KR101759382B1 - Method for Diagnosis of Bladder Cancer using PRAC methylation and A Use thereof - Google Patents

Abstract

The present invention relates to a novel biomarker for the diagnosis of bladder cancer and a use thereof, and more particularly to a biomarker for the diagnosis of bladder cancer, which comprises a non-muscle invasive bladder cancer (Non-muscle invasive bladder cancer including recurrence, etc.) utilizing the expression pattern according to a methylation pattern of a prostate cancer susceptibility candidate -muscle Invasive Bladder Cancer (NMIBC) prognostic markers.

Description

Methods for Diagnosing Bladder Cancer Using PRAC Methylation and its Use {

The present invention relates to a novel biomarker for the diagnosis of bladder cancer and a use thereof, and more particularly, to a biomarker for non-muscle invasive bladder cancer using methylation expression pattern according to a methylation pattern of a prostate cancer susceptibility candidate (PRAC) ; NMIBC) prognostic markers.

Bladder cancer is the most frequently occurring cancer in the urinary tract, with 16.5 cases per 100,000 population in the West, compared with 4.5 cases in Korea. In addition, the incidence rate is higher than in the Western world, and the incidence rate is increasing year by year. In Korea, it is known to be the most common cancer among urological cancers (Lee C, et al., 1992)

Bladder cancer is classified into non-muscle invasive bladder cancer and invasive bladder cancer depending on the degree of invasion. Non-muscle invasive bladder cancer is a relatively localized lesion in the mucosa without involvement of the muscle layer, and can be treated with transurethral resection of bladder tumor or intravesical chemotherapy or BCG. However, cancer recurrence and invasive cancer Is a problem. On the other hand, invasive bladder cancer refers to a state in which cancer has penetrated into the muscle layer. In order to treat the bladder cancer, complicated urinary diversion should be performed along with radical cystectomy, and the result may be fatal to the patient. Therefore, prediction and early detection and prevention of relapse and progression after primary treatment is very important

Recently, methods for diagnosing cancer through DNA methylation measurement have been proposed. DNA methylation mainly occurs in the cytosine of the CpG island (CpG island) of the promoter region of a specific gene, thereby preventing the binding of the transcription factor And the methylation of the CpG islands of the promoter of the tumor suppressor gene is highly useful for cancer research. The methylation specific PCR (hereinafter referred to as "MSP ") or the methylation specific PCR An attempt has been actively made for the diagnosis and screening of cancer by an automatic base analysis or the like.

Methylation of tumor-related genes is an important indicator of cancer, and it can be used in many aspects such as diagnosis and early diagnosis of cancer, prediction of carcinogenesis risk, prediction of cancer prognosis, follow-up after treatment, and prediction of response to chemotherapy. Recently, attempts have been made to examine the methylation of tumor-associated genes in blood, sputum, saliva, stool, urine, etc. and to use them in various cancer treatments (Esteller, M. et al., Cancer Res., 59:67, 1999; Sanchez-Cespedez, M. et al., Cancer Res., 60: 892, 2000; Ahlquist, DA et al., Gastroenterol., 119: 1219, 2000).

Therefore, the present inventors have investigated the relationship between PRAC (prostate cancer susceptibility candidate) methylation level and clinicopathological factors from non-muscle invasive bladder cancer (NMIBC) tissues, and found that non-muscle invasive bladder cancer Bladder Cancer (NMIBC)) was confirmed to be a useful marker for the prediction of prognosis.

1. Korean Patent Publication No. 10-2011-0036554 2. Korean Patent Publication No. 10-2013-0121467 3. Korean Patent Publication No. 10-2014-0064899

The present invention relates to a novel use of a novel methylation level of a prostate cancer susceptibility candidate (PRAC)

It is an object of the present invention to provide a composition for a marker for the diagnosis of bladder cancer prognosis, which contains the PRAC.

Another object of the present invention is to provide various uses for the diagnosis of bladder cancer prognosis of the PRAC.

It is still another object of the present invention to provide a method for screening a substance having a therapeutic function of bladder cancer using the PRAC gene.

In order to solve the above problems, the present invention provides a marker composition for diagnosing bladder cancer prognosis, which contains a PRAC gene. In particular, it is effective in the diagnosis of non-muscle invasive bladder cancer (NMIBC).

Since the PRAC gene is hypermethylated in bladder cancer tissues, it is possible to diagnose bladder cancer prognosis. In particular, the PRAC gene can be used as an independent predictor of bladder cancer prognosis. The bladder cancer prognosis includes progression and recurrence.

The PRAC gene preferably comprises the sequence shown in SEQ ID NO: 5, for example, the sequence consisting of SEQ ID NO: 6.

Based on the function of the gene, the present invention provides, as one embodiment, a composition for diagnosing bladder cancer prognosis, which comprises an agent for measuring the level of methylation of a PRAC gene.

Determining the level of expression of said gene comprises measuring the level of methylation, mRNA or protein, most preferably measuring the level of methylation of said gene.

The methylation level can be measured by known PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, pyrosequencing, And bisulfite sequencing may be used. Preferably pyrosequencing and bisulfite sequencing methods.

In addition, the present invention provides, as another embodiment, a kit for prognosis of bladder cancer comprising the composition for a marker or the diagnostic composition. At this time, the kit may be an RT-PCR kit, a DNA chip kit, or a protein chip kit.

The present invention also provides, as yet another embodiment, a method for providing information for the diagnosis of bladder cancer, preferably non-muscle invasive bladder cancer (NMIBC), comprising:

Measuring the level of methylation of the PRAC gene from a biological sample isolated from the patient; And

Comparing the level to the methylation level of a corresponding gene of a normal control sample.

At this time, when the PRAC gene is hypermethylated, it can provide information that the risk of progression and recurrence of bladder cancer is great.

In addition, the present invention provides a screening method of bladder cancer, particularly non-muscle invasive bladder cancer treatment material, comprising the following steps as another embodiment utilizing the gene function of the biomarker.

(a) hypermethylating the PRAC gene in the presence of a candidate agent; And

(b) selecting a candidate substance for treating bladder cancer when the methylation of the gene is suppressed, as compared to when the PRAC gene is hypermethylated without a candidate substance.

As described above, the present invention provides a variety of uses based on novel functions of PRAC genes as bladder cancer, most preferably non-muscle invasive bladder cancer (NMIBC) prognostic factors.

As described above, the present invention relates to a novel use of a specific gene for PRAC (prostate cancer susceptibility candidate) methylation, and is effective for the diagnosis of bladder cancer, particularly non-muscle invasive bladder cancer (NMIBC). The present invention may also provide a method for screening a therapeutic agent for bladder cancer by screening a substance that inhibits PRAC and methylation. Therefore, the present invention may be useful for diagnosis and prognosis prediction and treatment in bladder cancer.

1 is a graph showing Kaplan-Meier curves for predicting recurrence and progression according to the methylation state in NMIBC patients.

The terms used in the present invention are defined as follows.

"Diagnosis" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to predict or confirm a bladder cancer prognosis. It is particularly useful for the diagnosis of non-muscle invasive bladder cancer (NMIBC).

"Diagnostic markers or diagnostic markers are substances that can distinguish bladder cancer cells from normal cells and include polypeptides or nucleic acids (eg, mRNA) that show increased patterns in bladder cancer cells compared to normal cells, Etc. For the purpose of the present invention, the cancer marker for prognosis of bladder cancer is a prostate cancer susceptibility candidate (PRAC) gene, which is expressed in NMIBC tissues as a prostate cancer susceptibility candidate (PRAC) gene. Methylated.

"Prognosis" refers to the course of disease and prediction of outcome. In the present invention, it includes prediction of recurrence and progression of bladder cancer. Therefore, it is very important to accurately predict recurrence and progression of bladder cancer, and it is necessary to have factors that can predict the response of treatment while supplementing clinical indicators such as tissue differentiation and stage. And can be used as a prognostic factor for bladder cancer. That is, the measurement of the expression characteristics of these genes can be used as a prognostic indicator (diagnostic marker) useful for predicting the differentiation, stage, and progression of bladder cancer.

"Cancer "," tumor ", or "malignant" refers to or represents the physiological condition of a mammal that is generally characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma.

"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included.

"Tissue or cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.

"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule. The nucleic acid used in the present invention is preferably a CpG-containing nucleic acid such as a CpG island.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

The term "gene expression" generally refers to a cellular process in which a biologically active polypeptide is produced from a DNA sequence and exhibits biological activity in the cell. In this sense, gene expression includes post-transcriptional and post-transcriptional processes that not only involve transcription and translation processes, but can also affect the biological activity of the gene or gene product. Such procedures include, but are not limited to, RNA synthesis, processing and transport as well as polyp peptide synthesis, transport and post-translational modification of the polypeptide. The present invention encompasses all cases of gene methylation, mRNA expression, and protein expression as a mode of gene expression.

"Coding region" or "coding sequence" refers to a nucleic acid sequence, a complement thereof, or a portion thereof, which encodes a particular gene product or fragment thereof that is required to be expressed, according to a common base pairing and codon usage relationship. Coding sequences include exons in genomic DNA or immature primary RNA transcripts that are joined together by a biochemical machinery of cells to provide mature mRNA. The antisense strand is a complement of the nucleic acid, and the coding sequence can be deduced therefrom. The coding sequences are placed in the relationship of transcriptional regulatory elements and translation initiation and termination codons such that transcripts of appropriate length are generated and translated in the appropriate reading frame to produce the desired functional product.

"Primer" refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA-targeted polynucleotide and serves as a starting point for the stepwise synthesis of a polynucleotide from a mononucleotide by the action of, for example, the nucleotidyltransferase that occurs in the polymerase chain reaction .

"Protein" also includes fragments, analogs, and derivatives of proteins that retain essentially the same biological activity or function as the reference protein

"Label" or "label " means a compound or composition that facilitates the detection of a reagent, such as a reagent conjugated, conjugated, conjugated, or fused to a nucleic acid probe or antibody. The label may itself be detected (e. G., A radioactive isotope label or a fluorescent label), in the case of an enzyme label, to catalyze the chemical modification of the detectable substrate compound or composition.

"Antibody" is used in its broadest sense and specifically includes intact monoclonal (monoclonal) antibodies, polyclonal antibodies, multispecific antibodies (e. G. Bispecific antibodies) formed from at least two intact antibodies, and And antibody fragments exhibiting the desired biological activity.

"Epigenetics" refers to the transfer of genes to the offspring without altering the DNA sequence. A study of the concept that abnormal genetic information due to changes in four base sequences (deletion, substitution, amplification, etc.) is accumulated in tumorigenic genes or tumor suppressor genes and their function is amplified or lost, , But this alone did not explain the development, growth and metastasis of cancer. In recent years, hosiology, which regulates only the expression pattern of genes without mutations, is developing into a new field of cancer-related research. Welfare changes occur through processes such as DNA methylation, histone modification and genomic imprinting.

"Treatment" means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction Also, "treatment" may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. &Quot; Palliating " a disease may reduce the extent of the disease state and / or undesirable clinical symptoms and / or delay or slow the time course of the progression, It means to lose.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The disease to be diagnosed in the present invention is a disease associated with bladder cancer, particularly non-muscle invasive bladder cancer (NMIBC).

It is known that bladder cancer is the most frequent urological tumor in Korea. It is known that the mechanism and progression of bladder cancer occur through various causes and stages. Recent studies on chromosomes and gene abnormalities, The prognostic factors that can predict the prognosis are actively researched.

Bladder cancer can be classified as superficial cancer and invasive cancer at the time of initial diagnosis, and it is superficial in about 75% of patients. In the case of superficial tumors, 30% of recurrent superficial tumors are more malignant or progressing to stage than malignant tumors, and 10% of them are involved in muscle layer. Recurrence in 50-70%, progression to invasive bladder cancer in 5%, and recurrence in more than 80% of highly malignant T1 lesions and invasive bladder cancer in 50% of patients within 3 years. Among the factors that are considered to affect the progression of these tumors are bladder cancer, multiple intraepithelial cancer, high recurrence rate, residual tumor after BCG therapy in bladder, and expression of p53 gene in T1 staging and G3 malignant pathway (T1G3) .

In patients with invasive bladder cancer, bladder ablation is the standard treatment, and the prognosis after invasive bladder cancer is T stage, N stage, lymph node density, malignancy, tumor size, number, morphology, lymphatic / And the like. Approximately 80-90% of myoclonic bladder cancers are those of primary, near-invasive bladder cancer without a history of previous superficial bladder cancer, and about 15% of cases have previously progressed from superficial bladder cancer to invasive bladder cancer. The first diagnosis is invasive bladder cancer (primary invasive cancer), and the first diagnosis is superficial bladder cancer. However, recurrence of invasive bladder cancer (progressive invasive cancer) is also present.

Therefore, these frequent recurrences and progression of the staging are problems frequently encountered in bladder cancer, and it is necessary to develop an indicator to find an indicator that can effectively predict recurrence and invasiveness of bladder cancer or to develop a treatment method.

As a result of the study on gene abnormality, it has been reported that gene amplification such as 1q21-24, 3p24, 6p22, 8q21-22, 10q22-23, 12q15-21, 17q11-21, 17q22 is frequently found in bladder cancer (Int J Oncol 2000 ; Prostate cancer susceptibility candidate (PRAC) gene and bladder cancer (PRAC) gene; There is no known information about the association with prognosis.

The present inventors have found that PRAC (prostate cancer susceptibility candidate) gene is hypermethylated in bladder cancer tissue. In particular, clinicopathological relevance of the bladder cancer prognosis and the methylation status of the specific gene PRAC was first identified.

Accordingly, the present invention relates to the use of the PRAC gene as a marker for bladder cancer prognosis, and it can be diagnosed by predicting bladder cancer prognosis through confirmation of hypermethylation of the gene and reduction in expression thereof. In particular, it is very useful for the prognosis of non-muscle invasive bladder cancer (NMIBC).

The prognosis of bladder cancer includes recurrence and progression.

Recurrence is defined herein as recurrence of primary NMIBC at a lower or equivalent pathological stage and progression is defined as muscle infiltration (TNM stage T2 or higher stage or metastatic disease).

Thus, the present invention relates, in one aspect, to the use of the PRAC gene as a bladder cancer prognostic marker.

The selection and application of significant diagnostic markers determines the reliability of diagnostic results. Significant diagnostic markers are those markers that are highly reliable with high validity and consistency in repeated measurements. The bladder cancer prognostic marker of the present invention shows the same results in repeated experiments with genes whose expression changes either directly or indirectly with the onset of bladder cancer and the difference in expression level is very large when compared with the control group, Are highly reliable markers. Therefore, the diagnosis result based on the result of measuring the expression level of the significant diagnostic marker of the present invention can be reasonably reliable.

The specific gene PRCA of the present invention is an independent predictor of bladder cancer recurrence and progression and is characterized in that the time required for recurrence and progression of hypermethylation in bladder cancer tissues is significantly reduced. In other words, PRCA and methylation are useful as a significant marker of recurrence and progression. In other words, confirmation of hypermethylation of PRCA gene can predict the prognosis of bladder cancer.

From the same viewpoint, the present invention relates to a composition for diagnosing bladder cancer prognosis comprising an agent for measuring the PRAC gene methylation level.

Here, the 'gene expression level measurement' includes both the methylation or the measurement of the level of the mRNA or its protein by the methylation, and known methods can be used. The methylation level is most preferably determined in the present invention.

"DNA methylation" is a DNA methyl transferase (DNMT) that binds a methyl group (CH3) at the 5-carbon site of CpG, which occurs in the promoter CpG island, which causes cell cycle or apoptosis Regulates, restores DNA, and is involved in cell adhesion and intercellular interactions.

A CpG island is a region with a C + G content of 50% or more and a CpG ratio of 3.75% or more and a length of 0.2 to 3 kb. The human genome has about 45,000 CpG islands, most of which are found in promoter regions that regulate gene expression. Indeed, the CpG isoform is found in the promoter of housekeeping genes that amount to about 50% of the human gene (Cross, S. and Bird, A., Curr. Opin. Gene Develop., 5: 309, . The CpG is a site where most vestigial changes occur frequently in mammalian cells.

In particular, when the PRAC of the present invention is methylated, such methylation inhibits the expression and function of the gene in the same manner as a mutation of a coding sequence, thereby promoting the development, recurrence, and progression of bladder cancer.

Therefore, as a specific example, a method for detecting whether methylation of the PRAC gene can include the following steps:

(a) separating sample DNA from a clinical sample;

(b) amplifying the separated DNA using a primer capable of amplifying a PRAC gene fragment; And

(c) determining whether the promoter is methylated based on whether or not the amplified product is produced in step (b).

For sample preparation, tissues or cell samples from mammals (typically human patients) can be used. Samples may be obtained by a variety of procedures known in the art including, but not limited to surgical excision, aspiration, or biopsy. The tissue can be fresh or frozen. The tissue sample can be fixed (i.e., preserved) by conventional methods. Those skilled in the art will appreciate that the fixative may be selected for purposes in which the sample is histologically stained or otherwise analyzed. Those skilled in the art will also appreciate that the fixed length is determined by the size of the tissue sample and the fixative used.

The PRCA gene of the present invention is preferably derived from all animals, preferably human. The PRCA includes wild type, mutants by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. For example, PRCA polypeptide fragments, chemically modified derivatives thereof, as well as nucleic acids and / or amino acid sequence variants thereof. Since the PRCA sequence is known, the sequence of the gene can be obtained by chemical synthesis (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or by using a recombinant technology. PRCA used in one embodiment of the present invention is represented by SEQ ID NO: 6. In particular, the PRAC gene fragment is characterized in that it comprises the region represented by SEQ ID NO: 5, more preferably SEQ ID NO: 4. As a preferred example, in one embodiment of the present invention, the PRCA fragment was amplified using primer pairs of SEQ ID NOS: 1 and 2 to confirm methylation.

The DNA methylation level can be determined by various known methods. For example, the methylation measurement method can be performed by PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, pyrosequencing, and bi- And sulfite sequencing. In one embodiment of the present invention, pyrosequencing was used.

Methylation specificity PCR  ( 메틸레이션 specific PCR ) Way

When the genomic DNA is treated with bisulfite, the cytosine in the 5'-CpG'-3 region is methylated and remains cytosine as it is, and when it is unmethylated, it changes into uracil. Therefore, a PCR primer corresponding to the 5'-CpG-3 'nucleotide sequence was prepared from the base sequence after bisulfite treatment. At this time, PCR primers corresponding to methylation and two types of primers corresponding to unmethylated primers were prepared. When pyroDNA is converted into bisulfite and then PCR is performed using the above two types of primers, PCR products are produced by using primers corresponding to the methylated nucleotide sequence when methylated, and in the case of unmethylated PCR products are produced using primers corresponding to unmethylated sequences. The methylation can be qualitatively confirmed by agarose gel electrophoresis.

Real-time methylation specificity PCR  ( real time 메틸레이션 specific PCR )

Real-time methylation specific PCR is the conversion of methylation-specific PCR method to real-time measurement method. The PCR primer corresponding to methylation of bisulfite treated with genomic DNA is designed and real-time PCR is performed using these primers . At this time, there are two methods of detection using a TanMan probe complementary to the amplified nucleotide sequence and detection using Sybergreen. Therefore, real-time methylation-specific PCR can quantitatively analyze only methylated DNA. At this time, a standard curve can be prepared using an in vitro methylated DNA sample, and the methylation degree can be quantitatively analyzed by amplifying a gene having no 5'-CpG-3 'sequence in the nucleotide sequence as a negative control for standardization.

Pyrosequencing

The pyrosequencing method is a method of converting the bisulfite sequencing method into quantitative real-time sequencing. Similar to bisulfite sequencing, the genomic DNA was transformed by treatment with bisulfite, and then a PCR primer corresponding to the region lacking the 5'-CpG-3 'nucleotide sequence was prepared. The genomic DNA was treated with bisulfite, amplified with the PCR primer, and subjected to real-time sequencing using a sequencing primer. The amount of cytosine and thymine in the 5'-CpG-3 'region was quantitatively analyzed and the degree of methylation can be expressed as a methylation index. In one embodiment of the present invention, the pyrosequencing method is used.

Methylation DNA  Using specific binding proteins PCR  Or quantification PCR  And DNA  chip

In PCR or DNA chip method using methylated DNA-specific binding protein, when a protein specifically binding to methylated DNA is mixed with DNA, only the methylated DNA can be selectively isolated because the protein specifically binds to the methylated DNA . Genomic DNA was mixed with methylated DNA-specific binding protein and only methylated DNA was selectively isolated. These separated DNAs can be amplified using a PCR primer corresponding to the promoter site, and then methylated by agarose electrophoresis.

In addition, methylation can be measured using a quantitative PCR method. The methylated DNA separated by a methylated DNA-specific binding protein is labeled with a fluorescent dye and hybridized to a complementary probe-integrated DNA chip to measure methylation .

Detection of differential methylation - Bisulfite  How to Sequence

Another method for detecting a nucleic acid containing methylated CpG comprises contacting a sample containing nucleic acid with an agent for modifying unmethylated cytosine and amplifying the CpG-containing nucleic acid of the sample using a CpG-specific oligonucleotide primer . Here, the oligonucleotide primer may be characterized in that methylated nucleic acid is detected by distinguishing modified methylated and unmethylated nucleic acids. The amplification step is optional, but is not necessary. The method relies on PCR reactions to distinguish between modified (e. G., Chemically modified) methylated and unmethylated DNA. Such a method is disclosed in U.S. Patent 5,786,146, which is described in connection with bisulfite sequencing for the detection of methylated nucleic acids.

Important probes can be labeled for detection and can be labeled, for example, as radioactive isotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. It is well known in the art to appropriately label such a probe, and can be carried out by a conventional method.

In a specific embodiment of the present invention, by measuring the level of DNA methylation of the PRAC gene using the above-mentioned method or the like, it is possible to provide an application for diagnosing that it is hypermethylated to indicate the prognosis of bladder cancer.

In a similar aspect, the present invention relates to a kit for the diagnosis of human bladder cancer prognosis.

The kit is used to determine the prognosis of bladder cancer, preferably Non-muscle Invasive Bladder Cancer (NMIBC), by analyzing the expression of PRAC gene, preferably methylation level.

For this purpose, the kit may comprise, for example, a primer or probe specifically binding to a PRAC gene sequence; Or PRAC, which specifically binds to at least one protein selected from the group consisting of:

That is, when the human bladder cancer diagnostic kit of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally include a reagent necessary for PCR amplification, such as a buffer, a DNA polymerase (for example, Thermus aquaticus thermostable DNA polymerases obtained from Thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs. And, when the human bladder cancer diagnostic kit of the present invention is applied to immunoassay, the kit of the present invention may optionally include a secondary antibody and a labeling substrate.

The kit of the present invention may be made from a number of separate packaging or compartments containing the above reagent components.

As described above, another aspect of the present invention provides a bladder cancer diagnostic kit comprising the composition for diagnosing bladder cancer according to the present invention. Preferably, the diagnostic kit may further comprise one or more other component compositions, solutions or devices suitable for the assay method.

In one embodiment, the kit of the present invention comprises a first container containing a partitioned carrier means for containing a sample, a preparation delicately cleaving unmethylated cytosine, a second container containing a primer for amplifying a CpG containing nucleic acid, And a third container containing a means for detecting the presence of the uncut nucleic acid.

In another aspect, the present invention provides a method for diagnosing a bladder cancer prognosis, which comprises measuring the level of expression of a PRAC gene, preferably a methylation level, based on such findings, or information for the diagnosis of bladder cancer prognosis, for example, recurrence or progression and a method for providing information on progression.

In one embodiment, the method of the present invention comprises

Measuring the level of methylation of the PRAC gene from a biological sample isolated from the patient; And

And comparing the expression level of the gene or the level of the protein encoded by the gene with the expression of the gene of interest in a normal control sample. Other details are as described above.

That is, the method for diagnosing a bladder cancer prognosis relates to examining the level of methylation of a particular marker PRAC, in particular according to Non-muscle Invasive Bladder Cancer (NMIBC), and the methods disclosed herein are useful for the treatment of bladder cancer patients Effective, and cost-effective means of obtaining useful data and information when evaluating appropriate or effective therapies. In particular, it can provide useful information on recurrence or progress.

In another aspect, the present invention relates to a method for screening a substance for treating bladder cancer that inhibits hypermethylation of PRAC, based on the facts described above.

The method comprises, in one embodiment,

(a) hypermethylating the PRAC gene in the presence of a candidate agent; And

(b) selecting a candidate substance for treating bladder cancer when the methylation of the gene is inhibited, as compared with the case where the PRAC gene is hypermethylated without a candidate substance.

The present invention extends the genetic approach to the upregulation or downregulation of the expression of genes involved in the methylation of PRAC.

As described above, in the present invention, the methylation increase and expression decrease profile characteristics of the PRAC gene were shown in bladder cancer, preferably non-muscle invasive bladder cancer (NMIBC) patients. These results indicate that the gene It is suggested that the gene identified by the present invention as a target gene for developing or screening a bladder cancer diagnosis and treatment agent can be used.

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 for illustrating the present invention only and that the scope of the present invention is not construed as being limited by these embodiments.

Materials and methods

1. Collecting objects and samples

A total of 136 human bladder tissues (8 normal controls [NC] and 128 NMIBCs) were used for pyrosequencing (PSQ) analysis (Table 1). NMIBC tissues were obtained from primary NMIBC patients undergoing TUR (transurethral resection) diagnosed with transitional cell carcinoma between 1995 and 2010. To rule out the possibility of incomplete limitations or factors that could affect the analysis, patients who experienced disease relapse within 6 months or who were followed up for less than 6 months were excluded. NC tissues were obtained from individuals with benign prostatic hyperplasia or bladder injury

All tumors were macroscopically cut within 15 min of surgical resection. Each NMIBC sample was identified by pathological analysis of sections of tissues obtained from TUR specimens and immediately frozen in liquid nitrogen and stored at -80 ° C. The above samples were received from Chungbuk National University Hospital. The collection and analysis of all samples were approved by the Chungbuk National University Hospital Institutional Review Board (GR2010-12-010) and asked for consent from the patients.

Tumor stages were classified according to the 2002 TNM classification and the 1973 World Health Organization grading systems. If the bladder cancer sample does not contain adequate muscle or high - grade tumors were detected, 2 ^nd TUR was performed after the initial resection.

Patients with moderate- or high-risk NMIBC received one cycle of intravesical instillation therapy. Each patient was managed according to standard recommendations.

Recurrence was defined as return of primary NMIBC at a lower or equivalent pathological stage (Ta / T1) and progression was defined as muscle invasion (TNM stage or higher) or nodal / distant metastatic disease.

2. DNA extraction and PSQ analysis

Genomic DNA was extracted by standard methods using a Wizard genomic DNA purification system (Promega). Disulfide conversion of genomic DNA was performed using the EZ DNA methylation kit (Zymo Research). PRAC DNA methylation status was assessed by PSQ using PyroMark Q96 ID (Qiagen, Valencia, Calif.). Primer sequences and amplification conditions are listed in Table 1.

Primera was designed using NCBI Reference Sequence Construction Version 36.1. PCR reactions included 0.01 μM each primer and Bioneer Taq (Bioneer, Daejeon, Korea) and 20 ng bisulfite-treated DNA. The thermocycling parameters were as follows: denaturation at 94 占 폚 for 5 minutes, followed by 45 cycles at 94 占 폚 for 30 seconds, annealing at 55 占 폚 for 30 seconds, and 72 占 폚 for 30 seconds, and last extension at 72 占 폚 for 5 minutes .

[Table 1]

Example  1: Reference value characteristic

The baseline characteristics of patients with NC and NMIBC are shown in Table 2.

The mean age of patients with NMIBC was 64.0 ± 13.4 years. The mean recurrence-free survival and progression-free survival were 47.2 ± 40.4 months (mean, 35.8; range, 6.1 ~ 183.3) and 61.1 ± 41.7 months (mean, 50.9; range, 6.6 ~ 183.3).

[Table 2]

Example  2: Relationship between methylation level and clinicopathologic variables

As shown in Table 3, the level of methylation of the PRAC gene was significantly higher in the samples of NMIBC patients than NC patients (p <0.001).

To assess the relationship between methylation pattern and clinical pathology, methylation levels were examined in relation to well-known prognostic factors such as number of tumors, grade and stage. High levels of PRAC methylation were significantly associated with tumor grade and stage.

[Table 3]

Example  3: prognosis As a predictor  Methylation state ( Methylation status as  a predictor of prognosis )

The methylation level of PRAC was significantly higher in the poor prognostic group (recurrence or progression) than in the good prognostic group (Table 3). To determine whether methylation markers are associated with prognosis, the methylation level of the PRAC gene was differentiated (low methylation or hypermethylation) to a defined median as a cut-off point.

Kaplan-Meier estimates showed that the PRAC and methylation groups had significantly less recurrence and progression times than the PRAC low methyl group (Fig. 1, log-rank test, p <0.05).

Univariate and multivariate Cox regression analysis showed that PRAC gene methylation was associated with recurrence (risk ratio [HR], 2.652; 95% confidence interval [CI], 1.241-5.667; p = 0.012) and progression (HR, 9.531; 95% CI, 1.172-77.497; p = 0.035) (Table 4).

[Table 4]

These results suggest that PRAC gene methylation is a useful predictor of NMIBC recurrence and progression.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Method for Diagnosis of Bladder Cancer using PRAC methylation and          A Use thereof <130> PN1501-009 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 ggattttggt ttttattttt gtaga 25 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 ctcacccttc ccttatttc 19 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 3 tgtttttttt tttaataagg taaat 25 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PRCA gene fragment <400> 4 tgatcggtgg gccaagggcg ttatcgacgg atcg 34 <210> 5 <211> 243 <212> DNA <213> Artificial Sequence <220> <223> PRAC gene fragment <400> 5 ggattctggt ccccaccttt gcagagagaa cagcgatgtt gtgcgcccat ttctcagatc 60 aaggaccggc ccatcttact acctccaaga gtgcttttct ctctaataag gtaaactgat 120 cggtgggcca agggcgttat cgacggatcg ctcagtatgg tagctgcatc aggaggccct 180 gggagagggt ctcccaggaa tttgggagcc ttcagaagtt ttgggaaaca agggaagggt 240 gag 243 <210> 6 <211> 802 <212> DNA <213> Artificial Sequence <220> <223> PRCA <400> 6 gtccttcctc tcctagccta aggcgtgcaa acagagcgcc actgggaggc tgaaaccttt 60 aggccgatgc ttgcttgcaa ggtcaggcaa gctggattct ggtccccacc tttgcagaga 120 gaacagcgat gttgtgcgcc catttctcag atcaaggacc ggcccatctt actacctcca 180 agagtgcttt tctctctaat aaggtaaact gatcggtggg ccaagggcgt tatcgacgga 240 tcgctcagta tggtagctgc atcaggaggc cctgggagag ggtctcccag gaatttggga 300 gccttcagaa gttttgggaa acaagggaag ggtgagcagc aggctttcac cgataccacc 360 tggcgggagc acctactcgc ggttcctgga gagaccggca gccgccctgg gcagaaaggg 420 acaaagaaaa tgtttcgcag cacgcgggac ctgcaggacc taggcggggg agaggcttgg 480 gagtgggaac tagcacccgc tgtaaggtct gccataagac ttaatgtttg tctccaatgg 540 gatggagtcc tggcataagc aaaattaata ttgataacgt tattattatt taaaatttgt 600 aaatattgta aaatatttta gaaaacatct actttgaaac atctactggg cgagaccagg 660 agtgatggct cagcctgtaa ttctggaatt tcgggaggcc gaggcaggaa gattccttga 720 gcacaggagt tccagaccag cctgggcaat gtagcaagac gctgtctcta tttatacaat 780 aaaatttttt taaaaaagga aa 802

Claims (18)

A composition for markers for the diagnosis of bladder cancer prognosis, which contains the PRAC gene. 2. The marker composition according to claim 1, wherein the PRAC gene is hypermethylated in bladder cancer tissues. The method according to claim 1,
Wherein the bladder cancer is non-muscle invasive bladder cancer (NMIBC). The method according to claim 1,
Wherein the bladder cancer prognosis includes progression and recurrence. The method according to claim 1,
Wherein the PRAC gene comprises the sequence represented by SEQ ID NO: 5. The method according to claim 1,
Wherein the PRAC gene comprises the sequence represented by SEQ ID NO: 6. A composition for diagnosing bladder cancer prognosis, comprising a preparation for measuring the methylation level of a PRAC gene. 8. The method of claim 7,
Wherein the bladder cancer is non-muscle invasive bladder cancer (NMIBC). 8. The method of claim 7,
Wherein the bladder cancer prognosis includes progression and recurrence. A kit for the diagnosis of non-muscle invasive bladder cancer prognosis comprising the composition according to claim 1 or 7. Measuring the level of methylation of the PRAC gene from a biological sample isolated from the patient; And
And comparing said level with a methylation level of a corresponding gene of a normal control sample. 12. The method of claim 11,
Wherein the risk of progression and recurrence of bladder cancer is high when the PRAC gene is hypermethylated. 13. The method of claim 12,
Wherein the bladder cancer is a non-muscle invasive bladder cancer (NMIBC). 12. The method of claim 11,
Wherein the methylation level is measured using pyrosequencing or bisulfite sequencing methods. 15. The method of claim 14,
Wherein the sequencing method uses a pair of primers represented by SEQ ID NO: 1 and SEQ ID NO: 2. A screening method of bladder cancer progression and recurrence inhibiting substance comprising the steps of:
(a) hypermethylating the PRAC gene in the presence of a candidate agent; And
(b) selecting a candidate substance for inhibiting methylation of the gene, as compared with the case where the PRAC gene is hypermethylated without a candidate substance, as a substance for inhibiting progression and recurrence of bladder cancer. 17. The method of claim 16,
Wherein the bladder cancer is a non-muscle invasive bladder cancer (NMIBC). delete

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