WO2016108403A1 - Use of rsph9 as bladder cancer prognosis diagnostic marker - Google Patents

Abstract

The present invention relates to a novel biomarker for bladder cancer prognosis diagnosis, and a use thereof and, more specifically, to a use as a non-muscle invasive (superficial) bladder cancer (NMIBC) diagnosis and bladder cancer prognosis diagnostic marker using expression characteristics according to the methylation pattern of RSPH9 gene.

Description

Use of RSPHP as a diagnostic marker for bladder cancer prognosis

The present invention relates to a novel biomarker for the diagnosis of bladder cancer and its use, and more specifically to non-muscle invasive bladder cancer (NMIBC) prognosis using expression characteristics according to the methylation pattern of the RSPH9 gene It relates to the use as a marker.

Bladder cancer is the most frequent cancer in the urinary system. It is reported that 16.5 cases per 100,000 population occur in the West and 4.5 cases occur in Korea. Although the incidence rate is lower than in the West, the incidence rate is increasing year by year, and it is known as the most frequent cancer among the urinary system cancers in Korea (Lee C, et al., 1992).

Bladder cancer is classified into non-muscle invasive bladder cancer and invasive bladder cancer according to the degree of invasion. Noninvasive bladder cancer is a lesion in which the cancer is confined to the mucous membrane without invasion of the muscle layer and can be treated relatively simply by injecting an anticancer agent or BCG into the bladder, depending on the risk factors after transurethral resection of bladder tumor. Relapse and progression to invasive cancer are problematic. Invasive bladder cancer, on the other hand, refers to a condition in which the cancer has infiltrated into the muscle layer. For the treatment of the invasive bladder cancer, in addition to radical bladder extraction, complex urinary diversion may be performed, and the patient may have fatal results. Therefore, prediction and early detection and prevention of recurrence and progression after primary treatment are very important.

Recently, methods for diagnosing cancer by measuring DNA methylation have been proposed. DNA methylation mainly occurs in the cytosine of CpG island of the promoter region of a specific gene, whereby the binding of transcription factors interferes. As the expression of certain genes is blocked, detection of the methylation of the promoter CpG island of tumor suppressor genes is very helpful for cancer research, which is called methylation specific PCR (hereinafter referred to as "MSP") or Attempts have been made to use such methods as automatic base analysis to diagnose and screen cancer.

Promoter methylation of tumor-related genes is an important indicator of cancer and can be used in many ways, including the diagnosis and early diagnosis of cancer, the prediction of carcinogenic risk, the prognosis of cancer, the follow-up of treatment, and the prediction of response to anticancer therapy. Attempts have recently been made to investigate the promoter methylation of tumor-related genes in blood, sputum, saliva, feces, and urine and 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).

Accordingly, the present inventors have found that RSPH9 gene, which is specifically methylated in bladder cancer cells, is hypermethylated, and the gene expression is down-regulated through microarray analysis from non-muscle Invasive Bladder Cancer (NMIBC) tissue. The present invention was completed by confirming.

The present invention relates to a novel use of the particular gene RSPH9,

An object of the present invention is to provide a composition for markers for the diagnosis of bladder cancer, containing one or more of the above genes.

Another object of the present invention to provide a variety of uses for the diagnosis of bladder cancer and bladder cancer prognosis of the genes.

Still another object of the present invention is to provide a method for screening a substance having a therapeutic function of bladder cancer using the genes.

In order to solve the above problems, the present invention provides a biomarker of RSPH9 gene and a composition for the marker for diagnosing bladder cancer containing them. In particular, it is effective for the diagnosis of non-muscle invasive bladder cancer (NMIBC) prognosis.

Since the gene is hypermethylated in bladder cancer and its expression is reduced, bladder cancer can be diagnosed.

In addition, the bladder cancer diagnosis according to the present invention may also include a diagnosis of bladder cancer prognosis, which may include the progression and recurrence of bladder cancer.

Based on the function of the gene, the present invention provides a composition for diagnosing bladder cancer, in one embodiment, comprising an agent for measuring RSPH9 gene expression level.

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

At this time, the measurement of methylation level is known PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, pyro sequencing and Methods such as bisulfite sequencing can be used.

The agent for measuring the level of the gene mRNA may comprise a primer specifically binding to the RSPH9 gene, the agent for measuring the level of the protein may comprise an antibody specific for RSPH9 protein.

In another aspect, the present invention provides a kit for diagnosing bladder cancer prognosis comprising the diagnostic composition. In this case, the kit may be an RT-PCR kit, a DNA chip kit or a protein chip kit.

In another aspect, the present invention provides a method for providing information for diagnosing the prognosis of non-muscular invasive bladder cancer, including:

Measuring the methylation level of the RSPH9 gene from a biological sample isolated from the patient; And

Comparing the expression level of the gene or the level of the protein encoded by the gene with the methylation level of the gene of the normal control sample.

The sample may be selected from a crowd consisting of tissue, cells, blood, serum, plasma, saliva and urine.

In another aspect, the present invention utilizes the gene function of the biomarker, and provides a method for screening a material for treating bladder cancer, in particular, non-invasive (superficial) bladder cancer, comprising the following steps.

(a) methylating the RSPH9 gene in the presence of a candidate substance; And

(b) selecting a candidate for treating bladder cancer when methylation of the genes is inhibited, as compared with methylation of RSPH9 gene without a candidate.

As described above, the present invention provides a variety of uses based on the novel function of the RSPH9 gene as a prognostic factor for bladder cancer, most preferably Non-muscle Invasive Bladder Cancer (NMIBC).

As described above, the present invention relates to a novel function of a particular gene, and has the effect of providing a composition and method for diagnosing the prognosis of bladder cancer, especially 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 hypermethylation of the RSPH9 gene.

Accordingly, the present invention relates to RSPH9 gene function in bladder cancer, and may be usefully used for early diagnosis and treatment of bladder cancer.

1 shows (A) methylation level of BARHL2, (B) methylation level of RSPH9, and (C) methylation level of RAB37 in bladder tissue according to the patient's prognosis.

2 and 3 are graphs showing the Kaplan-Meier curve for predicting the prognosis according to the methylation status of each gene in NMIBC patients. 2 (A) shows the survival without relapse according to the methylation state of BARHL2, (B) shows the survival without relapse according to the RSPH9 methylation state, (C) of Figure 3 (C) shows the progression-free survival according to the methylation state of BARHL2, ( D) shows progression free survival according to RSPH9 methylation status.

4 shows tumor cell number changes and RSPH9 methylation levels in 5-Aza-CdR treated bladder cancer cell lines (J82, T24).

Definitions of terms used in the present invention are 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 the prognosis of bladder cancer. Especially, it is useful for the diagnosis of non-muscle invasive bladder cancer (NMIBC) prognosis.

"Diagnostic markers or diagnostic markers are substances that can differentiate bladder cancer cells from normal cells. Polypeptides or nucleic acids (e.g. mRNA, etc.) show an increased pattern in cells with bladder cancer compared to normal cells, Organic biomolecules such as lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, polysaccharides, etc.), etc. For the purposes of the present invention, bladder cancer prognostic markers are hypermethylated in bladder cancer tissue with RSPH9 to reduce their expression. Genes.

"Prognosis" means the prediction of the course and outcome of a disease, and the present invention encompasses the prediction of recurrence and progression of bladder cancer. In order to accurately predict the recurrence and progression of bladder cancer, the indicators are very important, and factors that can predict the response of treatment while complementing clinical indicators such as tissue differentiation and stage are needed. It can be used as a prognostic factor for bladder cancer. That is, the expression characteristics of these genes can be used as a prognostic indicator (diagnosis marker) useful for predicting the degree of differentiation, stage, and progression of bladder cancer.

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

"Subject" or "patient" means any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included are any subjects who participated in clinical research trials showing no disease clinical findings or subjects who participated in epidemiologic studies or who used as controls.

"Tissue or cell sample" means a collection of similar cells obtained from a tissue of a subject or patient. Sources of tissue or cell samples may include solid tissue from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; Blood or any blood component; Cells at any time of pregnancy or development in the subject. Tissue samples may also be primary or cultured cells or cell lines.

"Nucleic acid" is meant to include any DNA or RNA, eg, chromosomes, mitochondria, viruses, and / or bacterial nucleic acids present in tissue samples. One or both strands of a double stranded nucleic acid molecule and any fragment or portion of an 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 in protein coding or transcription or in the regulation of other gene expression. The gene may consist of any nucleic acid encoding a functional protein or only a portion of a nucleic acid encoding or expressing a protein. Nucleic acid sequences may include gene abnormalities in exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or unique sequences adjacent to genes.

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 a cell. In that sense, gene expression includes not only transcriptional and translational processes, but also posttranscriptional and posttranslational processes that can affect the biological activity of a gene or gene product. The processes include, but are not limited to, RNA synthesis, processing and transport, as well as post-translational modifications of polypeptide synthesis, transport and polypeptide. In the present invention, aspects of gene expression include all cases of methylation of gene promoter, mRNA expression and protein expression.

A "coding region" or "coding sequence" refers to a nucleic acid sequence, complement thereof, or portion thereof that encodes a particular gene product or fragment thereof that requires expression, depending on the conventional base pair and codon usage relationship. Coding sequences include exons in genomic DNA or immature primary RNA transcripts that are linked together by a cell's biochemical machinery to provide mature mRNA. Antisense strands are complements of the nucleic acids, and coding sequences can be estimated from them. The coding sequence is placed in a relationship with transcriptional regulatory elements and translation initiation and termination codons such that transcripts of the appropriate length are produced and translated in the appropriate reading frame to produce the desired functional product.

An “primer” is an oligonucleotide sequence that hybridizes to complementary RNA or DNA target polynucleotides and functions as a starting point for the stepwise synthesis of polynucleotides from mononucleotides, for example by the action of nucleotidyltransferases that occur in polymerase chain reactions. Means.

A “protein” is also to include fragments, analogs and derivatives of a protein that possess essentially the same biological activity or function as the reference protein.

By "label" or "label" is meant a compound or composition that directly or indirectly facilitates the detection of a reagent conjugated to, fused, conjugated or fused to a reagent, eg, a nucleic acid probe or antibody. The label may itself be detected (eg, a radioisotope label or a fluorescent label) or, in the case of an enzyme label, may catalyze the chemical modification of the detectable substrate compound or composition.

The expression “down-regulation” refers to a significant decrease in the expression level of a particular gene into mRNA or protein by intracellular transcription or translation compared to normal tissue cells. it means.

"Antibody" is used in its broadest sense and specifically refers to intact monoclonal (monoclonal) antibodies, polyclonal antibodies, multispecific antibodies (eg bispecific antibodies) formed from at least two intact antibodies and Antibody fragments that exhibit the desired biological activity.

"Epigenetics" refers to changes in gene expression that are inherited by offspring without alteration of DNA sequences. Mostly, researches on the concept that abnormal genetic information due to changes in four nucleotide sequences (loss, substitution, amplification, etc.) accumulate in tumorigen genes or tumor suppressor genes, and their function is amplified or lost, affecting cancer development. But this alone has not been enough to explain the development, growth, and metastasis of cancer. Recently, epidemiology that regulates the expression of genes without mutations is developing into a new field of cancer-related research. Epigenetic changes occur through processes such as DNA methylation, histone modification, and genomic imprinting.

"Treatment" means an approach to obtain beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (partially or wholly), detectable or not detected. "Treatment" may also mean increasing survival compared to expected survival when untreated. Treatment refers to both therapeutic treatment and prophylactic or preventive measures. Such treatments include not only the disorders to be prevented but also the treatments required for already occurring disorders. "Palliating" a disease may reduce the extent of the disease state and / or undesirable clinical signs and / or slow or lengthen the time course of progression as compared to untreated treatment. It means losing.

"About" means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for reference quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths. , Amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length, varying by about 3, 2 or 1%.

Throughout this specification, the terms “comprises” and “comprising”, unless otherwise indicated in the context, include a given step or element, or group of steps or elements, but any other step or element, or step or element It should be understood that this group is not excluded.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated concretely.

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

Bladder cancer is the most frequent urinary tract tumor in Korea, and the mechanism and progression of bladder cancer are known to occur through various causes and stages. Recent studies on chromosomes and genetic abnormalities and the occurrence, recurrence and progression of bladder cancer There are many studies on prognostic factors that can predict.

Bladder cancer can be divided into superficial and invasive cancers at the time of initial diagnosis and appears superficial in about 75% of patients. Superficial tumors are common with recurrence, with about 30% of recurrent superficial tumors showing higher malignancy or stage progression and, at about 10%, invasion into the muscle layer. Low malignant Ta lesions recur in 50-70%, progress to invasive bladder cancer in about 5%, and high malignant T1 lesions relapse in more than 80% and within 3 years in 50% of patients with invasive bladder cancer Proceed. Factors recognized to affect the progression of these tumors include bladder cancer found in T1 stage and G3 malignancy (T1G3), multiple epithelial cancer, high recurrence rate, residual tumor after bladder BCG therapy, and expression of p53 gene. Reported.

In invasive bladder cancer, bladder extraction is known as a standard treatment. The prognosis after invasive bladder cancer is T stage, N stage, lymph node density, malignancy, tumor size, number, type, lymph node / vascular involvement, and urinary tract epithelium. It is known to be related to the state of. Eighty to ninety percent of invasive bladder cancers are primary invasive bladder cancers without a history of previous superficial bladder cancers, but about 15% of those have previously progressed from superficial bladder cancers to invasive bladder cancers. There were also cases of primary bladder cancer diagnosed as invasive bladder cancer at the first diagnosis and superficial bladder cancer at the first diagnosis, but recurred and advanced to invasive bladder cancer (advanced invasive cancer).

Therefore, such frequent recurrences and progression of stages are a frequent problem in bladder cancer, and it is necessary to find indicators or develop treatments that can effectively predict the progression of recurrence and invasiveness of bladder cancer.

As a result of studies on genetic abnormalities, there are reports 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 ; 17: 1025-9, Korean J Urol 2005; 46: 211-20, Cancer Genet Cytogenet 1999; 110: 87-93, Cancer Res 1998; 58: 3555-60), on the association between RSPH9 gene and bladder cancer prognosis The contents are unknown.

We found that the RSPH9 gene was hypermethylated in bladder cancer tissue. In other words, the relationship between the expression of the bladder cancer and the specific gene was first identified.

Accordingly, the present invention relates to the use of RSPH9 gene as a diagnostic marker for bladder cancer prognosis, and can predict and diagnose bladder cancer prognosis by confirming whether the gene is hypermethylated and thus reduced expression. In particular, it is very useful for prognostic diagnosis of non-muscle Invasive Bladder Cancer (NMIBC). Also specifically, the RSPH9 gene according to the present invention may be composed of a nucleotide sequence represented by SEQ ID NO: 1.

Prognosis of the bladder cancer includes recurrence and progression.

In this specification, recurrence is defined as the relapse of primary NMIBC at lower or equivalent pathological stage, and progression is defined as muscle infiltration (TNM stage T2 or higher) or metastatic disease.

<Use of bladder cancer prognosis diagnostic marker>

Accordingly, the present invention relates in one aspect to the use of the RSPH9 gene as a diagnostic marker for prognosis of bladder cancer and bladder cancer.

The selection and application of significant diagnostic markers determines the reliability of the diagnostic results. Significant diagnostic markers mean markers of high reliability such that the results obtained by diagnosis are accurate, have high validity, and show consistent results in repeated measurements. Bladder cancer prognosis diagnostic marker of the present invention shows the same result in repeated experiments with genes whose expression changes by direct or indirect factors with the onset of bladder cancer, and the difference in expression level is very large when compared with the control group, resulting in incorrect results. Highly reliable markers with little probability. Therefore, the result of diagnosis based on the result obtained by measuring the expression level of the significant diagnostic marker of the present invention can be reasonably reliable.

Certain genes of the present invention are characterized in that they are hypermethylated and downregulated in bladder cancer tissues.

Thus, bladder cancer can be predicted through methylation and / or downregulation of the RSPH9 gene.

In particular, the present invention can be used individually as a diagnostic or predictive marker, or in combination with several marker genes in the form of a panel display, where several marker genes are reliable through an overall expression pattern or a list of methylated genes. And to improve the efficiency. The genes identified in the present invention can be used individually or as a set of genes in which the genes mentioned in this example are combined. Alternatively, genes can be ranked, weighted, and selected for the level of likelihood of developing cancer, depending on the number and importance of the genes methylated together.

In the same aspect, the present invention relates to a composition for diagnosing bladder cancer prognosis comprising an agent for measuring RSPH9 gene expression level.

At this time, the 'gene expression level measurement' includes both measuring the level of methylation, mRNA or protein thereof. Most preferably the methylation level is measured.

1. Measurement of DNA Methylation Level

"DNA methylation" refers to the binding of methyl groups (CH3) to the 5 carbon sites of CpG by DNA methyl transferase (DNMT), which occurs in the promoter CpG islands, and this DNA methylation is responsible for cell cycle or apoptosis. Regulate, repair DNA, and are involved in cell adhesion and intercellular interaction.

CpG islands are sites of 0.2-3kb in length with a C + G content of at least 50% and a CpG ratio of at least 3.75%. There are about 45,000 CpG islands in the human genome, most of which are found at promoter sites that regulate gene expression. Indeed, the CpG islands are found in promoters of housekeeping genes, about 50% of human genes (Cross, S. and Bird, A., Curr. Opin. Gene Develop., 5: 309, 1995). . The CpG is a site where most epigenetic changes occur frequently in mammalian cells.

In particular, when the RSPH9 promoter CpG island of the present invention is methylated, such methylation inhibits expression and function of the gene in the same manner as mutation of the coding sequence, thereby promoting cancer development, recurrence and progression.

Therefore, as a specific example, a method for detecting whether the gene is promoter methylated may include the following steps:

(a) isolating sample DNA from clinical samples;

(b) amplifying the isolated DNA using a primer capable of amplifying a fragment comprising a CpG island of an RSPH9 gene promoter; And

(c) determining whether the promoter is methylated based on the presence or absence of the amplified product produced in step (b).

The measurement of the DNA methylation level can use various known methods. For example, the methylation measurement method may be PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, pyrosequencing and vi It may be characterized in that it is selected from the group consisting of sulfite sequencing.

Methylation specific PCR method

When bisulfite is treated on genomic DNA, cytosine at the 5'-CpG'-3 site remains cytosine when methylated, and uracil when unmethylated. Therefore, PCR primers corresponding to the sites where the 5'-CpG-3 'nucleotide sequence exists were prepared for the converted nucleotide sequence after bisulfite treatment. In this case, PCR primers corresponding to methylation and two types of primers corresponding to unmethylated were prepared. When pyro DNA is converted to bisulfite, and PCR is performed using the two kinds of primers, PCR products are produced by using the primers corresponding to the methylated sequences when methylated. In the PCR product is produced by using a primer corresponding to unmethylation. Methylation can be qualitatively confirmed by agarose gel electrophoresis.

Real time methylation specific PCR

Real-time methylation-specific PCR converts the methylation-specific PCR method into a real-time measurement method. After treating bisulfite on genomic DNA, a PCR primer corresponding to methylation is designed and real-time PCR is performed using these primers. It is. At this time, there are two methods of detection using a TaqMan probe complementary to the amplified base sequence, and two methods of detection using Sybergreen. Thus, real-time methylation specific PCR can selectively quantitate only methylated DNA. In this case, a standard curve may be prepared using an in vitro methylated DNA sample, and the standardization may be quantitatively analyzed by amplifying a gene without a 5'-CpG-3 'sequence in a nucleotide sequence into a negative control group.

Pyrosequencing

The pyro sequencing method is a method of converting the bisulfite sequencing method into quantitative real-time sequencing. As in bisulfite sequencing, genomic DNA was converted by bisulfite treatment, and PCR primers corresponding to sites without the 5'-CpG-3 'sequence were prepared. The genomic DNA was treated with bisulfite, amplified with the PCR primers, and then subjected to real-time sequencing using the sequencing primers. Quantitative analysis of the amount of cytosine and thymine at the 5'-CpG-3 'site can indicate the degree of methylation as the methylation index.

PCR or quantitative PCR and DNA chips using methylated DNA specific binding proteins

In the PCR or DNA chip method using methylated DNA-specific binding proteins, when a protein that specifically binds to methylated DNA is mixed with DNA, only methylated DNA can be selectively separated because the protein specifically binds to methylated DNA. . After genomic DNA was mixed with methylated DNA specific binding proteins, only methylated DNA was selectively isolated. After amplifying these separated DNA using a PCR primer corresponding to the promoter site, it can be determined whether or not methylation by agarose electrophoresis.

In addition, methylation can also be determined by quantitative PCR.Methylated DNA separated by methylated DNA-specific binding proteins can be labeled with a fluorescent dye and hybridized to DNA chips having complementary probes to measure methylation. Can be.

Detection of Differential Methylation-Bisulfite Sequencing Method

Other methods of detecting nucleic acids containing methylated CpG include contacting a sample containing nucleic acid with an agent that modifies unmethylated cytosine and amplifying the CpG-containing nucleic acid of the sample using CpG-specific oligonucleotide primers. It includes. Here, the oligonucleotide primer may be characterized by detecting the methylated nucleic acid by distinguishing the modified methylated and unmethylated nucleic acid. The amplification step is optional and desirable but not necessary. The method relies on a PCR reaction that distinguishes between modified (eg, chemically modified) methylated and unmethylated DNA. Such methods are disclosed in US Pat. No. 5,786,146, which is described in connection with bisulfite sequencing for the detection of methylated nucleic acids.

That is, in one specific aspect of the present invention, by measuring the DNA methylation level of RSPH9 genes by using the above method and the like can provide a use for diagnosing the prognosis of bladder cancer when hypermethylated.

2. mRNA expression level measurement

"Measurement of mRNA expression level" is to measure the amount of mRNA in the process of confirming the presence and expression of mRNA of the marker genes in a biological sample to diagnose bladder cancer.

Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real time reverse transcriptase (Realtime RT-PCR), and RNase protection assay (RPA). , Northern blotting, DNA chips, etc., but are not limited to these.

At this time, the primers used may initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffers and temperatures. Primers of the invention are sense and antisense nucleic acids having 7 to 50 nucleotide sequences as primers specific for each marker gene. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis. The primers can be chemically synthesized using other well known methods and can be modified using many means known in the art. The nucleic acid sequence can also be modified with a label that can provide a detectable signal directly or indirectly. Examples of labels include radioisotopes, fluorescent molecules, biotin, and the like.

Thus, in one specific aspect of the present invention, it is possible to provide a bladder cancer diagnostic marker composition comprising a primer sequence specific for the RSPH9 gene. At this time, the down-regulation by measuring the mRNA expression level of the genes may provide a use for diagnosing the prognosis of the bladder cancer.

3. Determination of protein expression level

In addition, protein expression level measurement is a process of confirming the presence and expression level of a protein expressed from a bladder cancer marker gene in a biological sample in order to diagnose bladder cancer. Preferably, the antibody specifically binds to the protein of the gene. You can check the amount of protein using.

An antibody means a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes all polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

Analytical methods for this purpose include Western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc., but are not limited to these. .

Thus, as another aspect, the present invention can provide a bladder cancer diagnostic marker composition comprising an antibody specific for RSPH9 protein. At this time, when down-regulated by measuring the protein expression level of the genes can provide a use for diagnosing the prognosis of bladder cancer.

<Sample>

The following protocol related to the detection of specific markers in a sample is shown for illustration.

For sample preparation, tissue or cell samples from mammals (generally human patients) can be used. Samples can be obtained by a variety of procedures known in the art, including but not limited to surgical excision, aspiration or biopsy. The tissue may be fresh or frozen.

Tissue samples can be fixed (ie preserved) by conventional methods. Those skilled in the art will appreciate that the fixative may be selected depending on the purpose for which the sample is histologically stained or otherwise analyzed. One skilled in the art will also understand that the length of fixation is determined by the size of the tissue sample and the fixative used.

The method further includes a protocol for examining methylation levels, mRNA or protein expression in tissue or cell samples. As described above, methods for evaluating methylation, mRNA, protein in cells are well known in the art.

The method may also include a protocol for investigating or detecting methylation and mRNA in tissue or cell samples by microarray technology.

Microarray methods can simultaneously study the RNA expression of thousands or even tens of thousands of genes in a tumor, making it possible to more effectively gain comprehensive insight into the molecular basis of human disease. It can also be used to assess gene expression patterns, clinical outcomes and responses to chemotherapy in tumor classification.

As such, prognostic diagnosis of bladder cancer is possible by measuring RSPH9 gene expression by the above protocol using a sample. As mentioned above, the gene is preferably measured in many different combinations.

Important probes can be labeled so that they can be detected, for example, with radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. Proper labeling of such probes is a technique well known in the art and can be carried out by conventional methods.

In a similar aspect, the present invention relates to a kit for diagnosing 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 the RSPH9 gene, which can be done in two ways: genetically Genetic analysis and immunoassay.

To this end, the kit may comprise, for example, a primer or probe that specifically binds to the RSPH9 gene sequence; Or an antibody that specifically binds to RSPH9 protein.

That is, if 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 contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus). thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. In addition, when the kit for diagnosing human bladder cancer of the present invention is applied to an immunoassay, the kit of the present invention may optionally include a secondary antibody and a substrate of a label.

Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.

As such, 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 kits of the invention comprise a compartmentalized carrier means for holding a sample, a first container containing an agent that sensitively cleaves unmethylated cytosine, a second container containing a primer for amplifying CpG containing nucleic acid and a truncated or It may include one or more containers including a third container containing means for detecting the presence of the uncleaved nucleic acid.

Bladder cancer prognosis diagnosis method

In another aspect, the present invention provides a method for diagnosing bladder cancer prognosis or a method for providing information for diagnosing bladder cancer prognosis comprising measuring the expression level of the RSPH9 gene based on such finding.

Since the 'gene expression level measurement' includes both measuring the level of methylation, mRNA or protein thereof, information about the prognosis of bladder cancer can be predicted through methylation and / or downregulation of RSPH9 gene.

In one embodiment, the method of the present invention

Measuring the expression level of RSPH9 gene from a biological sample isolated from the patient; And

Comparing the expression level of the gene or the level of the protein encoded by the gene with the expression of the gene in the normal control sample. In particular, it is characterized by measuring the methylation level of the genes. Other details are as described above.

That is, the expression of the RSPH9 gene is characterized in that it is an indicator for bladder cancer.

In other words, the method for diagnosing bladder cancer prognosis relates to investigating the expression of specific markers according to non-muscle Invasive Bladder Cancer (NMIBC), in particular the method disclosed herein It may provide a convenient, efficient and cost effective means for obtaining useful data and information when evaluating appropriate or effective therapies.

<Screening method>

On the other hand, in another aspect, the present invention relates to a method for screening a substance for bladder cancer that inhibits the methylation of RSPH9 based on the above-described fact.

The method is an embodiment,

(a) expressing an RSPH9 gene in the presence of a candidate substance; And

(b) compared to the case of expressing the RSPH9 gene without a candidate, it may comprise the step of selecting a candidate material for the treatment of bladder cancer when methylation of the genes is inhibited.

 The present invention extends to a genetic approach to upregulation or downregulation of expression of genes involved in methylation of RSPH9.

As such, the present invention showed an increased methylation and decreased expression profile of the RSPH9 gene in patients with bladder cancer, preferably Non-muscle Invasive Bladder Cancer (NMIBC), and this result is a diagnostic marker for bladder cancer prognosis. It is suggested that the genes characterized by the present invention can be used as target genes for developing or screening bladder cancer diagnosis and treatment utilizing the use of the genes.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Target and Sample Collection

A total of 136 human bladder samples were used for pyrosequencing (PSQ) analysis; Eight normal controls (NC) and 128 NMIBCs were used. NMIBC samples were obtained from 128 primary NMIBC patients who underwent transurethral resection (TUR) for histologically diagnosed transitional cell carcinoma between 1995 and 2010. To exclude confounding variables that may have an incomplete resection or overly impact analysis, patients who relapsed within six months or less than six months were excluded from the study. Normal bladder urinary epithelial samples were obtained from individuals with benign prostatic hyperplasia or bladder injury and used as controls.

All tumors were macro-dissected within 15 minutes of surgical resection. Each NMIBC sample was confirmed by pathological analysis of the tissue sample (ie, sections obtained from TUR samples were snap-frozen in liquid nitrogen and stored at -80 ° C). The sample used for the experiment was provided by Chungbuk National University Hospital. All samples were collected and analyzed with the approval of the Institutional Review Board of Chungbuk National University Hospital with the consent of each patient.

Tumors were staged according to the 2002 TNM classification and the 1973 WHO grading system. If the bladder cancer sample did not contain adequate muscle or a highly differentiated tumor was detected, a second TUR was performed within 2 to 4 weeks after the initial resection. Moderate and high-risk NMIBC patients underwent one cycle of urinary bladder therapy. Each patient underwent follow-up and examination according to standard recommendations.

"Recurrence" is defined as relapse of primary NMIBC at lower or equivalent pathological stage (Ta / T1), and "progression" is defined as muscle infiltration (TNM stage T2 or higher) or metastatic disease. It was.

DNA methylation profile

24 human bladder samples (NMIBC = 18, NC = 6) for microarray methylation data were used. Methylation patterns were analyzed using a genome-wide Infinium array (Illumina Inc., San Diego, Calif.) Capable of interrogation of 27,578 CpG dinucleotides covering 14,495 genes. β values represent a quantitative measure of the DNA methylation level of specific CpG islands from 0 (completely unmethylated) to 1 (completely methylated).

PSQ  analysis

DNA methylation status of NMIBC-specific hypermethylated CpG sites was analyzed by PSQ using PyroMark Q96 ID (Qiagen, Valencia, Calif.) According to manufacturer's instructions. PSQ primers were designed to include CpG sites analyzed on Illumina Infinium array. The primer sequences and amplification conditions are shown in Table 1 and Table 2 below.

Human bladder cancer cell lines T24 (KCLB 30004) and J82 (KCLB 30001) were purchased from Korean Cell Line Bank (Seoul, South Korea). All cells were maintained in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% FBS (Gibco BRL, Grand Island, NY) and L-glutamine, and the cells were maintained at 37 ° C., 5% CO ₂ wet air conditions. Incubated at. Cell lines were incubated until confluent and then treated with PBS (Sigma Aldrich, St. Louis, MO) or 0.3 μmol / L 5-Aza-CdR (Sigma Aldrich, St. Louis, MO). The medium was changed daily, and cells were harvested and counted once every 3 days, 7 days and thereafter.

Statistical analysis

DNA methylation data was normalized using quantile normalization in the R language environment (version 2.10.0, available at http://www.r-project.org/). To detect NMIBC-specific candidate methylation markers, genes with different levels of methylation between NMIBC and NC (Δβ-values> 0.5) were selected.

To evaluate the genes identified in this experiment, the Δβ-value and two microarray data sets from the Western population were compared: (1) 32 bladder tissues (NMIBC = 26, NC = 6), (2) 70 bladder tissues (NMIBC = 64, NC = 6). Continuous variables between groups were analyzed using two-sample t-test or ANOVA trend analysis using polynomial control.

To minimize the bias for any cut-off point, patients were divided into subgroups (hypomethylated or hypermethylated) and median applied, and survival function of candidate genes was evaluated. The Kaplan-Meier method was used to estimate time-to-recurrence or progression based on methylation status, and differences were assessed using log-rank statistics.

For multivariate Cox proportional hazards regression models, the prognostic values of methylation sites are individually assessed and well-known clinicopathology (gender, age, tumor size, tumor count, urinary tract). Intra-bladder treatment, degree of differentiation and stage).

Statistical analysis is performed by IBM SPSS Statistics ver. 21.0 (IBM Co., Armonk, NY). P <0.05 was considered statistically significant.

<Example 1>

Baseline characteristics

Baseline characteristics of NC and NMIBC patients are shown in Table 3 below. The mean relapse-free survival and progression-free survival duration were 44.1 ± 39.1 months (median, 30.6; range, 6.0-205.3) and 60.8 ± 40.7 months (median, 54.1; range, 6.4-205.3), respectively.

<Example 2>

Identification of Differently Methylated and Expressed Genes in NMIBC and NC

Genome-wide methylation profiles were obtained from 18 NMIBC patients and analyzed by comparison with 6 NCs. A complete set of microarray data from human bladder tissue is available online at http://www.ncbi.nlm.nih.gov/geo/ with the data series access number GSE37817. To select NMIBC-specific methylation markers, very stringent selection criteria (Δβ-value> 0.5) were applied. Compared with NC, 25 unique CpG island loci were identified in 23 genes hypermethylated in NMIBC, which are shown in Table 4. Independent sets of Infinium microarray methylation data from Western populations were used to assess the effectiveness of candidate genes as methylation markers for NMIBC. Because of limited data available, only partial verification was possible. Δβ values are almost identical to those in other experiments. To identify candidate markers, genes representing the top 25 Δβ values were selected for their clinically appropriate evaluation in a validation cohort.

[Revision 25.01.2016 under Rule 91]

< Example  3>

PSQ  analysis

To confirm the clinical relevance of candidate methylation markers, PSQ analysis was performed using bisulfite-modified genomic DNA obtained from 136 human bladder samples (NMIBC = 128, NC = 8). PSQ analysis of three of the five candidate genes (BarH-like homeobox 2 (BARHL2), radial spoke head 9 homolog (RSPH9), and member RAS oncogene family (RAB37)) was technically possible and these genes were determined by PSQ. Analyzed.

< Example  4>

Correlation Between Methylation Levels and Clinical Pathology Variables

As shown in Table 5, the methylation levels of candidate genes were significantly higher in the NMIBC patient sample compared to the normal sample (P <0.001). To assess the correlation between methylation patterns and clinicopathological factors, methylation levels were observed in terms of well-known prognostic factors such as tumor number, tumor size and tumor differentiation and stage.

As a result, high methylation levels of BARHL2 and RSPH9 were closely related to tumor size, differentiation and stage.

Example 5

Methylation status as a prognostic predictor

The methylation levels of BARHL2 and RSPH9 were significantly higher in the poor prognosis group (relapse or progression) than in the good prognosis group (see Table 5).

NMIBC patients were divided into three prognostic groups (non-relapsed and non-progressive, relapsed, and advanced), showing that methylation of BARHL2 and RSPH9 is associated with poor prognosis (see FIG. 1). In addition, to confirm the relevance of candidate gene methylation status as predictors, methylation levels of each gene were divided into two groups (hypomethylation or hypermethylation) using median cut-off points. Kaplan-Meier evaluation confirmed a significant difference in time-dependent relapse or progression according to the methylation status of BARHL2 and RSPH9 (see FIG. 2, log-rank test: P <0.05). In univariate and multivariate Cox regression analysis, RSPH9 methylation status was independent predictive of relapse (Hazard ratio (HR), 3.02; P = 0.001) and progression (HR, 8.25; P = 0.028, Table 6) in primary NMIBC patients. It appeared to be a factor. However, BARHL2 methylation did not reach statistical significance for prognostic prediction (P> 0.05).

<Example 6>

Reversibility of RSPH9 Methylation Following AZA Treatment

To investigate the potential reversibility of RSPH9 methylation, cells from two bladder cancer cell lines (T24 and J82) were treated with 0.3 μM 5-Aza-CdR for 24 hours, the cell numbers were changed sequentially, and the RSPH9 methylation levels were respectively The cell line was evaluated. Compared to untreated cells, cell number decreased one day after 5-Aza-CdR treatment and reached maximum levels on day 3 in J82 and T24 cells. After 1 week of 5-Aza-CdR treatment, the cell number began to increase gradually, and reached a constant value after 6 weeks in both cell lines. Consistent with the change in cell number, RSPH9 methylation levels decreased 3 days after treatment in J82 and T24 cell lines and gradually increased after 1 week (FIG. 3).

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (11)

A composition for bladder cancer diagnostic markers containing the RSPH9 gene. The method of claim 1, Said gene is hypermethylated in bladder cancer and has a low expression composition for markers. The method of claim 1, The bladder cancer is a composition for markers, characterized in that non-muscle Invasive Bladder Cancer (NMIBC). The method of claim 1, Bladder cancer diagnosis is a composition for markers, characterized in that it comprises the progression and recurrence of bladder cancer. Bladder cancer diagnostic composition comprising an agent for measuring the expression level of the RSPH9 gene. The method of claim 5, The expression level of the gene is bladder cancer diagnostic composition, characterized in that for measuring the level of methylation, mRNA or protein. The method of claim 5, The bladder cancer is a composition for diagnosing bladder cancer, characterized in that the non-muscle Invasive Bladder Cancer (NMIBC). Bladder cancer diagnostic kit comprising the composition according to claim 5. Measuring the methylation level of the RSPH9 gene from a biological sample isolated from the patient; And Comparing the expression level of the gene or the level of the protein encoded by the gene with the methylation level of the gene of the normal control sample. The method of claim 9, The bladder cancer is characterized in that the non-muscle Invasive Bladder Cancer (NMIBC). The method of claim 9, The sample is selected from the group consisting of tissue, cells, blood, serum, plasma, saliva and urine.

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