-δ inhibitor of the present invention comprises the following steps:

(a) obtaining a biological sample isolated from the subject outside the body;

(b) measuring the expression level of the nucleic acid sequence encoding the PI3K-δ protein or the concentration of the PI3K-δ protein from the biological sample; And

(c) determining whether the expression level of the nucleic acid sequence encoding the PI3K-δ protein contained in the biological sample of the subject or the concentration of the PI3K-δ protein is higher than the expression level contained in the biological sample of the normal group.

A method for providing information for predicting or diagnosing a reverse papilloma of the present invention described above in the description of a method for providing information on the expression level of PI3K-δ in a reverse papilloma to determine whether to use the PI3K-δ inhibitor The same part as the description of will be referred to that part.

It was confirmed that the PI3K-δ protein expression level was increased in tissues for patients with reverse papilloma according to the present invention, and substances capable of inhibiting or inhibiting the PI3K-δ protein activity and expression can be used as a therapeutic agent for reverse papilloma. have.

However, since PI3K-δ protein is involved in the growth, movement, and survival of cells other than reverse papillomas, the use of PI3K-δ inhibitors is determined by comparing the level of PI3K-δ protein in the subject and the level of PI3K-δ protein in the normal person. You can provide information to make a decision.

According to a preferred embodiment of the present invention, when the PI3K-δ expression level is higher than that of a normal person in a reverse papilloma, it is preferable to use a PI3K-δ inhibitor.

The PI3K-δ inhibitor may be any one selected from the group consisting of small molecule compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to PI3K-δ protein, but is not particularly limited thereto.

In addition, the PI3K-δ inhibitor is composed of an antisense nucleotide complementarily binding to an mRNA of a gene encoding a PI3K-δ protein, a small interfering RNA (siRNA), and a short hairpin RNA (shRNA). It may be any one selected from the group, but is not particularly limited thereto.

The method of screening for candidates for prevention, treatment or improvement of reverse papilloma of the present invention includes the following steps:

(1) a treatment step of treating the test sample with PI3K-δ protein; And

(2) An analysis step of selecting a test sample that inhibits the activity of the PI3K-δ protein.

In addition, the screening method for a candidate substance for preventing, treating, or improving anti-reversible papilloma of the present invention includes the following steps.

(I) a treatment step of treating the test sample to the PI3K-δ protein-expressing cells; And

(II) Analysis step of selecting a test sample that inhibits the expression of PI3K-δ protein in the cells.

Among the descriptions of the method of providing information for predicting or diagnosing a reverse papilloma, the same part as the description of the screening method of the present invention described above will be referred to.

In the present invention, "test sample" refers to an unknown substance used in screening to test whether it affects the activity or expression level of the PI3K-δ protein or the expression level of the gene encoding the PI3K-δ protein.

The test sample may include a chemical substance, a nucleotide, an antisense-RNA, a small interference RNA (siRNA), and a natural product extract, but is not limited thereto.

Thereafter, from the PI3K-δ protein treated with the test sample, its activity is measured, or the expression level of the PI3K-δ protein or the activity of the PI3K-δ protein or the gene encoding the PI3K-δ protein in the cells treated with the test sample The expression level of can be measured. As a result of the measurement, if it is determined that the expression of the PI3K-δ protein or the expression of the gene encoding the PI3K-δ protein or the activity of the PI3K-δ protein is inhibited, the test sample is a candidate material capable of preventing, treating or improving reverse papilloma. It can be determined as.

In the above, the analysis step may be performed through various methods known in the art, but is not particularly limited thereto. The analysis step is preferably reverse transcriptase-polymerase chain reaction, real time-polymerase chain reaction, western blot, ELISA (enzyme linked immunosorbent assay), radioimmunity. Analysis (radioimmunoassay, RIA), radioimmunodiffusion, and immunoprecipitation can be used.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a composition for predicting or diagnosing reverse papilloma comprising an agent measuring the expression level of PI3K-δ (Phosphatidylinositol 3-kinase delta).

(Ii) In addition, the present invention provides a method of providing information for predicting or diagnosing a reverse papilloma and a method of screening a candidate substance capable of preventing, improving or treating a reverse papilloma.

(Iii) The PI3K-δ (Phosphatidylinositol 3-kinase delta) biomarker provided by the present invention can improve the accuracy of diagnosis of the patient's reverse papilloma by more accurately determining whether or not the onset of reverse papilloma has been developed, and the condition for the lesion And it can be used to better predict the clinical outcome of the patient after treatment, to determine an appropriate treatment method or to increase the survival rate after the onset of reverse papilloma.

FIG. 1 shows the results of confirming the change in protein expression of PI3k-δ in normal nasal mucosa (HC) and seminal papilloma nasal mucosa (IP) by Western blot.
FIG. 2 shows the results of confirming the change in protein expression of pAKT, a precursor of PI3k-δ, in normal nasal mucosal tissue (HC) and seminal papilloma nasal mucosa (IP) by Western blot.
Figure 3 is a confocal microscope visualizing the localization of PI3K-δ (green) and VEGF (red) in normal nasal mucosa (HC) and semi-papillary nasal mucosa (IP).
FIG. 4 is a graph showing the expression level of VEGF according to the expression level of PI3K-δ in the non-mucosal tissue (IP) of a reverse papilloma.
Figure 5 is a confocal microscope visualizing the localization of PI3K-δ (green) and HIF-α (red) in human normal nasal tissues (HC) and human semi-inducible nasal tissues (IP).
6 is a graph showing the expression level of HIF-α according to the expression level of PI3K-δ in the nasal tissue (IP) of a patient with semi-transparent papilloma.
7 is a confocal microscope visualizing the localization of VEGF (green) and HIF-α (red) in human normal nasal tissues (HC) and human semi-inducible nasal tissues (IP).
8 is a graph showing the expression level of HIF-α according to the expression level of VEGF in the nasal tissue (IP) of a patient with reverse papilloma.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

<Experiment method>

1. Selection of research targets

This study was conducted under the approval of the Medical Research Ethics Committee of Chonbuk National University Hospital, and recruited patients with reversible papilloma. All of them were tested for blood eosinophil count, neutrophil count, blood immunoglobulin E level, allergic reaction test, X-ray test and Examined by computed tomography, only 10 patients with precisely reversed papillomas were included in this study.

2. Biological sample separation

Inverted papilloma (IP) was obtained (n=10) from the nasal mucosal tissues of patients identified as reverse papilloma (n=10), and non-mucosal tissue (Healthy control, HC) of normal patients was obtained (n=10). The recovered tissue was separated into two pieces, one was immediately frozen and used for Western blotting for measuring PI3K-δ (delta) expression, and the other tissue was fixed in 4% paraformaldehyde. It was used for immunofluorescence staining.

<Example 1> Comparative analysis of protein expression in nasal mucosal tissues of patients with semi-transparent papilloma and non-mucosal tissues of normal subjects

The nasal mucosal tissue (IP) and the non-mucosal tissue (HC) of a patient with reversible papilloma obtained through Experimental Methods 1 and 2 were prepared. To perform Western blot, each tissue was crushed into cells, and the cells were then subjected to cell disruption buffer (150 mM NaCl, 1.0% nonidet-P 40 (NP40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, pH 8.0). It was crushed with 50 mM Tris and pH 7.4 protease inhibitor cocktail (including Roche Applied Science, Vienna, Austria). General Western blot analysis was performed using the lysate as a sample. Specifically, anti-PI3K delta, phospho AKT, and anti-β-actin (1:1000; Cell Signaling Technology Inc.) as primary antibodies were used as secondary antibodies. Horseradish peroxidase-conjugated secondary antibodies (1:1,0000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used, and the immune response band was confirmed by enhanced chemiluminescence (ECL).

1 shows the result of confirming the protein expression change of PI3k-δ in the normal nasal mucosa (HC) and the seminal papilloma nasal mucosa (IP) by Western blot, and FIG. 2 is the normal nasal mucosa (HC) and the seminal papilloma nasal mucosa. It shows the result of confirming the change in protein expression of pAKT, a precursor of PI3k-δ, in tissue (IP) by Western blot.

In addition, raw dates of PI3k-δ and pAKT (phosphoAKT) measured by Western blot from normal nasal mucosa (HC) and semi-papillary nasal mucosa (IP) are shown in Tables 1 and 2, respectively.

HC IP 1.264575 6.416464 1.368409 2.071595 One 2.618456 One 1.739777 1.034203 1.35842 One 5.064943 One 2.47117 1.569114 8.505771

HC IP 1.32655 2.129646 One 10.10873 1.04 2.90288 One 31.4938

1, 2, Tables 1 and 2, the expression levels of PI3K-δ and phospho AKT, a precursor thereof, were higher in the nasal mucosal tissue of the semi-transferred papilloma than in the normal nasal mucosa, which showed a significant result.

<Example 2> Comparative analysis of PI3K-δ and VEGF protein expression in nasal mucosal tissues of patients with reverse papilloma and non-mucosal tissues of normal subjects

The nasal mucosal tissue (IP) and the non-mucosal tissue (HC) of a patient with reversible papilloma obtained through Experimental Methods 1 and 2 were prepared. For confocal analysis, tissues fixed with 4% paraformaldehyde from nasal mucosal tissues (IP) of patients with semi-transparent papilloma and non-mucosal tissues (HC) of normal subjects were used. Then, 1% Triton X-100 (in PBS) was permeabilized at room temperature for 20 minutes. Bovine serum albumin (BSA, Sigma-Aldrich, MO, USA) was blocked at room temperature for 4 hours. To reduce non-specific protein binding, the blocked tissues were incubated overnight at 4° C. with primary antibodies such as anti-PI3K-δ (Santa Cruz) and VEGF (Abcam). Each of the tissues was washed 3 times with PBS and incubated with Alexa Flour 488 or Alexa Flour 594 conjugated secondary antibody for 4 hours at 4°C. Next, the cell nuclei were counter-stained with DAPI (4',6-diamidino-2-phenylindole dihydrochloride, Invitrogen, CA, USA), and analyzed with NIH image J software. It was observed with a confocal laser-scanning fluorescence microscope.

3 is a confocal microscope visualizing the localization of PI3K-δ (green) and VEGF (red) in normal nasal mucosal tissue (HC) and semi-transparent papilloma nasal mucosal tissue (IP), and FIG. 4 is It is a graph showing the expression level of VEGF according to the expression level of PI3K-δ in (IP).

According to FIG. 3, it was confirmed that the expression of PI3K-δ (green) and VEGF (red) (vascular permeable material) was significantly higher in the semi-transferred papilloma nasal mucosa (IP) than in the normal nasal mucosa (HC). In addition, it can be seen that PI3K-δ (green) and VEGF (red) are located in the same cell part.

According to FIG. 4, it can be seen that the expression of PI3K-δ and the expression of VEGF are closely related to each other (p<0.05).

<Example 3> Comparative analysis of PI3K-δ and HIF-1α protein expression in nasal mucosal tissues of patients with reverse papilloma and non-mucosal tissues of normal subjects

For confocal analysis, tissues fixed with 4% paraformaldehyde in human normal nasal tissue (HC) and human semi-derived nasal tissue (IP) were used. Then, 1% Triton X-100 (in PBS) was permeabilized at room temperature for 20 minutes. Bovine serum albumin (BSA, Sigma-Aldrich, MO, USA) was blocked at room temperature for 4 hours. To reduce non-specific protein binding, the blocked tissues were incubated overnight at 4° C. with primary antibodies such as anti-PI3K-δ (Santa Cruz) and HIF-1α (Abcam). Each of the tissues was washed 3 times with PBS and incubated with Alexa Flour 488 or Alexa Flour 594 conjugated secondary antibody for 4 hours at 4°C. Next, the cell nuclei were counter-stained with DAPI (4',6-diamidino-2-phenylindole dihydrochloride, Invitrogen, CA, USA), and analyzed with NIH image J software. It was observed with a confocal laser-scanning fluorescence microscope.

FIG. 5 is a visualization of the localization of PI3K-δ (green) and HIF-α (red) in human normal nasal tissue (HC) and human semi-inducible nasal tissue (IP) with a confocal microscope, and FIG. 6 is It is a graph showing the expression level of HIF-α according to the expression level of PI3K-δ in the nasal tissue (IP) of a patient with reverse papilloma.

According to FIG. 5, it was confirmed that the expression of PI3K-δ (green) and HIF-α (red) (vascular permeable substance) was significantly higher in human semi-inducible nasal tissue (IP) than in normal nasal tissue (HC). According to FIG. 6, it can be seen that PI3K-δ expression and HIF-1α are closely related to each other (p<0.05).

<Example 4> Comparative analysis of VEGF and HIF-1α protein expression in nasal mucosal tissues of patients with reverse papilloma and non-mucosal tissues of normal subjects

For confocal analysis, tissues fixed with 4% paraformaldehyde in human normal nasal tissue (HC) and human semi-derived nasal tissue (IP) were used. Then, 1% Triton X-100 (in PBS) was permeabilized at room temperature for 20 minutes. Bovine serum albumin (BSA, Sigma-Aldrich, MO, USA) was blocked at room temperature for 4 hours. To reduce non-specific protein binding, the blocked tissues were incubated overnight at 4° C. with primary antibodies such as VEGF (Abcam) and HIF-1α (Abcam). Each of the tissues was washed 3 times with PBS and incubated with Alexa Flour 488 or Alexa Flour 594 conjugated secondary antibody for 4 hours at 4°C. Next, the cell nuclei were counter-stained with DAPI (4',6-diamidino-2-phenylindole dihydrochloride, Invitrogen, CA, USA), and analyzed with NIH image J software. It was observed with a confocal laser-scanning fluorescence microscope.

Figure 7 is a confocal microscope visualizing the localization of VEGF (green) and HIF-α (red) in human normal nasal tissue (HC) and human semi-inducible nasal tissue (IP). It is a graph showing the expression level of HIF-α according to the expression level of VEGF in the nasal tissue (IP) of a papilloma patient.

According to FIG. 7, it can be seen that the expression of VEGF (green) and HIF-α (red) (vascular permeable substance) is significantly higher in the human semi-inducible nasal tissue (IP) than in the normal nasal tissue (HC). In addition, it can be seen that the protein expression of VEGF (green) and HIF-1α (red) is increased at similar sites.

According to FIG. 8, it can be confirmed that VEGF and HIF-1α have a close relationship with each other (p<0.05).

In sum, PI3K-δ expression and enzyme activity of the present invention were increased in nasal tissues of semi-reversible papilloma compared to normal nasal tissues, and through this, PI3K-δ of the present invention is a biomarker in effectively diagnosing and predicting reverse papilloma. It was confirmed that there is efficacy. In addition, as it was confirmed that VEGF (vascular endothelial growth factor) and HIF-1α (hypoxia induced factor-1α), which are important for improving vascular permeability and angiogenesis, are also increased with PI3K-δ, For accurate prediction or diagnosis, it is desirable to simultaneously check the expression of PI3K-δ as well as VEGF and HIF-1α.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (11)

PI3K-δ (Phosphatidylinositol 3-kinase delta) composition for predicting or diagnosing reverse papilloma comprising an agent for measuring the expression level. The method of claim 1,
A composition for predicting or diagnosing reverse papilloma, characterized in that it further comprises an agent for measuring one or more expression levels selected from the group consisting of vascular endothelial growth factor (VEGF) and HIF-1α (Hypoxia-Inducible Factor-1 alpha). The method of claim 1,
The agent for measuring the expression level of PI3K-δ is an antibody capable of specifically binding to the PI3K-δ protein or a fragment thereof; Or a primer or probe that specifically binds to the gene encoding the PI3K-δ; a composition for predicting or diagnosing reverse papilloma, characterized in that. The method of claim 1,
The PI3K-δ protein is a composition for predicting or diagnosing reverse papilloma, characterized in that derived from a biological sample isolated from the specimen. The method of claim 4,
The biological sample is a composition for predicting or diagnosing reverse papilloma, characterized in that obtained from nasal mucosal tissue. A method of providing information for predicting or diagnosing reverse papilloma comprising the following steps:
(a) obtaining a biological sample separated from the subject outside the body;
(b) measuring the expression level of the PI3K-δ protein or the gene encoding the PI3K-δ protein from the biological sample; And
(c) determining whether the PI3K-δ protein expression level contained in the biological sample of the subject is higher than the PI3K-δ protein expression level contained in the biological sample of the normal group. The method of claim 6,
(d) measuring the expression level of at least one selected from the group consisting of VEGF and HIF-1α in the biological sample obtained from step (a); And
(e) additionally checking whether the expression level of at least one selected from the group consisting of VEGF and HIF-1α contained in the biological sample of the subject is higher than the expression level contained in the biological sample of the normal group; further comprising Method for providing information for predicting or diagnosing reverse papilloma, characterized in that. The method of claim 6,
The method of providing information for predicting or diagnosing reverse papilloma, characterized in that the biological sample is obtained from a nasal cavity or sinus tissue. A method for providing information on the level of PI3K-δ expression in a reverse papilloma to determine whether to use a PI3K-δ inhibitor comprising the following steps:
(a) obtaining a biological sample isolated from the subject outside the body;
(b) measuring the expression level of the nucleic acid sequence encoding the PI3K-δ protein or the concentration of the PI3K-δ protein from the biological sample;
(c) determining whether the expression level of the nucleic acid sequence encoding the PI3K-δ protein contained in the biological sample of the subject or the concentration of the PI3K-δ protein is higher than the expression level contained in the biological sample of the normal group. A method for screening for candidates for prevention, treatment or improvement of reverse papilloma comprising the following steps:
(1) a treatment step of treating the test sample with PI3K-δ protein; And
(2) An analysis step of selecting a test sample that inhibits the activity of the PI3K-δ protein. A method for screening for candidates for prevention, treatment or improvement of reverse papilloma comprising the following steps:
(I) a treatment step of treating the test sample to the PI3K-δ protein-expressing cells; And
(II) Analysis step of selecting a test sample that inhibits the expression of PI3K-δ protein in the cells.

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