TWI496891B - Novel epigenetic biomarkers for bladder cancer detection and method thereof - Google Patents

Description

Novel epigenetic biomarker for detecting bladder cancer and method thereof

The invention relates to a novel epigenetic biomarker for detecting bladder cancer and a method thereof, in particular to a method for screening bladder cancer with methylated DNA as a biomarker.

Bladder cancer is the sixth most common cancer in the world and has a particularly high incidence in southwestern Taiwan. About 90% of bladder cancers are urothelial carcinoma (UC), also known as transitional cell carcinoma (TCC). Another 8% of bladder cancers are squamous cell carcinomas, and about 2% are adenocarcinomas. . Although bladder cancer patients have a low mortality rate, long-term follow-up and repeated cystoscopy are necessary because bladder cancer is a highly recurrent tumor. For detailed bladder examinations, detecting bladder abnormalities must be performed directly through the patient's urethra using a cystoscope, and most patients are subject to these invasive tests.

Genomic deletions are considered to be important factors in tumor formation. For a long time, we have been accustomed to the idea that the coding in the genome depends on the four bases of ATCG. Knudson proposed the double-invasive theory as early as 1975. Two-hit theory), indicating that mutations or deletions accompanying some homologous tumor suppressor genes may cause or cause cancer; however, other information that affects phenotype may exist in the modified base 5-A In 5-methylcytosine, 5-methylcytosine was found in the palindromic sequence 5'-CpG-3' in mammalian cells, except for some called "CpG islands" in mammalian cells. Outside the region of (CpG islands, CGIs), most CpG dinucleotide pairs are methylated, and CpG islands refer to a large number of GC- and CpG in a region of approximately 1000 base pairs (1 Kb). -, usually located near the gene, and found near the promoter of a widely expressed gene. Methylation of cytosine occurs after DNA synthesis, from monomethyl donor s-adenosine methionine (S-adenosylmethionine, SAM) transfers monomethyl groups to the 5th carbon position of cytosine, which is carried out by DNA methyltransferase (DNMTs), the main arm of mammals. The basal transferase, which is responsible for post-replicative restoration to permethylation, is called maintenance methylation; otherwise, DNMT3A and DNMT3B are considered to be primarily responsible for A. To base the new position, perform a step called de novo methylation.

Loss of methylation of CpG dinucleotide, meaning general low methylation, is the first epigenetic abnormality in cancer cells; however, in the past few years Studies have shown that site-specific hypermethylation at specific locations (eg, some tumor suppressor genes) is associated with loss of function, which may provide selective advantages in cancer production; in promoter regions High methylation of the upper CpG island can cause chromatin remodeling by histone modification followed by successive gene silencing; in addition to chromosomal deletions and gene mutations In addition, epigenetic silencing of tumor suppressor genes caused by hypermethylation of promoters is also common in human cancers.

Therefore, a sensitive and non-invasive assay is urgently needed for patients with bladder cancer, and DNA methylation is an ideal biomarker for cancer detection. Due to the nature of the interaction between genetics and the environment, the degree of methylation of tumor suppressor genes varies with different genes and different ethnic groups. Different diseases may have different methylator phenotypes; however, in bladder cancer What specific genes are methylated and how many genes are needed to meet the needs of clinical applications are still issues that need to be identified in the future.

There are still many shortcomings in the above-mentioned conventional bladder cancer screening methods, which is not a good designer, and needs to be improved.

The object of the present invention is to provide a method for detecting a novel epigenetic biomarker of bladder cancer, the method comprising: providing a test subject; detecting a CpG sequence of at least one target gene in the genomic DNA of the test subject In the assimilation state, the target gene is selected from the group consisting of ZNF671, PTPRR, SOX1, PAX1, ZNF582, AJAP1, SOX17, EDN3, ST6GAL2, ZNF614, PTGDR, SYT9, SOX8, HS3ST2, POU4F3, ADRA1D, At least one of a group consisting of MAGI2, EPO, NEFH, POU4F2, STC2, and THRB; and determining whether the specimen has cancer or precancerous lesions according to the presence or absence of the methylation status of the target gene, or as an indicator of prognosis.

To achieve the foregoing object, wherein the target gene is ZNF671 and another target gene is selected from the group consisting of PTPRR, SOX1, PAX1, ZNF582, AJAP1, SOX17, EDN3, ST6GAL2, ZNF614, PTGDR, SYT9, SOX8, HS3ST2, POU4F3, ADRA1D, At least one of a group consisting of MAGI2, EPO, NEFH, POU4F2, STC2, and TIRB.

To achieve the foregoing object, the test sample comprises urine, feces, blood, ascites, sputum, oral mucosal cells, gastric juice, bile, or an ex vivo sample of bladder cancer tissue after surgery.

In order to achieve the above object, the CpG sequence methylation state detection method of the target gene comprises methylation-specific polymerase chain reaction (MSP), quantitative methylation-specific polymerase chain reaction (quantitative) Methylation-specific PCR (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer, denaturing high-performance liquid chromatography (DHPLC), And at least one of pyrosequencing.

In order to achieve the above object, the state of methylation of the target gene is detected by the primer pair sequence, and the primer pair of each target gene is: ZNF671 primer pair is SEQ ID No: 1-2, PTPRR primer pair is SEQ ID No: 3-4, the SOX1 primer pair is SEQ ID No: 5-6, the PAX1 primer pair is SEQ ID No: 7-8, the ZNF582 primer pair is SEQ ID No: 9-10, and the AJAP1 primer pair is SEQ ID No. :11-12, SOX17 primer pair is SEQ ID No: 13-14, EDN3 primer pair is SEQ ID No: 15-16, ST6GAL2 primer pair is SEQ ID No: 17-18, ZNF614 primer pair is SEQ ID No: 19 -20, the PTGDR primer pair is SEQ ID No: 21-22, the SYT9 primer pair is SEQ ID No: 23-24, the SOX8 primer pair is SEQ ID No: 25-26, and the HS3ST2 primer pair is SEQ ID No: 27-28. The POU4F3 primer pair is SEQ ID No: 29-30, the ADRA1D primer pair is SEQ ID No: 31-32, the MAGI2 primer pair is SEQ ID No: 33-34, and the EPO primer pair is SEQ ID No: 35-36, NEFH The pair of primers is SEQ ID No: 37-38, The POU4F2 primer pair is SEQ ID No: 39-40, the STC2 primer pair is SEQ ID No: 41-42, and the THRB primer pair is SEQ ID No: 43-44; wherein the primer comprises at least 50% sequence identity, complementary Sex or at least ten consecutive nucleotides identical in sequence.

Another object of the present invention is to provide a biomarker for detecting a novel epigenetic gene of bladder cancer, wherein the biomarker comprises a CpG site of a target gene and the site is methylated; wherein the target gene is CpG The site sequence is selected from the group consisting of ZNF671 (SEQ ID No: 1-2), PPTRR (SEQ ID No: 3-4), SOX1 (SEQ ID No: 5-6), PAX1 (SEQ ID No: 7-8), ZNF582 (SEQ ID No: 9-10), AJAP1 (SEQ ID No: 11-12), SOX17 (SEQ ID No: 13-14), EDN3 (SEQ ID No: 15-16), ST6GAL2 (SEQ ID No: 17-18), ZNF614 (SEQ ID No: 19-20), PTGDR (SEQ ID No: 21-22), SYT9 (SEQ ID No: 23-24), SOX8 (SEQ ID No: 25-26), HS3ST2 (SEQ ID No: 27-28), POU4F3 (SEQ ID No: 29-30), ADRA1D (SEQ ID No: 31-32), MAGI2 (SEQ ID No: 33-34), EPO (SEQ ID No: 35) -36), consisting of NEFH (SEQ ID No: 37-38), POU4F2 (SEQ ID No: 39-40), STC2 (SEQ ID No: 41-42), and THRB (SEQ ID No: 43-44) At least one group.

In order to achieve the above object, wherein the CpG site sequence of the target gene is ZNF671 (SEQ ID No: 1-2) and the CpG site sequence of another target gene is selected from PTPRR (SEQ ID No: 3-4), SOX1 (SEQ ID No: 5-6), PAX1 (SEQ ID No: 7-8), ZNF582 (SEQ ID No: 9-10), AJAP1 (SEQ ID No: 11-12), SOX17 (SEQ ID No: 13-14), EDN3 (SEQ ID No: 15-16), ST6GAL2 (SEQ ID No: 17-18), ZNF614 (SEQ ID No: 19-20), PTGDR (SEQ ID No: 21-22), SYT9 (SEQ ID No: 23-24), SOX8 (SEQ ID No: 25-26), HS3ST2 (SEQ ID No: 27-28), POU4F3 (SEQ ID No: 29-30), ADRA1D (SEQ ID No: 31) -32), MAGI2 (SEQ ID No: 33-34), EPO (SEQ ID No: 35-36), NEFH (SEQ ID No: 37-38), POU4F2 (SEQ ID No: 39-40), STC2 ( At least one of the groups consisting of SEQ ID Nos: 41-42) and THRB (SEQ ID No: 43-44).

Another object of the present invention is to provide a use of a target gene for preparing a vaccine for bladder cancer screening, which is a state in which a CpG sequence in a target genome of a subject is methylated, wherein the target gene is selected. Free ZNF671, PTPRR, SOX1 At least one of a group consisting of PAX1, ZNF582, AJAP1, SOX17, EDN3, ST6GAL2, ZNF614, PTGDR, SYT9, SOX8, HS3ST2, POU4F3, ADRA1D, MAGI2, EPO, NEFH, POU4F2, STC2, and THRB.

To achieve the above object, wherein the target gene is ZNF671 and another target gene is selected from the group consisting of ZNF671, PTPRR, SOX1, PAX1, ZNF582, AJAP1, SOX17, EDN3, ST6GAL2, ZNF614, PTGDR, SYT9, SOX8, HS3ST2, POU4F3, At least one of a group consisting of ADRA1D, MAGI2, EPO, NEFH, POU4F2, STC2, and THRB.

Figure 1 shows the degree of methylation of ZNF671 gene in bladder cancer and normal human urine samples by qMSP.

Figure 2 shows the degree of methylation of PTPRRR, SOX1, PAX1 and ZNF582 genes in bladder cancer and normal human urine samples by qMSP.

Figure 3 is a graph showing the degree of methylation of AJAP1, SOX17, EDN3 and ST6GAL2 genes in bladder cancer and normal human urine samples by qMSP.

Figure 4 is a graph showing the degree of methylation of ZNF614, PTGDR, SYT9 and SOX8 genes in bladder cancer and normal human urine samples by qMSP.

Figure 5 is a graph showing the degree of methylation of HS3ST2, POU4F3, ADRA1D and MAGI2 genes in bladder cancer and normal human urine samples by qMSP.

Figure 6 shows the degree of methylation of EPO, NEFH and POU4F2 genes in bladder cancer and normal human urine samples by qMSP.

Figure 7 is a graph showing the degree of methylation of STC2 and THRB genes in bladder cancer and normal human urine samples by qMSP.

Figure 8 is a graph showing the degree of methylation of the HOXB3 gene in bladder cancer and normal human urine samples by qMSP.

The present invention relates to a method for detecting a novel epigenetic biomarker of bladder cancer, the method comprising: providing a test subject; detecting at least one of the genomic DNA of the test subject The methylation status of the CpG sequence of the target gene selected from ZNF671, PTPRR, SOX1, PAX1, ZNF582, AJAP1, SOX17, EDN3, ST6GAL2, ZNF614, PTGDR, SYT9, SOX8, HS3ST2, POU4F3, ADRA1D, MAGI2 At least one of a group consisting of EPO, NEFH, POU4F2, STC2, and THRB; and determining whether the sample has cancer or precancerous lesions according to the presence or absence of the methylation status of the target gene, or as an indicator of prognosis.

The invention also discloses a biomarker for detecting a novel epigenetic gene of bladder cancer, wherein the biomarker comprises a CpG site of a target gene and the site is methylated.

All of the technical and scientific terms described in this specification, unless otherwise defined, are intended to be common to those of ordinary skill in the art.

The terms "detection" and "screening" are not definitive diagnostic methods, according to the medical knowledge in the prior art and the information obtained by detecting the state of methylation of target genes in various types of CpG sequences disclosed in the present patent application. It is not possible to directly derive the diagnosis or health status, that is, whether or not there is a diagnosis of cancer, but only to "process or detect tissue, body fluids or excretions that have been removed from the human or animal body to obtain an intermediate result. The method of information, or the method of processing the information, is "an invention that is not a diagnostic method." According to those who have common knowledge in the field (for example, doctors, medical researchers), it is necessary to perform pathological biopsy to diagnose cancer. In other words, the present invention is a method for detecting and screening bladder cancer, rather than a definitive diagnosis method. Therefore, the present invention is not a legally unpatented diagnostic method.

The term "test subject" refers to a test sample that is isolated from the body, and includes the aforementioned ascites, blood, urine, feces, sputum, oral mucosal cells, gastric juice, bile, or post-operative cancer tissue. Sample sample. The cancer screening method of the present invention is for detecting the state of methylation of a target gene in the isolated samples as a screening index for various types of cancer. The cancer screening method and the screening index provided by the invention can be tested by the testing researchers in the laboratory.

The terms "treatment," "in treatment," and the like, mean a method of delaying, ameliorating, reducing, or reversing a condition that is currently afflicting a patient or any condition associated with the condition and preventing the condition or any symptoms that are present. Methods.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples. The drugs and biological materials used in the present invention are commercially available and easy to obtain, the following are only An example of a pipeline that can be obtained.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

Cell line information

Human bladder urinary epithelial cells (HUC) are cultured in urothelial cell culture fluid (UCM).

Patient sample

From bladder cancer patients in Chiayi Christian Hospital, Taiwan, 100 bladder cancer (UC) samples (Table 1) and 9 adjacent normal regions were collected. Urine samples from 27 bladder cancer patients were collected and urine samples from 19 non-cancer individuals were used as a control group.

Target gene

The inventors of the present invention found that there are 22 CpG sites highly correlated with bladder cancer in the whole genome DNA, which can be used as a biological indicator for detecting bladder cancer. The 22 CpG points are from 22 target genes, and the relevant information is as follows: Table 2: Target gene information

Immediate quantitative methylation-specific polymerase chain reaction (QMSP)

To detect the presence or absence of gene promoter methylation in urine samples, a more sensitive quantitative methylation-specific polymerase chain reaction (qMSP) was used. We used real-time PCR system for qMSP, total volume of 20μl containing 10μl 2X SYBR Green real-time fluorescent quantitative PCR premix (Toyobo, Osaka, Japan), each primer was 0.25μM and 4μl of bisulfite-transformed DNA at 95 10 minutes at ° C, 40 cycles at 95 ° C for 15 seconds, 30 seconds at 60 ° C, and 30 seconds at 72 ° C. The qMSP primer (Table 3) is specific for each target gene. In qMSP, β-actin (ACTB) was used as a standard to input the content of DNA. The degree of methylation is determined by the number of threshold cycles (Ct), which is determined by the standard curve generated for each sample relative to the IVD-MSP fragment. of. The percentage of methylation is calculated by dividing the quantified target gene to ACTB ratio by dividing the same amount of IVD and multiplying by 100.

Methylation Microarray

Methylated microarrays are a high-throughput screening tool. The present invention performs methylation analysis using an illumina infinium 27K methylation array, 7 human bladder cancer tissues were used as a test group, and normal bladder urinary tract epithelial cells (HUC) were used as a control group.

Statistical Analysis

A comparative evaluation of nonparametric variables was performed using the Mann-Whitney method. All statistical analyses, including the ROC curve, were performed by SPSS software (version 18.0). A P value of less than 0.05 is considered to be a significant difference.

Embodiment 1

The methyl array identified new methylation target genes in bladder cancer patients: we used the illumina infinium 27K methylation array to select a higher methylation ratio score ( β value >0.5 for methylation), comparing the normal bladder urinary tract The CpG island methylation region of epithelial cells and seven human bladder cancer samples, in which the ZNF671 target gene shows its promoter sequence, is highly methylated in bladder cancer samples and was therefore selected for further analysis.

Embodiment 2

Methylation of ZNF671 gene in urine can be used as a test tool:

To assess the feasibility of using DNA methylation as a marker for cancer detection and recurrence, methylation status was detected using a more sensitive quantitative MSP (QMSP) method from the urine of 28 bladder cancer patients and 19 non-cancer patients. (figure 1). The mean number of ZNF671 in the normal sample was 1.69, and the average number of cancer samples was 19.24. Compared with the non-cancer control group, the ZNF671 gene had a higher degree of methylation in the cancer samples (P < 0.001). The sensitivity and specificity of the ZNF671 gene in cancer detection were 42.3% and 100%, respectively.

Embodiment 3

Methylation of other target genes in urine can be used as a test tool:

We also used qMSP to assess the methylation status of the target gene in the urine of patients with bladder cancer. The target genes are: PPTRR, SOX1, PAX1, ZNF582 (Fig. 2); AJAP1, SOX17, EDN3, ST6GAL2 (Fig. 3); ZNF614, PTGDR, SYT9, SOX8 (Fig. 4); HS3ST2, POU4F3, ADRA1D, MAGI2 (Fig. 5); EPO, NEFH, POU4F2 (Fig. 6); STC2, THRB (Fig. 7). Table 4 shows the average number of target gene normal and bladder cancer samples in each group. The results showed that these target genes have a higher degree of methylation in the urine samples of bladder cancer patients compared to normal non-cancer urine samples.

Embodiment 4

The invention combines 22 genes in a combination of one or more sites, and is used for screening bladder cancer, which can improve the sensitivity of detection. This part of the data is divided into 20 groups of 20 bladder cancer urine samples, each of which is combined. This time, only ZNF671 is used with the other 21 target genes as an example, and the combined target genes are more sensitive (Table 5).

Embodiment 5

Figure 8 shows the degree of methylation of HOXB3 gene in bladder cancer and normal human urine samples by qMSP. The results show that both normal and cancer samples HOXB3 are highly methylated, and there is no statistically significant difference between them. Screen for bladder cancer target genes. It is not that the genes are highly methylated, and they are all associated with bladder cancer.

The method for screening cancer screening provided by the present invention has the following advantages when compared with the conventional techniques described above:

1. Compared to the traditional method of invasive detection, the present invention is a sensitive and non-invasive detection method, which is a great boon for bladder cancer patients, and DNA methylation is an ideal for cancer detection. Biomarker. We have identified a novel target gene, ZNF671, in bladder cancer, whose surface gene silencing is due to DNA methylation.

2. Methylation analysis confirmed that the degree of methylation in bladder cancer patients showed a highly significant association with cancer histological grade and recurrence-free survival.

3. Urine analysis of target genes ZNF671, PPRRR, SOX1, PAX1, ZNF582, AJAP1, SOX17, EDN3, ST6GAL2, ZNF614, PTGDR, SYT9, SOX8, HS3ST2, POU4F3, ADRA1D, MAGI2, EPO, NEFH, POU4F2 The degree of methylation of STC2 and THRB serves as a tool for detecting bladder cancer.

The detailed description above is a detailed description of a possible embodiment of the present invention, but is not intended to limit the scope of the invention, and the equivalents or modifications, such as: Equivalent embodiments of the manner in which the degree of methylation of each target gene in the sample is determined, etc., should be included in the patent scope of the present application.

The above-mentioned multiple functions are in fact the statutory invention patents that fully meet the novelty and progressiveness. If you apply in accordance with the law, you are requested to approve the application for this invention patent to encourage invention.

<110> National Chung Cheng University Dederson Medical Foundation, Chiayi Christian Hospital

<120> Novel epigenetic biomarkers for detecting bladder cancer and methods thereof

<160> 44

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 1

<210> 2

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 2

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 3

<210> 4

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 4

<210> 5

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 5

<210> 6

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 6

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 7

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 8

<210> 9

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 9

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 10

<210> 11

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 11

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 12

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 13

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 14

<210> 15

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 15

<210> 16

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 16

<210> 17

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 17

<210> 18

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 18

<210> 19

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 19

<210> 20

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 20

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 21

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 22

<210> 23

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 23

<210> 24

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 24

<210> 25

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 25

<210> 26

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 26

<210> 27

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 27

<210> 28

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 28

<210> 29

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 29

<210> 30

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 30

<210> 31

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 31

<210> 32

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 32

<210> 33

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 33

<210> 34

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 34

<210> 35

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 35

<210> 36

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 36

<210> 37

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 37

<210> 38

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 38

<210> 39

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 39

<210> 40

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 40

<210> 41

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 41

<210> 42

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 42

<210> 43

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> Forward introduction

<400> 43

<210> 44

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 44

Claims (10)

A method for detecting a novel epigenetic biomarker of bladder cancer, the method comprising: providing a test subject; detecting a methylation state of a CpG sequence of a genomic DNA target gene ZNF671 of the test subject; and according to the target gene The presence or absence of the methylation state, whether the specimen has cancer or precancerous lesions, or as an indicator of prognosis. The method for detecting novel epigenetic biomarkers of bladder cancer according to claim 1, wherein the target gene is ZNF671 and another target genes SOX1, PAX1, AJAP1, SOX17, EDN3, ST6GAL2, ZNF614, SYT9, Any of SOX8, HS3ST2, POU4F3, ADRA1D, MAGI2, EPO, NEFH, STC2 or THRB. The method for detecting a novel epigenetic biomarker for bladder cancer according to claim 1, wherein the test sample comprises urine, feces, blood, ascites, sputum, oral mucosal cells, gastric juice, bile, or In vitro samples of bladder cancer tissue after surgery. The method for detecting a novel epigenetic biomarker for bladder cancer according to the first aspect of the invention, wherein the CpG sequence methylation state detection method of the target gene comprises a methylation-specific polymerase chain reaction (methylation-specific) PCR, MSP), quantitative methylation-specific PCR (QMSP), bisulfite sequencing (BS), microarrays, mass spectrometer At least one of denaturing high-performance liquid chromatography (DHPLC) and pyrosequencing. The method for detecting a novel epigenetic biomarker of bladder cancer according to claim 1, wherein the target gene ZNF671 is methylated by a primer pair sequence SEQ ID No: 1-2 Detected. The method for detecting a novel epigenetic biomarker for bladder cancer according to claim 2, wherein the methylation status of the target gene is detected by a primer pair sequence, and the primer pair of each target gene is: ZNF671 primer pair is SEQ ID No: 1-2, SOX1 primer pair is SEQ ID No: 5-6, PAX1 primer pair is SEQ ID No: 7-8, AJAP1 primer pair is SEQ ID No: 11-12, SOX17 primer For the SEQ ID Nos: 13-14, the EDN3 primer pair is SEQ ID No: 15-16, the ST6GAL2 primer pair is SEQ ID No: 17-18, and the ZNF614 primer pair is SEQ ID No: 19-20, and the SYT9 primer pair is SEQ ID No: 23-24, SOX8 primer pair is SEQ ID No: 25-26, HS3ST2 primer pair is SEQ ID No: 27-28, POU4F3 primer pair is SEQ ID No: 29-30, ADRA1D primer pair is SEQ ID No: 31-32, the MAGI2 primer pair is SEQ ID No: 33-34, the EPO primer pair is SEQ ID No: 35-36, the NEFH primer pair is SEQ ID No: 37-38, and the STC2 primer pair is SEQ ID No: 41-42, THRB primer pair is SEQ ID No: 43-44. A biomarker for detecting a novel epigenetic gene of bladder cancer, wherein the biomarker comprises a CpG site of a target gene and the site is methylated; wherein the CpG site sequence of the target gene is ZNF671 (SEQ ID No) :1-2). The biomarker for detecting a novel epigenetic gene of bladder cancer according to claim 7, wherein the CpG site sequence of the target gene is ZNF671 (SEQ ID No: 1-2) and the CpG position of another target gene. Dot sequence SOX1 (SEQ ID No: 5-6), PAX1 (SEQ ID No: 7-8), AJAP1 (SEQ ID No: 11-12), SOX17 (SEQ ID No: 13-14), EDN3 (SEQ ID No: 15-16), ST6GAL2 (SEQ ID No: 17-18), ZNF614 (SEQ ID No: 19-20), SYT9 (SEQ ID No: 23-24), SOX8 (SEQ ID No: 25-26), HS3ST2 (SEQ ID No: 27-28), POU4F3 (SEQ ID No: 29-30), ADRA1D (SEQ ID No: 31-32), MAGI2 (SEQ ID No: 33-34), EPO (SEQ ID No: 35-36), NEFH (SEQ ID No: 37-38), STC2 (SEQ ID No: 41-42) or THRB (SEQ ID No: Any of 43-44). Use of a target gene for the preparation of a reagent for bladder cancer screening, which is a state in which a CpG sequence in a DNA of a target genome in a subject is methylated, wherein the target gene is ZNF671. The use of the target gene according to claim 9 in the preparation of a reagent for bladder cancer screening, wherein the target gene is ZNF671 and another target genes SOX1, PAX1, AJAP1, SOX17, EDN3, ST6GAL2, ZNF614, Any of SYT9, SOX8, HS3ST2, POU4F3, ADRA1D, MAGI2, EPO, NEFH, STC2 or THRB.