KR102331138B1 - A Composition for Diagnosing Painful Bladder Syndrome - Google Patents

Abstract

The present invention relates to a composition for diagnosing or predicting painful bladder syndrome and a composition for preventing or treating painful bladder syndrome. The present invention can be effectively used in developing a rational treatment plan for interstitial cystitis, which is an intractable disease, by providing a reliable molecular diagnostic marker for painful bladder syndrome for which clear diagnostic criteria are not established and, more specifically, for interstitial cystitis. According to one aspect of the present invention, the present invention provides a composition for diagnosing or predicting painful bladder syndrome, comprising, as an active ingredient, a preparation for measuring the expression level of one or more genes selected from the group consisting of CD5, CD38, ITGAL, IL7R, and PSMB9.

Description

A composition for diagnosis of painful bladder syndrome {A Composition for Diagnosing Painful Bladder Syndrome}

The present invention relates to a diagnostic composition for painful bladder syndrome, specifically, interstitial cystitis, comprising a genetic marker.

Painful Bladder Syndrome is a disease of suprapubic pain associated with bladder filling without urinary tract infection or other obvious causes, and is often accompanied by daytime frequency, nocturia, and urgency. Among them, interstitial cystitis (IC) refers to bladder pain syndrome showing characteristic cystoscopic findings such as glomerulation and Hunner lesions and histological findings of mast cell clusters.

Interstitial cystitis is a chronic, wasting inflammation of the urinary tract characterized by a persistent inflammatory process within the bladder tissue, and its symptoms vary, but generally range from mild discomfort to pressure, tenderness, or severe pain in the pelvic area, and a feeling of urgency and frequent urination. It may also be accompanied by numbness and urge incontinence. The prevalence of interstitial cystitis varies from 67 to 230 per 100,000 women with clinically confirmed disease, and diagnostic criteria were established by the National Institute of Arthritis, Diabetes, Digestive and Kidney Disease (NIDDK) in 1988 and 2002. There have been many confusions and changes in the diagnostic criteria since its presentation. Although there are many opinions that interstitial cystitis can be diagnosed with clinical symptoms alone if other diseases are excluded, many clinicians argue that characteristic cystoscopy and histological findings should be accompanied.

In the case of the potassium sensitivity test, which was introduced as one of the diagnostic methods for interstitial cystitis, Parsons et al. ( J Urol 159:1862-1866(1998)) reported that 75% of interstitial cystitis patients responded to the potassium sensitivity test. However, this is not a recommended test method due to controversy over specificity and sensitivity. Recently, as an easier and more objective test, research on biomarkers or urine markers has been active. Representatively, antiproliferative factor (APF), Tamm-Horsfall protein (THP), heparin binding-epidermal growth factor (HB-EGF), bladder surface Bladder surface factor and IL-6 were reported as significant diagnostic markers. However, along with the continuous change of diagnostic criteria, the criteria for the extent to which interstitial cystitis can be confirmed remains unclear.

Numerous papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

Non-Patent Literature 1. REVIEWS IN UROLOGY 2002; 4 (Suppl 1): S3-S8.

The present inventors made intensive research efforts to develop an accurate and objective molecular diagnostic method for painful bladder syndrome, specifically interstitial cystitis, for which clear diagnostic criteria have not been established due to various and complex etiology and symptoms. As a result, by discovering that the six genes listed above are highly expressed in patients with interstitial cystitis compared to normal individuals in bladder tissue or urine sediment, and also have a high protein-protein interaction, the present invention is completed. became

Accordingly, an object of the present invention is to provide a composition for diagnosis or prediction of painful bladder syndrome.

Another object of the present invention is to provide a composition for preventing or treating painful bladder syndrome and a screening method thereof.

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

According to one aspect of the present invention, the present invention provides a painful bladder syndrome (painful bladder syndrome) comprising, as an active ingredient, an agent for measuring the expression level of one or more genes selected from the group consisting of CD5, CD38, ITGAL, IL7R, KLRB1 and PSMB9. To provide a composition for diagnosis or prediction of bladder syndrome).

The present inventors made intensive research efforts to develop an accurate and objective molecular diagnostic method for painful bladder syndrome, specifically interstitial cystitis, for which clear diagnostic criteria have not been established due to various and complex etiology and symptoms. As a result, the six genes listed above are not only highly expressed in patients with interstitial cystitis compared to normal individuals in bladder tissue or urine sediment, but also have a high protein-protein interaction, so that these genes are associated with painful bladder syndrome. It was found that it can function as a reliable diagnostic marker of

As used herein, the term “painful bladder syndrome” refers to a chronic inflammatory disease caused by inflammation of the bladder and sensory system abnormalities due to causes other than bacterial infection, accompanied by bladder fullness, humerus pain, and frequent urination during the day and night. , also called bladder pain syndrome. Painful bladder syndrome belongs to a group of diseases that are difficult to diagnose clearly and objectively, as the definition and diagnostic criteria of the disease are constantly changing, and objective diagnostic criteria have not been established, and the diagnostic criteria provided by NIDDK also do not meet some cases. . According to a specific embodiment of the present invention, the painful bladder syndrome that can be diagnosed or predicted by the composition of the present invention is interstitial cystitis.

As used herein, the term “diagnosis” refers to determining a subject's susceptibility to a specific disease or disorder, determining whether an object currently has a specific disease or disorder (eg, of hypoxic cancer). identification), determining the prognosis of a subject afflicted with a particular disease or condition, or therametrics (e.g., monitoring a subject's condition to provide information about the efficacy of treatment). .

According to a specific embodiment of the present invention, the agent for measuring the expression level of the gene may be a nucleic acid molecule that specifically binds to the gene, for example, a primer or a probe.

As used herein, the term “nucleic acid molecule” refers to deoxyribonucleotides or ribonucleotides that exist in single-stranded or double-stranded form, and includes analogs of natural nucleotides unless otherwise specified (Scheit, Nucleotide Analogs, John Wiley). , New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

As used herein, the term “primer” refers to an oligonucleotide, and conditions that induce the synthesis of a primer extension product complementary to a nucleic acid chain (template), that is, the presence of nucleotides and a polymerase such as a DNA polymerase, and It can serve as a starting point for synthesis under conditions of suitable temperature and pH. Preferably, the primer is a deoxyribonucleotide and is single-stranded. The primer used in the present invention may include naturally occurring dNMP (ie, dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides. In addition, the primer may also include ribonucleotides.

In the present invention, the term “complementary” means that a primer or probe is sufficiently complementary to selectively hybridize to a target nucleic acid sequence under predetermined annealing or hybridization conditions, and is substantially complementary and perfectly complementary. complementary), and preferably means completely complementary. As used herein, the term "substantially complementary sequence" used in relation to a primer sequence is not only a completely identical sequence, but also partially anneals to a specific sequence to function as a primer, and partially to a sequence to be compared. It is meant that sequences that do not match are also included.

As used herein, the term “probe” refers to a natural or modified monomer or a linear oligomer including deoxyribonucleotides and ribonucleotides capable of hybridizing to a specific nucleotide sequence. Preferably, the probe is single-stranded for maximum efficiency in hybridization. The probe is preferably a deoxyribonucleotide.

As the probe used in the present invention, a sequence that is completely complementary to a sequence of a target site on a gene of the present invention may be used, but a sequence that is substantially complementary within a range that does not interfere with specific hybridization is may be used. Preferably, the probe used in the present invention contains a sequence capable of hybridizing to a target site sequence on the gene of the present invention. More preferably, the 3'-end or 5'-end of the probe has a base complementary to the target site sequence on the gene of the present invention.

Suitable conditions for hybridization are Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach , It can be determined with reference to the information disclosed in IRL Press, Washington, DC (1985).

According to a specific embodiment of the present invention, the agent for measuring the expression level of the gene is an antibody or aptamer that specifically binds to a protein encoded by the gene. According to the present invention, the expression level of the CD5, CD38, ITGAL, IL7R, KLRB1 or PSMB9 gene is detected at the protein level according to an immunoassay method using an antigen-antibody reaction and used to diagnose painful bladder syndrome. can be This immunoassay may be performed according to various previously developed immunoassays or immunostaining protocols. For example, when the method of the present invention is performed according to a radioimmunoassay method, ^{an antibody labeled with a radioisotope (eg, C 14} , I ¹²⁵ , P ³² and S ³⁵ ) detects a polypeptide of the present invention. can be used to The diagnostic antibody that can be used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.

Antibodies to the polypeptides of the present invention can be prepared by methods commonly practiced in the art, for example, fusion methods (Kohler and Milstein, European Journal of Immunology , 6:511-519 (1976)), recombinant DNA methods (USA). 4,816,567) or by phage antibody library methods (Clackson et al, Nature , 352:624-628 (1991) and Marks et al, J. Mol. Biol. , 222:58, 1-597 (1991)). can be manufactured. General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley/Greene, NY, 1991.

The diagnostic composition of the present invention may use an aptamer that specifically binds to the protein of the present invention instead of an antibody. As used herein, the term “aptamer” refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule, which binds to a specific target substance with high affinity and specificity to exhibit action. For general information on aptamers, see Bock LC et al., Nature 355(6360):564-6(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):426-30(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24):14272-7 (1998).

As used herein, the term “composition for diagnosing or predicting painful bladder syndrome” refers to an integrated method for measuring the expression level of the marker gene of the present invention or the protein it encodes in order to predict or determine whether a subject develops painful bladder syndrome. It refers to a mixture or device, and may be expressed as “a kit for diagnosing or predicting painful bladder syndrome”.

According to a specific embodiment of the present invention, the composition of the present invention includes an agent for measuring the expression level of PSMB9 as an active ingredient.

More specifically, an agent for measuring the expression level of ITGAL is additionally included, and most specifically, an agent for measuring the expression level of KLRB1 is additionally included.

According to another aspect of the present invention, the present invention comprises the steps of measuring the expression level of one or more genes selected from the group consisting of CD5, CD38, ITGAL, IL7R, KLRB1 and PSMB9 in a biological sample isolated from an individual; And it provides a method of providing information necessary for diagnosis or prediction of painful bladder syndrome, comprising the step of comparing the expression level with the expression level measured in a normal control sample.

Since the genetic markers used in the present invention, the means for measuring their expression levels, and specific diseases to be diagnosed or predicted through them have already been described above, their description will be omitted to avoid excessive duplication.

According to another aspect of the present invention, the present invention provides for the treatment of painful bladder syndrome comprising an inhibitor of one or more genes selected from the group consisting of CD5, CD38, ITGAL, IL7R, KLRB1 and PSMB9 as an active ingredient. A composition for prevention or treatment is provided.

As used herein, the term “inhibitor” refers to a substance that causes a decrease in the activity or expression of a target gene or protein, whereby the activity or expression of the target gene or protein becomes undetectable or exists at an insignificant level. , refers to a substance that reduces activity or expression to the extent that its biological function can be significantly reduced.

Inhibitors of CD5, CD38, ITGAL, IL7R, KLRB1 and PSMB9 genes are, for example, shRNA, siRNA, miRNA, ribozyme, peptide nucleic acid (PNA) that inhibit the expression of these genes whose nucleotide sequences are known in the art. acids) CRISPR systems containing antisense oligonucleotides or guide RNAs that recognize target genes, and antibodies or aptamers that inhibit these genes at the protein level, as well as compounds, peptides and natural products that inhibit their activity, Without being limited thereto, any means of suppressing gene and protein levels known in the art may be used.

As used herein, the term “shRNA (small hairpin RNA)” is a single strand of 50-70 nucleotides forming a stem-loop structure in vivo. An RNA sequence that creates a tight hairpin structure. Usually, a long RNA of 19-29 nucleotides is base-paired on both sides of a loop of 5-10 nucleotides to form a double-stranded stem, and in order to always be expressed, it is introduced into a cell through a vector containing a U6 promoter. It is transduced and is usually passed on to daughter cells so that suppression of the expression of the target gene is inherited.

As used herein, the term “siRNA” refers to a short double-stranded RNA capable of inducing an RNAi (RNA interference) phenomenon through cleavage of a specific mRNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, most preferably 20 to 70 bases, and if the expression of the target gene can be inhibited by the RNAi effect, blunt ends or cohesive ends Both ends are possible. The structure of the adhesive end can be both a structure in which the three-terminal protrudes and a structure in which the five-terminal side protrudes.

As used herein, the term “miRNA (microRNA)” refers to a single-stranded RNA molecule that is not expressed in cells, has a short stem-loop structure, and inhibits the expression of a target gene through complementary binding to the mRNA of the target gene. do.

As used herein, the term “ribozyme” is a type of RNA and refers to an RNA molecule having the same function as an enzyme that recognizes the base sequence of a specific RNA and cuts it by itself. A ribozyme is a complementary nucleotide sequence of a target mRNA strand and consists of a region that binds with specificity and a region that cuts the target RNA.

As used herein, the term “peptide nucleic acid (PNA)” refers to a molecule capable of complementary binding to DNA or RNA while having both nucleic acid and protein properties. PNA is not found in nature but is artificially synthesized by a chemical method, and forms a double strand through hybridization with a natural nucleic acid of a complementary base sequence to control the expression of a target gene.

As used herein, the term “antisense oligonucleotide” refers to a nucleotide sequence complementary to a sequence of a specific mRNA, which binds to a complementary sequence in a target mRNA and translates its protein into a protein, translocation into the cytoplasm, maturation, or any other It refers to a nucleic acid molecule that inhibits an essential activity for an overall biological function. Antisense oligonucleotides may be modified at one or more bases, sugars or backbone positions to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol. , 5(3):343-55, 1995). . The oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyls, cycloalkyls, short chain heteroatomics, heterocyclic sugarscholphonates, and the like.

As used herein, the term “gRNA (guideRNA)” refers to an RNA molecule used in a gene editing system that recognizes a target gene and specifically cuts the recognized loci by inducing a nuclease. A typical example of such a gene editing system is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.

According to the present invention, the expression inhibitor of the present invention may be a specific antibody that inhibits the activity of the protein encoded by the genes. The antibody specifically recognizing the target protein is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.

As used herein, the term “prevention” refers to inhibiting the occurrence of a disease or disease in a subject who has never been diagnosed with a disease or disease, but is likely to have the disease or disease.

As used herein, the term “treatment” refers to (a) inhibiting the development of a disease, disorder or condition; (b) alleviation of the disease, condition or condition; or (c) eliminating the disease, condition or symptom. When the composition of the present invention is administered to a subject, the expression level or activity of CD5, CD38, ITGAL, IL7R, KLRB1 and PSMB9 is reduced, thereby inhibiting the progression of symptoms of painful bladder syndrome, eliminating it, or alleviating it. Accordingly, the composition of the present invention may be a composition for treating painful bladder syndrome by itself, or may be administered with other pharmacological components and applied as a therapeutic adjuvant for the disease. Accordingly, as used herein, the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic adjuvant” or “therapeutic adjuvant”.

As used herein, the term “administration” or “administering” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the subject's body.

In the present invention, the term “therapeutically effective amount” refers to the content of the composition in which the pharmacological component in the composition is sufficient to provide a therapeutic or prophylactic effect to an individual to whom the pharmaceutical composition of the present invention is to be administered. prophylactically effective amount”.

As used herein, the term “subject” includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Specifically, the subject of the present invention is a human.

According to another aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating painful bladder syndrome, comprising the following steps:

(a) contacting a test substance with a biological sample comprising at least one gene selected from the group consisting of CD5, CD38, ITGAL, IL7R, KLRB1 and PSMB9 or a protein encoding them; and

(2) measuring the expression level or activity of the gene or protein in the sample;

When the expression level or activity of the gene or protein is decreased, the test substance is determined as a composition for preventing or treating painful bladder syndrome.

Since the disease to be prevented or treated by the composition screened in the present invention has already been described above, the description thereof will be omitted to avoid excessive duplication.

As used herein, the term “biological sample” refers to any sample obtained from a mammal, including a human, containing cells expressing the above-listed genes, including, but not limited to, tissues, organs, cells, cell cultures or body fluids. . More specifically, the biological sample is a biological sample including bladder tissue, bladder tissue-derived cells, or urine.

The term “test substance” used while referring to the screening method of the present invention is added to a sample containing a cell expressing the gene of the present invention to test whether the activity or expression level of the gene or the protein they encode is affected. It means an unknown substance used in screening for The test substance includes, but is not limited to, compounds, nucleotides, peptides and natural extracts. The step of measuring the expression level or activity of the gene or protein in the biological sample treated with the test substance may be performed by various methods for measuring expression level and activity known in the art. As a result of the measurement, when the expression level or activity of these genes or proteins is reduced, the test substance can be determined as a composition for preventing or treating painful bladder syndrome.

As used herein, the term “reduction in expression level or activity” refers to the level of expression of CD5, CD38, ITGAL, IL7R, KLRB1 or PSMB9-mediated pain bladder syndrome progression, symptom or severity to a level that is measurable. Or it means that the intrinsic function in vivo is reduced. Specifically, it may refer to a state in which the activity or expression level is decreased by 20% or more, more specifically, a state in which the activity or expression level is decreased by more than 40%, more specifically, a state in which the activity or expression level is decreased by more than 60% compared to the control group.

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

(a) The present invention provides a composition for diagnosing or predicting painful bladder syndrome and a composition for preventing or treating painful bladder syndrome.

(b) The present invention provides a reliable molecular diagnostic marker for painful bladder syndrome, specifically interstitial cystitis, for which clear diagnostic criteria have not been established, thereby establishing a rational treatment strategy for interstitial cystitis, an intractable disease, at an early stage. can be usefully used for

1 shows the results of analysis of significant genes in IC/BPS. The Volcano plots of FIGS. 1A-1D show transcript expression between normal and IC/BPS patients. Green: p-value < 0.05, logFC ≥ 1 or ≤ -1, orange: p-value > 0.05, logFC ≥ 1 or ≤ -1, red: logFC >1 or < -1, black: no significance.
2 is It is a figure showing the analysis results for common positive and negative genes in IC/BPS. The intersection region is a gene that is significantly expressed in common in the three datasets of the GEO2R database. Positive genes are shown in FIG. 2A, and negative genes are shown in FIG. 2B.
3 is Expression of common DEGs in the IC/BPS data set is shown. The heatmap shows the expression and trends of the intersecting genes within each data set. These data were analyzed through hierarchical clustering using MeV software. The color of the pattern depends on the gene expression with a threshold of -1 < z-score < 1, and high and low expression outside the criteria were designated as the highest and lowest values. GSE11783 (FIG. 3A), GSE28242 (FIG. 3B) and GSE57560 (FIG. 3C) are shown, respectively.
4 is It is a figure showing the predicted pathway and gene ontology (GO) for the common DEG of IC/BPS. Histograms were derived with the Enrichr platform obtained from KEGG, Reactome, NCI-Nature and Gene Ontology (p-value ≤ 0.05, top 10 pathways of significance). Red bars are high-expressed genes (Fig. 4a) and blue bars are low-expressed genes (Fig. 4b). The darker the color of the bar, the lower the significance of the p-value.
5 is a diagram showing the results of analyzing herb genes and their predicted pathways. Figure 5a shows the STRING predicting the interaction of the functional protein of a significant gene. An edge represents an interaction between nodes. The color of the nodes indicates high (red) or low (blue) expression in IC/BPS patients compared to controls. Figure 5b shows the interaction between the herb gene and the top six proteins. The redder the node color, the more significant interactions exist. Figure 5c shows the results of analyzing the hub gene-TF interaction using NetworkAnalyst's JASPAR. The edge means that the hub gene is associated with the putative TF. Green represents the herb gene and orange represents TF.
6 shows predicted pathways and GO of hub genes in IC/BPS. 6A-6F are bar graphs showing putative paths obtained from various path databases. These results are ranked by p-value, with gray bars for p-values >0.05 and dark short bars for has a higher p-value.
7 is a diagram showing the expression of herb genes in the IC/BPS dataset. Data were obtained from the GSE11783 ( FIG. 7A ) and GSE57560 ( FIG. 7B ) datasets, respectively. Expression comparison of six herb genes was performed between IC patients with HL and controls, and statistical analysis was performed through GraphPad prism. The p-values were analyzed using a two-sided independent t-test. Asterisks indicate *p < 0.05, **p < 0.01, and ***p < 0.001, respectively.
8 shows the results of measuring the relative mRNA expression of 6 genes through RT-qPCR using non-IC/BPS (n = 5) and IC/BPS (n = 7) tissue samples. Expression levels were normalized to GAPDH. *p < 0.05, ****p < 0.0001, NS: not significant.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled 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

Experimental method

Identification of DEGs from GEO datasets in IC/BPS

To identify DEG in IC/BPS, we used Gene expression Omnibus (GEO, NCBI, NIH, https://www.ncbi.nlm.nih.gov/geo/) to interstitial cystitis/cystitis Syndrome (IC/BPS) data were explored. Four datasets: GSE621 [19], GSE11783 [13], GSE28242 [5] and GSE57560 [10] were used for analysis. In addition, IC/BPS samples with normal and Hunner lesions were selected from each data set (Table 1-3). DEG was derived from a data set comparing IC/BPS and normal tissue. In order to select significant DEGs with high fold change, a volcano plot was produced using the iLINCS web (LINCS DCIC, http://ilincs.org) [26]. Significant DEG was considered if the a p-value threshold < 0.05 in IC/BPS was met and log2FC < 2 or log2FC > -2.

To identify DEGs in the GEO data set, Venn diagrams were drawn using increased or decreased DEGs obtained independently from three data sets, GSE11783, GSE28242 and GSE57560. Then, the expression of DEG was expressed as a heatmap of hierarchical cluster analysis using MeV software v4.9.0 (Multiple Experiment Viewer). The color of each pattern reflects gene expression with a threshold of -1 < z-score < 1. High and low expression out of the standard were designated as the highest and lowest values.

Information from the GSE28242 data set disease stage age gender ulcer normal
( n = 5) 51 F No 51 F No 38 M No 71 F No 75 F No IC/BPS
( n = 3) 82 M Yes 76 F Yes 55 F Yes

Information from the GSE11783 data set disease stage age gender ulcer normal
( n = 6) 59.2
[40.0-75.0] F No F No F No F No F No F No IC/BPS
( n = 5) 78
[68.0-85.0] F Yes F Yes F Yes F Yes F Yes

Information from the GSE57560 data set disease stage age gender glomerulization ulcer bladder resection normal
( n = 3) 76 F NA No No 43 F NA No No 53 F NA No No IC/BPS
( n = 2) 67 F severe Yes Yes 65 F Mild Yes Yes

Pathway analysis of gene ontology (GO) and DEG

Predicted pathways and gene ontology of high- or low-expression DEGs were analyzed using the Enrichr web (Mount Sinai Innovation Partners, https://amp.pharm.mssm.edu/Enrichr/) [7; 20]. In addition, the predicted pathways and GO of the hub gene were also analyzed using the Enrichr web. Bar graphs derived from the Enrichr web were ranked based on p-values calculated from several databases including KEGG, Reactome pathway, NCI-Nature pathway and GO (Biological process, Molecular Function, and Cellular Component).

Construction of Protein-Protein Interaction (PPI) and Identification of Herb Genes

To build the PPI network, Cytoscape v3.7.2 software (NRNB, NIH/NIGMS/BBCB) was used for DEG with the STRING application (©STRING consortium 2019, ELIXIR, https://string-db.org/). . Next, the top six hub genes that could be biomarkers for IC/BPS were searched using cytoHubba through Cytoscape v3.7.2 software [9; 24].

Construction of Hub Gene and Transcription Factor (TF) Network

To build a hub gene-TF network, a web-based tool, NetworkAnalyst (http://www.networkanalyst.ca/faces/home.xhtml) [34; 35] was used. This can visualize the network of TF-gene interactions. In the present invention, hub genes and their expected regulatory TF networks were constructed using the JASPAR TF binding site profile database and visualized with Cytoscape v3.7.2 software.

Collection of clinical samples from IC/BPS and normal bladder tissue

The study of the present invention was conducted under the approval of the Clinical Trial Ethics Committee of Konkuk University Hospital (IRB approval number: KUH1130060, KUMC 2019-07-009), in compliance with the Declaration of Helsinki, and consent was obtained from all patients participating in the study.

The present inventors strictly complied with the IC/BPS diagnostic criteria of the American Society of Urology [15]. Assessment of patient symptoms included the Visual Analogue Score (VAS) pain questionnaire, O'Leary-Sant Symptom (ICSI) and Problem Index (ICPI) (IC*?*Q), and Pelvic Pain and Urgency/Frequency Patient Symptom/Bother score (PUF). ) was included. In the present invention, patients with a PUF score of >13, an IC*?*Q score of >24, and a VAS score of >4 were selected. Cystoscopy, urine culture, cytology and physical examination were performed to classify IC/BPS subtypes, and patients with urinary tract infection, bladder cancer, urinary tract tuberculosis, urolithiasis, and other neurological diseases or endometriosis were excluded.

Based on the results of cystoscopy, patients with Hunner lesions were identified. Hydrodilatation, transurethral resection, and coagulation (TUR*?*C) were performed for the Huner lesion. IC/BPS patient-derived specimens (IC 1-7) were obtained from Huner lesions during TUR*?*C. For control samples (non-IC 1-5), tissue from the bladder during radical cystectomy (non-IC 1) or transurethral cystectomy (non-IC 2-5) for the patient were collected, and the tissue was finally confirmed not to be malignant. Not all non-IC patients experienced less abdominal pain when their bladder filled with urine, nor did they have less urinary tract symptoms. Inclusion-exclusion criteria for patient selection are listed in Table 4. After storing the bladder tissue sample obtained from the patient at -80°C, RNA extraction was performed.

Criteria for inclusion and exclusion of study patients inclusion criteria Persistent pelvic pain related to the bladder
exacerbation of pain in bladder storage
Symptoms persist for more than 6 weeks
Confirmation of Hunner lesions and glomeruli on cystoscopy Exclusion Criteria Untreated UTIs, STDs, or urinary stones
Neurological disorders affecting the pelvic nerves
history of endometriosis
urethral diverticulum
Suspected DRE or PSA ≥2.5 ng/ml

Real-time quantitative PCR (RT-qPCR)

Total RNA was isolated from clinical tissues using LaboPass ^TM Labozol reagent (COSMOGENTECH, CMRZ001, Seoul, Korea), and the extracted total RNA was cDNA with LaboPass ^TM M-MuLV Reverse Transcriptase Kit (COSMOGENTECH, CMRT010, Seoul, Korea). reverse transcribed with HiPi Real-Time PCR 2 x Master Mix (ELPISBIO, EBT-1802, Daejeon, Korea) was used to measure the relative expression between control and IC/BPS patient tissues of 6 herb genes. The primers used are listed in Table 5, and each procedure was performed according to the manufacturer's protocol. Relative expression was normalized to GAPDH expression, an internal control, and fold change in gene expression was calculated using the comparative ΔCT (cycle time) method.

Primer sequence used gene direction order GADPH forward GTC TCC TCT GAC TTC AAC AGC G reverse ACC ACC CTG TTG CTG TAG CCA A CD38 forward CAG ACT GGA GAA AGG ACT GC reverse TTT ACT GCG GGA TCC ATT GAG CD5 forward CGA GTT CTT GCC CTC CTT TGC T reverse TCC TGG CTG AAG AGC TGT CAC A IL7R forward ATC GCA GCA CTC ACT GAC CTG T reverse TCA GGC ACT TTA CCT CCA CGA G ITGAL forward CTG CTT TTG CCA GCC TCT CTG T reverse GCT CAC AGG TAT CTG GCT ATG G KLRB1 forward GTT CCA CCA AAG AAT CCA GCC TG reverse AAG AGC CGT TTA TCC ACT TCC AG PSMB9 forward CGA GAG GAC TTG TCT GCA CAT C reverse CAC CAA TGG CAA AAG GCT GTC G

statistical analysis

Data were analyzed using GraphPad Prism version 7 (GraphPad software, La Jolla California, USA). The herbal gene data obtained from the GEO dataset were transformed into histograms to analyze their statistical significance. These results were compared for the IC/BPS and normal groups, and statistical significance was calculated using a two-sided independent t-test. A p-value <0.05 was considered statistically significant (*p<0.05, **p<0.01, ***p<0.001).

Experiment result

Identification of IC/BPS in IC/BPS patient tissues

In the present invention, a GEO dataset was derived from microarray analysis of IC/BPS patient samples with Huner lesions using the GEO database. Then, several DEGs were identified from four GEO datasets through DEG analysis using the LINCS DCIC web. Analysis objects included 184 genes of GES28242, 6756 genes of GSE57560, 0 genes of GSE621, and 4702 genes of GSE11783 (FIGS. 1a-d). The GSE621 data set in which no DEGs meeting the criteria were found were excluded from subsequent analysis (Fig. 1a). From the three GEO datasets, 51 high-expression DEGs (Fig. 2a) and 2 low-expression DEGs (Fig. 2b) were derived by Venn diagrams. All DEGs are listed in Table 6. As a result of creating an expression heat map for overlapping DEGs using the GES28242, GSE57560 and GSE11783 datasets (FIGS. 3a-c), it can be seen that there is a significant difference in the expression of the selected gene between the IC/BPS group and the control group. there was.

Genes with significant expression differences (DEGs) GSE28242 GSE11783 GSE57560 gene p -value LogFC p -value LogFC p -value LogFC ABCD2 2.8.E-02 1.13 3.2.E-03 1.69 2.3.E-04 2.08 ADCY7 1.7.E-02 1.13 3.9.E-04 1.35 6.1.E-03 1.79 BTLA 2.8.E-02 1.18 3.6.E-03 1.25 2.8.E-02 2.99 CD180 4.0.E-03 1.12 8.9.E-05 2.38 2.7.E-02 2.68 CD38 4.8.E-02 1.48 8.5.E-07 4.33 1.9.E-03 4.31 CD5 3.0.E-02 1.00 9.0.E-04 1.63 6.9.E-03 3.49 CD84 3.3.E-02 1.32 4.8.E-04 1.09 1.2.E-02 1.33 CFB 8.6.E-03 2.08 1.1.E-06 2.61 4.2.E-02 2.90 CLIC2 1.5.E-02 1.63 2.2.E-04 1.22 3.5.E-03 1.36 CTSZ 8.1.E-03 1.02 2.9.E-03 1.11 7.0.E-03 1.13 CYSLTR1 2.4.E-02 2.30 3.7.E-05 1.54 2.6.E-02 1.26 DOCK10 1.2.E-02 1.54 4.2.E-04 1.79 1.2.E-02 2.01 EPSTI1 5.5.E-04 2.50 5.5.E-03 1.14 1.5.E-03 2.27 FYN 2.1.E-02 1.46 2.9.E-04 1.29 5.3.E-03 1.73 GIMAP4 2.6.E-02 1.95 2.1.E-03 1.06 1.7.E-03 2.02 GIMAP8 2.9.E-04 1.01 1.1.E-03 1.14 2.6.E-02 1.17 GLRX 2.9.E-02 1.68 3.5.E-04 1.15 2.7.E-02 1.31 GPR171 7.2.E-03 1.52 3.7.E-05 1.71 2.1.E-03 3.36 GPRIN3 6.1.E-03 1.03 8.2.E-06 1.70 4.2.E-04 2.60 GZMK 6.3.E-03 1.50 1.6.E-02 1.64 8.0.E-03 3.10 HLA-DMA 1.8.E-02 1.83 2.8.E-04 1.25 1.8.E-03 2.49 HLA-DMB 4.4.E-03 1.92 3.4.E-03 1.08 5.5.E-03 2.72 HLA-DOB 4.9.E-02 1.31 1.2.E-03 2.97 3.0.E-03 3.10 HLA-DPA1 2.9.E-02 1.76 1.2.E-02 1.54 4.5.E-03 2.32 IDO1 2.7.E-02 3.58 4.3.E-03 2.03 1.7.E-05 4.69 IFI27 3.0.E-02 1.51 1.4.E-04 1.69 1.7.E-03 2.07 IFITM3 1.8.E-02 1.57 7.2.E-04 1.14 2.3.E-02 1.29 IL32 2.0.E-03 1.45 2.4.E-03 1.34 5.9.E-03 2.55 IL7R 5.0.E-02 1.33 5.3.E-03 1.49 4.1.E-03 2.69 ITGA4 1.8.E-03 2.10 6.5.E-05 1.36 3.4.E-04 1.69 ITGAL 4.5.E-02 1.72 3.4.E-03 1.23 6.9.E-03 2.40 ITK 2.9.E-02 1.42 4.8.E-04 1.50 1.4.E-02 3.18 KCNA3 4.2.E-02 1.34 1.8.E-03 3.22 1.2.E-02 2.21 KLRB1 3.0.E-02 1.13 1.2.E-03 1.49 1.2.E-02 2.57 LEF1 8.5.E-03 1.06 5.1.E-03 1.11 1.4.E-02 2.19 MCOLN2 3.8.E-02 1.13 1.3.E-02 1.30 8.0.E-03 2.55 NCKAP1L 2.1.E-02 1.93 3.3.E-05 1.65 5.7.E-03 1.97 NLRC5 1.3.E-02 1.78 2.2.E-04 1.56 4.4.E-02 1.70 PLAC8 4.2.E-03 1.46 1.1.E-04 1.99 5.4.E-03 4.22 PSMB9 1.2.E-02 2.40 2.3.E-03 1.15 3.3.E-04 2.42 RASGRP1 3.7.E-02 1.06 3.5.E-05 1.86 6.4.E-03 2.65 SAMHD1 6.6.E-04 1.46 1.4.E-05 1.87 2.4.E-03 1.78 SEPT6 2.2.E-02 1.19 3.6.E-05 1.57 1.1.E-02 1.32 SH2D1A 1.2.E-02 1.21 9.9.E-04 1.95 2.1.E-03 3.11 SLAMF8 2.2.E-02 1.01 1.6.E-03 1.69 2.0.E-03 3.00 SLFN11 2.2.E-03 1.25 3.1.E-04 1.17 4.3.E-03 1.42 STAT1 1.4.E-02 1.85 2.9.E-02 1.46 6.4.E-03 2.91 TAP2 2.4.E-02 2.13 4.6.E-04 1.46 4.3.E-04 1.61 TGM2 1.3.E-03 2.53 5.7.E-05 1.64 1.6.E-03 1.60 TRAT1 1.1.E-02 1.43 1.6.E-03 1.71 4.6.E-03 3.04 WARS 1.8.E-02 2.14 3.3.E-04 1.45 3.7.E-03 2.41 CWH43 3.0.E-02 -1.45 9.5.E-07 -5.58 1.8.E-04 -5.92 CYP2J2 4.1.E-02 -1.02 6.8.E-06 -4.72 1.0.E-02 -1.52

Enrichment analysis for DEG in IC/BPS patient tissue

Next, amplification analysis for increased or decreased DEG was performed using the Enrichr web, and as a result, various important pathways, particularly those related to DEG with increased expression, were discovered. These include hematopoietic stem cell lineages of the KEGG 2019 pathway, viral myocarditis and staphylococcal infection (Fig. 4a(i)); Immune system, adaptive immune system and cytokine signaling within the immune system of Reactome pathway 2016 (Fig. 4a(ii)); and TCR signaling, integrin family cell-surface interactions and CXCR4-mediated signaling of CD4+ T cells of the NCI-nature pathway 2016 (Fig. 4a(iii)). Several pathways are associated with DEG with reduced expression, for example linoleic acid metabolism, ovarian steroid synthesis and arachidonic acid metabolism are pathways that are mainly increased in the KEGG 2019 pathway (Fig. 4b(i)); Synthesis of epoxy (EET) and dihydroxyeicosatrienoic acid (DHET), fatty acids and xenobiotics and Reactome pathway 2016 are associated with reduced expression of DEG (Fig. 4b(ii)).

Next, gene ontology (GO) analysis was performed using a common high/low-expression DEG through the Enrichr web. The highest GO terms (biological processes, molecular functions, and cellular components) are shown in FIG. 4 . In biological processes, common DEGs were most significantly associated with antigen receptor-mediated signaling pathways, whereas linoleic acid metabolic processes were most increased in common low-expressing DEGs (Figs. 4a(iv) and 4b(iii)). As a result of the subsequent molecular function amplification analysis, type II MHC protein complex was the most common term associated with high-expressing DEG, whereas low-expressing DEG was associated with arachidonic acid monooxygenase activity (Figs. 4a(v) and 4b (Fig. 4a(v) and 4b) iv)). In cellular components, high-expression DEG was most increased in type II MHC protein complex binding, but low-expression DEG was not increased in any cellular component (Fig. 4a(vi)).

PPI network analysis of DEG and identification of hub genes from IC/BPS patient tissues

To complete the PPI analysis, a PPI network was constructed with common high- and low-expression DEGs using Cytoscape v3.7.2 software together with the STRING plug-in. As shown in FIG. 5A , 53 nodes (51 high-expression DEGs and 2 low-expression DEGs) and 47 edges appeared in the network. Then, the top 6 hub genes were identified from the network using the cytoHubba plugin through the Cytoscape v3.7.2 software (Fig. 5b). These herb genes include cluster of differentiation 5 (CD5), cluster of differentiation 38 (CD38), integrin alpha L chain (ITGAL), interleukin 7 receptor (IL7R), killer cell lectin like receptor B1 (KLRB1), and proteasome subunit beta (PSMB9). 9).

Next, a hub gene-TF network was constructed through the NetworkAnalyst web using JASPAR, a TF binding site profile database. From the hub gene-TF network, 6 hub genes interacting with 26 regulatory TFs were identified (Fig. 5c). Among the herb genes, ITGAL was the most prominent gene regulated by 13 TFs, PSMB9 by 8 TFs, CD5 by 7 TFs; CD38 and IL7R were regulated by 5 TFs and KLRB1 by 4 TFs, respectively (Fig. 5c, left panel). Furthermore, several TFs have been shown to be associated with more than one herb gene. In particular, 10 TFs were observed to interact with two or more hub genes (Fig. 5c, right panel), suggesting that these TFs interact closely with the aforementioned hub genes. For example, GATA2 has been shown to regulate CD5, CD38, ITGAL, IL7R, and KLRB1, whereas FOXC1 regulates CD5, CD38 and KLRB1.

Amplification analysis of herb gene derived from IC/BPS patient tissue

To identify pathways and GO amplification of biomarkers, amplification analysis was performed again via Enrichr web using hub genes. As shown in FIG. 6 , in the pathway analysis, the hub genes are the hematopoietic stem cell lineage (KEGG 2019 pathway), the immune system (Reactome pathway 2016) and the integrin family cell-surface interaction (NCI-Nature pathway 2016) (FIGS. 6a-c) was remarkably amplified in In the GO assay, herb genes were identified in the regulation of immune responses (Biological Process 2018), hydrolase activity, hydrolysis of hydrolyzing N-glycosyl compounds (Molecular Function 2018) and clathrin-coated ER membrane (Cellular Component 2018). significantly increased (Fig. 6d-f).

Herbal gene expression analysis in GEO dataset and IC/BPS patient tissues

Finally, the expression of six herb genes in the GEO dataset was analyzed, followed by expression in IC/BPS patient tissues compared to adjacent normal tissues. For GEO analysis, the expression level of each gene was extracted from each GEO dataset, displayed as a boxplot, and statistical analysis was performed through a two-sided independent t-test. As a result of analysis of the GEO data sets (GSE11783 and GSE57560), all herb genes were significantly overexpressed in IC/BPS patients compared to normal patients (Fig. 7a and b). Among the herb genes, CD38, PSMB9, CD5, ITGAL and KLRB1 were significantly overexpressed in IC/BPS tissues, whereas IL7R was expressed in small amounts ( FIGS. 7A and 7B ).

Next, the mRNA expression of the herb gene was analyzed from IC/BPS patients and normal tissue samples provided by Konkuk University Hospital. The clinicopathological characteristics of non-IC/BPS and IC/BPS patients are summarized in Table 7. Although there was a relative expression level difference between IC/BPS samples, it was confirmed through RT-qPCR data that all six herb genes were expressed relatively high in IC/BPS tissues compared to control tissues ( FIG. 8 ). PSMB9, ITGAL and KLRB1 were significantly more overexpressed in IC/BPS samples compared to non-IC/BPS samples, suggesting that these genes were the most promising IC/BPS biomarkers among all herb genes ( FIG. 8 ). Expression of CD38 and IL7R also showed significant differences between non-IC/BPS and IC/BPS patients ( FIG. 8 ). In addition, CD5, another herb gene, showed an increased average expression pattern in the IC/BPS sample compared to the non-IC/BPS sample. These results suggest that all herb genes can be usefully used as diagnostic biomarkers and are related to the pathogenesis of IC/BPS.

Clinicopathological Characteristics of Participating Patients No. gender age VAS
score ICSI
score ICPI
score PUF Symptom Score PUF
bother score Stock Exchange Duration (Month) Non-IC 1 M 73 0 0 0 0 0 0 Non-IC 2 M 73 0 0 0 0 0 0 Non-IC3 M 73 0 0 0 0 0 0 Non-IC4 M 72 0 0 0 0 0 0 Non-IC5 M 72 0 0 0 0 0 0 IC 1 M 26 8 18 18 22 12 24 IC 2 F 47 10 20 20 22 15 30 IC 3 M 57 8 18 20 20 12 38 IC 4 F 73 10 20 20 20 15 36 IC 5 F 74 10 18 20 22 15 26 IC 6 F 72 8 16 20 20 15 30 IC 7 F 70 8 20 20 28 15 30

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation corp <120> A Composition for Diagnosing Painful Bladder Syndrome <130> HPC-9241 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GADPH F primer <400> 1 gtctcctctg acttcaacag cg 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GADPH R primer <400> 2 accaccctgt tgctgtagcc aa 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CD38 F primer <400> 3 cagactggag aaaggactgc 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CD38 R primer <400> 4 tttactgcgg gatccattga g 21 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CD5 F primer <400> 5 cgagttcttg ccctcctttg ct 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CD5 R primer <400> 6 tcctggctga agagctgtca ca 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL7R F primer <400> 7 atcgcagcac tcactgacct gt 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL7R R primer <400> 8 tcaggcactt tacctccacg ag 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ITGAL F primer <400> 9 ctgcttttgc cagcctctct gt 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ITGAL R primer <400> 10 gctcacaggt atctggctat gg 22 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> KLRB1 F primer <400> 11 gttccaccaa agaatccagc ctg 23 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> KLRB1 R primer <400> 12 aagagccgtt tatccacttc cag 23 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PSMB9 F primer <400> 13 cgagaggact tgtctgcaca tc 22 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PSMB9 R primer <400> 14 caccaatggc aaaaggctgt cg 22

Claims (14)

A composition for diagnosis or prediction of painful bladder syndrome, comprising as an active ingredient an agent for measuring the expression level of the KLRB1 gene.
The composition according to claim 1, wherein the composition further comprises an agent for measuring the expression level of one or more genes selected from the group consisting of CD5, CD38, ITGAL, IL7R and PSMB9.
The method of claim 1, wherein the agent for measuring the expression level of the gene is a primer or probe that specifically binds to the gene, or an antibody or aptamer that specifically binds to a protein encoded by the gene. composition.
The composition of claim 1, wherein the painful bladder syndrome is interstitial cystitis.
The composition according to claim 1, wherein the composition further comprises an agent for measuring the expression level of PSMB9.
The composition according to claim 5, wherein the composition further comprises an agent for measuring the expression level of ITGAL.
delete measuring the expression level of the KLRB1 gene in a biological sample isolated from an individual; and comparing the expression level with the expression level measured in a normal control sample. A method for providing information necessary for diagnosis or prediction of painful bladder syndrome.
The method of claim 8, wherein the painful bladder syndrome is interstitial cystitis.
delete delete delete delete delete

Images