KR20110080929A - Biomarker of urothelial cancer and the diagnostic method of the same - Google Patents

Abstract

PURPOSE: A marker and a method for diagnosing urothelial carcinoma are provided to ensure high sensitivity and specificity. CONSTITUTION: A method for diagnosing urothelial carcinoma using a marker comprises: a step of collecting urine from a test sample; a step of measuring expression level of one or more proteins among cofilin and P-cofilin; and a step of measuring expression level of the proteins with the expression level of a healthy person. The urothelial carcinoma is non-muscle invasive or muscle invasive. The method for measuring protein expression level is selected from immunoblotting, immunohistochemical assay, Boyden chamber assay, Real time polymerase chain reaction, MALDI TOF/TOF MS/MS, and ELISA.

Description

Biomarker of Urothelial Cancer and the Diagnostic Method of the Same}

The present invention relates to a UTI marker and a method for diagnosing UTI using the same, and more particularly, to a marker for normal, non-invasive and invasive UTI, and to a method for treating UTI and using it.

Bladder urinary tract cancers occur in the United States alone every year, 100,000 new patients, the most common among the urinary cancer in Korea, most non-invasive bladder cancer. Among the newly developed bladder urinary tract cancers, about 70% of muscle noninvasive bladder cancers and about 30% of muscle invasive bladder cancers are diagnosed. In particular, non-invasive bladder cancer is 70% stage Ta, 20% stage T1 and 10% carcinoma in situ (CIS), and the incidence of bladder cancer and the frequency of its operations are increasing.

Most of the non-invasive bladder cancers of the urinary tract cancers that develop in the bladder can be treated with urinary bladder tumor resection. There is a problem. Because of these problems, regular follow-up through urinary bladder and urinalysis is essential for the observation of recurrence and progression of non-invasive bladder cancer.

Urinalysis is a non-invasive test, it is a good marker for the detection of poorly differentiated bladder urinary tract carcinoma, the specificity is 90 ~ 95% has a very high advantage. However, overall sensitivity is as low as 35-40%, especially in low stage, differentiated bladder cancer. In addition, the possibility of sample deterioration and quantitative measurement is difficult due to the delay until the test is started after the test request, and the test results may be subjective, which may cause a difference in readings according to pathologists. There is a disadvantage that can depend on the skill of the.

The most useful cystoscopy for the follow-up of bladder urinary tract cancers is invasive pain and discomfort to patients, even with the most sensitive tests of the present test, the use of soft bladder, local anesthesia, and administration of pre-procedure analgesics. It is a test that has a limitation in diagnosing urothelial cell carcinoma of the upper urinary tract when the shape of the tumor is flat or in epithelial cancer.

The markers of bladder urinary tract cancers are more susceptible to disease than urinalysis. However, the specificity is lower than urinalysis. In recent decades, markers of new bladder cancer have been increasing, but the urine ThinPrepR test, BTA test, BAT Stat, BTA TRAK assay, NMP22 (Nuclear matrix proteins) test, NMP 22 BladderChekTM, UBTTM test and Accu-Dx test (fibrin degradation) Markers of various bladder cancers, such as products test), are not tumor-specific proteins for bladder urinary tract carcinoma, are nonspecific as tumor-associated proteins, and have low specificity due to positive reactions in other diseases and normals. Therefore, it is difficult to cope with low sensitivity urinalysis because it has big limitation in diagnosis and follow-up of bladder urinary tract cancer.

Bladder urothelial carcinoma plays an important role in inactivating tumor suppressor genes such as TP53, RB, P21, P27, and P16, activating oncogenes such as RAS, and overexpression of EGF (ERBB1, ERBB2) receptors.

Intraclinical immunotherapy and anticancer therapy are currently applied to prevent recurrence and progression of muscle non-invasive bladder cancer in clinical practice. However, about 50% of the most effective immunotherapy does not report relapse and progression.

Based on past studies, genes that act upon the development of bladder cancer have been actively studied to date, but there are no studies on the difference between the expression of protein expressed in muscle noninvasive and invasive bladder urothelial carcinoma and normal bladder tissue protein.

That is, the bladder urinary tract carcinoma markers currently being used in the clinic have been developed for the screening of changed proteins excreted in the urine, and thus have high sensitivity but low specificity as a diagnostic and follow-up tool.

Accordingly, the present inventors have completed the present invention as a result of diligent efforts to find markers with high sensitivity and specificity as a tool for diagnosis and follow-up of bladder urinary tract cancer.

After all, it is an object of the present invention to provide a urinary tract cancer marker and a method for diagnosing, treating, and prognosing a urinary tract cancer.

In order to achieve the above object, the present invention provides a urinary tract cancer marker comprising at least one protein of Cofilin and P-cofilin (P-cofilin).

In the present invention, the urinary tract cancer may be characterized in that it is non-invasive or invasive.

The present invention also comprises the steps of (a) collecting the urine of the diagnosis object; (b) measuring the expression level of any one or more proteins of cofilin and P-cofilin in the collected urine; And (c) comparing the expression level of any one or more proteins of cofilin and P-cofilin measured in step (b) with a normal person, and Prognostic methods are provided.

In the present invention, the urinary tract cancer may be characterized in that it is non-invasive or invasive.

In the present invention, the method of measuring the degree of protein expression (1) directly measuring the protein in the urine or (2) the method of examining protein expression by protein measurement and immunostaining in the erupted urinary epithelial cells in the urine It can be characterized by.

In the present invention, the method for measuring the expression level of the protein may be any one or more of immunoblotting, immunohistochemical assay, Boyden chamber assay, Real time polymerase chain reaction, MALDI TOF / TOF MS / MS, ELISA.

The present invention also provides a method for treating urinary tract cancer, characterized in that it inhibits any one of the proteins of Cofilin and P-cofilin.

The present invention also provides a diagnostic kit for examining risk factors of urinary tract cancer using the above method.

The present invention has the effect of providing a uniquely selected and secured urinary tract cancer markers through proteomics.

The urinary tract cancer marker of the present invention complements the problems of sensitivity and specificity of the bladder urinary tract cancer markers currently used in the clinic, and is applicable to the diagnosis, recurrence, and muscle invasiveness of the bladder urinary tract cancer.

Figure 1 shows the protein expression of normal, non-invasive and invasive bladder cancer tissue in 2-DE gel image, NMIBC is Non-muscle invasive bladder cancer, MIBC is muscle invasive bladder cancer Indicates.
Figure 2 shows the expression profile and quantitative analysis of upregulated or downregulated protein in normal, non-invasive and invasive bladder cancer tissue, NMIBC is non-muscle invasive bladder cancer, MIBC is muscle invasive bladder cancer (Muscle invasive bladder cancer).
Figure 3 shows the degree of expression and phosphorylation of kophyrin in normal, non-invasive and invasive bladder cancer tissue, NMIBC is Non-muscle invasive bladder cancer, MIBC is muscle invasive bladder cancer (Muscle invasive bladder cancer) Indicates.
4 shows EGF-induced phosphorylation of human bladder cancer T24 cells.
5 shows the effect of EGF on the proliferation and migration of human bladder cancer T24 cells.

Hereinafter, the present invention will be described in more detail.

Since the urinary bladder urinary carcinoma markers currently being applied in the clinic have been developed for the selection of changed protein from urine, the sensitivity and low specificity are problems as a diagnostic and follow-up tool. Unlike detection methods, differences in normal tissues, muscle non-invasive bladder urinary tract cancer, and muscle invasive bladder urinary tract carcinoma were identified based on the expression of protein in tissues during bladder urinary tract cancer. In other words, when urinary tract cancer develops, a difference in urine is identified based on a protein showing a marked difference.

The inventors of the present invention, by using proteomics to ensure the expression of protein of normal bladder tissue and muscle proteins differently expressed in non-invasive and invasive bladder cancer (cofilin) and p-cofilin (p-cofilin) to use The possibility was confirmed.

Immunohistochemical staining using cofilin and P-cofilin antibodies showed the same results as proteomics-based protein expression in normal bladder tissue, muscle noninvasive and invasive bladder urothelial carcinoma tissues. It is useful for the diagnosis of muscle noninvasive and muscle invasive bladder cancer through immunochemical staining in tissues after transurethral bladder tumor resection. In addition, the difference in the expression ratio of Cofilin and P-cofiln expressed in tissues can be applied to distinguish between muscle invasive and muscle-invasive bladder cancer.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Experimental Example  1. Preparation for experiment

The sources of materials used in the present invention are as follows:

a) Materials for 2-Dimension Electrophoresis (2-DE) and mass spectrometry (MS) -BioRad (Hercules, CA, USA) or Applied Biosystems (Foster City, CA, USA).

b) recombinant human epidermal growth factor (rhEGF) -R & D Systems (Minneapolis, MN, USA).

c) McCoy's 5 A medium-Welgene (Korea).

d) Fetal bovine serum (FBS), penicillin, streptomycin and chemicals-Hyclone (Logan, UT, USA) or Sigma (St. Louis, MO, USA).

e) Polyclonal anti-phosphorylated ^ser3 Kophyrins and anti-nonphosphorylated kophyrin antibodies-Cell Signaling (Beverly, MA, USA).

f) Polyclonal GAPDH Antibody-Sigma (St. Louis, MO, USA).

Experimental Example  2. Experimental method

All experiments were performed according to the guidelines of Konkuk University. This study was approved by the Institutional Review Board (IRB) of Chungju Hospital, Konkuk University. Urinary tract epithelium in normal and bladder cancer patients (non-invasive and invasive) was treated with bladder biopsy and physiological saline (in mM; NaCl 136.9, KCl 5.4, CaCl ₂ 1.5, MgCl ₂ 1.0, NaHCO ₃ 23.8, EDTA 0.01) It was removed by excision and histopathologically confirmed. The identified samples were flash frozen in liquid nitrogen (N ₂ ) for proteomic and western blot analysis. Samples for immunohistochemical analysis were soaked in 4% paraformaldehyde solution for 8 hours and buried in paraffin.

Experimental Example  3. Statistical Analysis

The data of the present invention is expressed as mean ± standard deviation (SEM), the statistical evaluation of the data using Student's t-test for comparison between groups, ANOVA for various comparisons. For p <0.05, it was considered a statistically different value.

Example  1. Secondary electrophoresis and MALDI - TOF Of TOF MS

Bladder tissue samples from normal and cancerous (invasive, noninvasive) patients were treated with 8M urea, 2M thiourea, 100 mM DTT, 4% CHAPS (3 [(3-Cholamidopropyl) dimethylammonio] -propanesulfonic acid) and 1 × complete protease inhibitor cocktail (Roche). Applied Science, Germany), it was homogenized in 2-DE lysis buffer (lysis buffer).

After incubating the homogenized suspension for 40 minutes, centrifugation was performed at 12,000 × g, 10 ° C. for 10 minutes, and then the supernatant was washed with 7M Urea, 2M thiourea, 100 mM DTT, 2% CHAPS 0.5% ampolyte (Bio-Rad). ) And 0.01% bromophenol blue, diluted with rehydration buffer and used as sample for 2-DE analysis.

2-DE analysis was performed by Lee CK et al ., Proteomics . 9: 4851, 2009; Lee CK et al ., Proteomics . 6: 6455, 2006; Lee CK, et al , Biochim Biophys Acta . 1774: 848, 2007) gels were stained using a photometer (Versa Doc Imagin System 1000 ^™ ) (see FIG. 1).

1 shows 2-DE gel images of protein expression in normal, non-invasive and invasive bladder cancer tissue. The numbers shown in FIG. 1 indicate the number of spots of the protein detected, and arrows indicate proteins that are expressed differently in the tissues of normal and cancer patients.

The gel was also leveled and statistically analyzed using PDQuest software (Version 7.1.1, Bio-Rad) (see FIG. 2).

That is, in-gel digestion and protein identification of the modified protein spot were performed in the same manner as the existing method (Lee CK). et al ., Proteomics. 9: 4851, 2009; Lee CK et al ., Proteomics . 6: 6455, 2006; Lee CK, et al, Biochim Biophys Acta . 1774: 848, 2007).

In brief, the protein spot was digested by trypsin and desalted by ZipTip C ₁₈ (Millipore).

The protein sample was mixed with CHCA substrate solution and then analyzed using MALDI-TOF / TOF (AB4700, Applied Biosystems) in reflector mode. Spectra were processed and analyzed with Global Protein Server Explorer 3.0 software (Applied Biosystems). An internal MASCORT program (Matrix Science, UK) was used to match database information with MS and MS / MS data. The result data is a survey of mouse and human databases downloaded from NCBI and the Swiss Prot / TrEMBL website (see FIGS. 2 and 3).

Figure 2 shows the expression profile and quantitative analysis of upregulated or downregulated protein in normal, non-invasive and invasive bladder cancer tissues. Arrows in the 2-DE gel indicate spots that differ between normal and cancer patients. Using PDQuest software (Version 7.1.1, Bio-Rad), statistical data were obtained from 2-DE gels, which performed four independent experiments. In the figures, the * indicates that there is a significant difference ( p <0.05) between normal, non-invasive and invasive cancer patients. In the graph, white bars represent bladder tissue of normal persons, striped bars represent bladder tissue of non-invasive cancer patients, and black bars represent bladder tissue results of invasive patients.

FIG. 3A is an enlarged view of kophyrin spots in 2-DE gels of bladder tissue in normal, non-invasive and invasive cancer patients (n = 4). Arrows indicate kopirin.

Example  2. Immunoblotting

For immunoblotting, proteins were extracted from bladder tissues and cells with cold lysis buffer. Protein concentrations were determined using protein assay reagents (Bio-Rad, Hercules, CAN, USA).

The protein samples were mixed and diluted 1: 1 with SDS sample buffer (v / v) and then modified by heating for 5 minutes. The samples (20-30 μg / lane) were separated by 14% SDS-PAGE and electrically transferred to polyvinylidene fluoride membranes (Millipore, Bedford, Mass., USA).

Thereafter, the membrane was immersed in a phosphate buffered saline (PBS) containing 0.05% Tween 20 and 5% fat-free dried milk at room temperature for 2 hours, and then blocked. Antibodies diluted to 1,000-5,000 were treated and reacted at 4 ° C. overnight.

The immune complex was reacted with horseradish peroxidase-conjugated antibody (Amersham Pharmacia, Piscataway, NJ, USA) at room temperature for 1 hour and then reacted with enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia) to identify it (Fig. 3). 3B shows Western blot analysis of bladder tissue of normal, non-invasive and invasive cancer patients. Protein samples were separated in 12% SDS-PAGE and were identifiable by kophyrin and p-kophyrin antibodies and chemiluminescent material. Quantitation software (Bio-Rad) was used for statistical analysis.

Example  3. Immunohistologic Analysis Immunohistochemical assay )

After cutting the paraffin containing bladder tissues of normal and cancer patients into 4-μm sections, the fragments were de-tharapinized and rehydrated, blocked with methanol containing 3% hydrogen peroxide, and then 1 Polyclonal anti-phosphorylated ^ser3 and anti-nonphosphorylated kophyrin antibodies diluted: 100 were prepared and incubated at room temperature for 60 minutes.

The sections were washed and incubated at room temperature for 30 minutes with a horseradish peroxidase (HRP) conjugated dextran polymer reagent kit. Peroxidase activity was observed using 3, 3'-diaminobenzidine tetrachloride. Control staining of the sections was carried out using hematocillin at room temperature, negative control was performed in the absence of primary antibody (see Figure 3). FIG. 3C shows immunostaining of kophyrin and p-kophyrin in bladder tissue of normal, non-invasive and invasive cancer patients. Paraffin containing bladder tissue from normal, non-invasive and invasive cancer patients was identified by polyclonal and py-copirine antibodies.

Example  4. Human bladder Cancer cell line T24  And siRNA - With kopirin  Transfection

Human bladder cancer T24 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in McCoy's 5 A medium containing 10% FBS, 100 U / ml penicillin, 100 g / ml streptomycin and 200 mM glutamine.

The cultured cells (8 × 10 ⁴ ) were replaced with FBS-free McCoy's 5 A medium, and the cells were then transfected with siRNA or nonsilencing control RNA using a transfection reagent (Welfect-QTM Gold; Welgene, Korea). The cells were transfected and transfected to a final concentration of 1,000 pM. The relative expression level of kophyrin was determined by immunoblotting analysis using an antibody.

Kophyrin siRNA was designed targeting the sequence of SEQ ID NO: 1 below.

SEQ ID NO: 5'-CCCAAACUGCUUUUGAUCU-3 '(Accession number: NM_005507; Bioneer, Korea).

Example  5. Human Bladder Cancer T24  Cell EGF Induction phosphorylation

In order to determine the amount of EGF and the degree of phosphorylation of kopirin according to the reaction time, EGF-induced phosphorylation of human bladder cancer T24 cells was examined (see FIG. 4).

4 shows EGF-induced phosphorylation of human bladder cancer T24 cells. Human bladder cancer T24 cells were activated by EGF (0-100 ng / ml) (A, C) and EGF (50 ng / ml) (B, D) during the indicated time. The cell lysate was immunoblot by anti-phosphorylated ^ser3 and non-phosphorylated ^kophyrin antibodies. The statistical results of (C) and (D) were quantified from (A) and (B), and the Kopirin phosphorylation reference level was expressed as 100% (n = 3). * Indicates that there is a significant difference ( p <0.05) from the reference level of phosphorylation.

Example  6. Proliferation assay

Proliferation of T24 cells was performed by XTT assay using WelCount ^™ cell viability kit (Welgene). T24 cells were applied to 96-well plates at a density of 1.5 × 10 ⁴ cells / well, incubated for 48 hours in McCoy's 5 A medium with or without EGF, and then treated with XTT dye (a tetrazolium salt). Next, the culture was carried out for 4 hours at 37 ° C. The amount of formazan crystal formation was quantified with an enzyme-linked immunosorbent assay reader (ELIAS) at 450 nm. Proliferation assays in kophyrin knock-downed cells were performed by the same method after incubation with siRNA-kophyrin for 48 hours (see FIG. 5). FIG. 5A shows the effect of EGF on proliferation of human bladder cancer T24 cells. Proliferation in quiescent state (n = 5) is expressed as 100%. * Indicates that the response at rest shows a significant difference ( p <0.05).

Example  7. Boyden chamber assay

Migration assays were performed in a 42-well microchemotaxis Boyden chamber (Neuro Probe, Cabin John, Md., USA). Polycarbonate membranes (8-m pores, Neuro Probe) were coated with 0.1-mg / ml type I collagen (BD Bioscience) and then dried for 60 minutes. Cells were harvested using trypsin-EDTA (Life Technologies, Paisley, UK) and resuspended in McCoy's 5 A medium containing 0.1% BSA with or without EGF. Thereafter, 3 × 10 ⁴ cells were loaded at the bottom of the chamber, and the microchambers were incubated at 37 ° C. for 80 minutes.

The membrane was fixed and stained using Diff-Quik (Baxter Healthcare, Miami, Fla., USA). The number of cells moving through the membrane was determined by counting four randomly selected points on the microscope (× 400) (see FIG. 5).

5B shows the effect of EGF on the migration of human bladder cancer T24 cells. Cells treated with EGF (1-100 ng / ml) for 80 minutes were quantified by Boyden chamber assay. The movement in quiescent state (n = 6) is expressed as 100%. * Indicates that the response at rest shows a significant difference ( p <0.05).

Migration assays were performed on cells transfected with siRNA-copyrin. All samples used in this experiment included 0.1% DMSO, but the presence of DMSO did not affect T24 cell migration.

As discussed in the above examples, Cofilin and phosphorylated cofilin (P-cofilin) can be investigated to predict the progression of bladder muscle non-invasive urothelial carcinoma into muscle-invasive urothelial carcinoma and induce inhibition of Cofilin and P-cofilin. Infusion of new agents can reduce the recurrence and progression of bladder urinary tract cancer and prevent progression to muscle-invasive urinary tract cancer.

The specific parts of the present invention have been described in detail above, and it should be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrual Cooperation Corp. <120> Biomarker of Urothelial Cancer and the Diagnostic Method of the          Same <130> P09-E444 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> RNA particle for designing of Cofilin siRNA <400> 1 cccaaacugc uuuugaucu 19

Claims (8)

Urinary epithelial cancer marker comprising a protein of any one or more of Cofilin and P-cofilin.
The method of claim 1,
Urinary epithelial cancer marker is characterized in that non-invasive or invasive.
Diagnosis and prognosis of UTIs comprising the following steps:
(a) collecting urine to be diagnosed;
(b) measuring the expression level of any one or more proteins of cofilin and P-cofilin in the collected urine; And
(c) comparing the expression level of any one or more of the proteins of Cofilin and P-cofilin measured in step (b) with a normal person.
The method of claim 3, wherein
The urinary tract cancer is a non-invasive or invasive method of diagnosing and prognostic methods of urinary tract cancer.
The method of claim 3, wherein
The method for measuring the degree of protein expression is characterized in that (1) the method of directly measuring the protein in the urine or (2) the method of testing the protein expression by immunoassay and protein measurement in the urinary tract epithelial cells in the urine Diagnosis and Prognostic Methods of Urinary Tract Cancer.
6. The method of claim 5,
The method of measuring the expression level of the protein is immunoblotting, immunohistochemical assay, Boyden chamber assay, Real time polymerase chain reaction, MALDI TOF / TOF MS / MS, and ELISA characterized in that the diagnostic method and prognosis of epithelial cancer Judgment method.
A method of treating urinary tract cancer, characterized in that it inhibits any one of the proteins of Cofilin and P-cofilin.
A diagnostic kit for testing risk factors for urinary tract cancer using the method of claim 3.

Images