KR101637338B1 - Method for detecting methylation of HBD-2 promoter and composition for diagnosing cancer using thereof - Google Patents

Abstract

The present invention relates to a composition for cancer diagnosis related to the methylation of HBD-2 gene promoter and its use, and more particularly, to a composition for cancer diagnosis through detection of methylation of a specific CpG in HBD-2 gene promoter, A method for detecting the methylation of a promoter, and a cancer diagnostic kit comprising the composition. Cancer can be diagnosed more accurately by detecting whether CpG is methylated in the HBD-2 gene promoter of a clinical sample through the method or composition of the present invention.

Description

TECHNICAL FIELD The present invention relates to a method for detecting methylation of HBD-2 gene promoter and a method for diagnosing cancer using the same,

The present invention relates to a composition for cancer diagnosis related to the methylation of HBD-2 gene promoter and its use, and more particularly, to a composition for cancer diagnosis through detection of methylation of a specific CpG in HBD-2 gene promoter, A method of detecting whether a promoter is methylated, a method of providing information for cancer diagnosis, and a cancer diagnostic kit containing the composition.

Even today, when medical science is developed, the 5-year survival rate is less than 50% for human cancers, especially solid tumors (other than cancer). Approximately two-thirds of all cancer patients are found at an advanced stage, most of whom die within two years of diagnosis. The treatment effect of such poor cancer is not only the problem of the treatment method, but it is not easy to diagnose cancer in an early stage and it is not easy to accurately diagnose advanced cancer and follow up after treatment.

At present, the diagnosis of cancer is based on history taking, physical examination, and clinical pathology. Once suspected, the cancer is progressed to radiation examination and endoscopy. Finally, it is confirmed by histological examination. However, the existing clinical test method, the number of cancer cells 1 billion, the diameter of cancer is more than 1 cm can be diagnosed. In this case, cancer cells already have the ability to metastasize, and more than half of the cancer has already metastasized. On the other hand, tumor markers that detect substances directly or indirectly produced by cancer in the blood are used for cancer screening. This is because the accuracy is limited, Even when it is not there, it often appears positively and causes confusion. Further, in the case of an anticancer agent mainly used for the treatment of cancer, there is a problem that the effect is exhibited only when the volume of cancer is small.

As described above, the diagnosis and treatment of cancer are all difficult because they are different from normal cells, and are very complex and diverse. Cancer continues to grow in excess of its will, freed from death, to survive, to invade surrounding tissues, and to spread to (distant) organs, leading to death. It also survives the attacks of immunological mechanisms and chemotherapy, and selectively proliferates cell clones that are constantly evolving and most beneficial to survival. Cancer cells are survivors with high viability due to mutation of multiple genes. In order to transform a single cell into a cancer cell and evolve into a malignant cancer mass seen in clinical practice, many genes must be mutated. Therefore, in order to diagnose and treat cancer in a fundamental way, it is necessary to approach it at the genetic level.

Recently, methods for diagnosing cancer through DNA methylation measurement have been proposed. DNA methylation occurs mainly in the cytosine of the CpG island of the promoter region of a specific gene, thereby interfering with the binding of the transcription factor, thereby causing gene silencing of the specific gene, In vivo, the function of the gene is lost without the mutation in the coding sequence of the protein coding sequence of the gene, and the function of tumor suppressor genes is lost in human cancer. . There is controversy as to whether the methylation of the promoter CpG island directly induces carcinogenesis or is secondary to carcinogenesis. However, this abnormal methylation / demethylation in CpG ischemia in a variety of cancer cells such as prostate cancer, colon cancer, uterine cancer, And hypermethylation of tumor suppressor genes, DNA repair genes, and cell cycle control genes in a variety of cancers has been shown to block the expression of these genes. It is a well-known fact that hypermethylation occurs at the promoter region of a specific gene at an early stage of cancer development. Thus, promoter methylation of tumor-associated genes is an important indicator of cancer, which can be used in many ways, including early diagnosis of cancer, prediction of carcinogenesis risk, prediction of cancer prognosis, follow-up after treatment, and prediction of response to chemotherapy. Recently, an attempt has been made to use this method for the diagnosis and screening of cancer by a method such as methylation specific PCR (hereinafter referred to as MSP), automatic base analysis or bisulfite pyrosequencing. Recently, Attempts have been made to use promoter methylation of tumor-associated genes for various cancer treatments.

Accordingly, the present inventors have conducted studies to develop a novel biomarker capable of accurately diagnosing cancer by a non-invasive method. As a result, the expression level of HBD-2 (human beta defensin-2) gene was decreased in cancer cells compared to normal cells, and the CpG site of HBD-2 gene promoter region in cancer cells was hypermethylated ), Confirming that the expression of HBD-2 gene and the methylation analysis of the promoter region can be used as markers which are very useful for the diagnosis of cancer, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a method for detecting the CpG methylation of the HBD-2 (human beta defensin-2) gene promoter from a clinical sample for cancer diagnosis.

Another object of the present invention is to provide a method for diagnosing cancer, comprising the steps of (a) detecting whether the HBD-1 and HBD-2 promoters are methylated and (b) comparing the methylation rate of HBD-1 and the HBD- And to provide a method for providing information for the user.

Another object of the present invention is to provide a cancer diagnostic composition comprising a substance capable of detecting whether or not methylation of the HBD-2 gene promoter CpG is detected.

Another object of the present invention is to provide a cancer diagnostic kit comprising a pair of primers for amplifying a fragment containing methylated CpG of the HBD-2 gene promoter.

Another object of the present invention is to provide a cancer diagnostic chip comprising a probe capable of hybridization with a fragment containing the methylation site of the HBD-2 gene promoter.

In order to accomplish the above object, the present invention provides a method for detecting the CpG methylation of a human beta-defensin-2 (HBD-2) gene promoter from a clinical sample for cancer diagnosis.

(A) detecting whether the HBD-1 and HBD-2 promoters are methylated and (b) comparing the methylation rate of HBD-1 with the HBD-2 promoter. And provides a method for providing information for cancer diagnosis.

Another object of the present invention is to provide a cancer diagnostic composition comprising a substance capable of detecting the methylation of the HBD-2 gene promoter CpG.

Another object of the present invention is to provide a cancer diagnostic kit comprising a pair of primers for amplifying a fragment containing methylated CpG of HBD-2 gene promoter.

In order to accomplish another object of the present invention, there is provided a cancer diagnostic chip comprising a probe capable of hybridization with a fragment containing a methylation site of an HBD-2 gene promoter.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for detecting the CpG methylation of the HBD-2 (human beta defensin-1) gene promoter from a clinical sample for cancer diagnosis.

HBD-2 is expressed mainly in mucous membranes of skin, urinary tract, airway, lung, etc. Unlike HBD-1 which is constitutively expressed, HBD-2 is an inflammatory cytokine such as IL-1β, TNF-α and IFN- Lt; / RTI > It is also a typical β-defensin, which is expressed by bacteria such as LPS, bacterial viral substances, and TLRs and NODR ligands. In OSCC, HBD-1 was expressed as a tumor suppressor gene and HBD-2 was expressed as a proto-oncogene (OSCC) in skin cancer and oral squamous cell carcinoma (OSCC) And the possibility of functioning as

In the present invention, the term "methylation" refers to a change in the gene expression pattern due to a methyl group attached to a base. In the present invention, methylation occurs at the promoter region of the HBD-2 gene. The methylation of the present invention specifically includes those occurring in the cytosine of CpG sites in which bases of C and G are consecutively present in the nucleotide sequence in the HBD-2 gene promoter, thereby interfering with the binding of transcription factors, 2 < / RTI > gene is blocked.

In the genomic DNA of mammalian cells, in addition to A, C, G, and T, there is a 5th base called 5-methylcytosine with a methyl group attached to the fifth carbon of the cytosine ring. 5-mC is attached only to C of the CG dinucleotide (5'-mCG-3 '), which is called CpG. C of CpG is mostly methylated by the contact of the methyl group. CpG methylation suppresses the expression of transposon and genomic repeat sequences. Furthermore, the CpG is a site where most of the widespread genetic changes occur in mammalian cells. 5-mC of CpG is naturally deaminated and is likely to become thymine (T).

The inventors of the present invention conducted a series of experiments to determine whether the methylation of the HBD-2 gene promoter is correlated with the onset or progression of the cancer. As a result, the inventors of the present invention found that the methylation of the CpG site, which is a specific region of the HBD- And prostate cancer patients, respectively. As a result, it was found that the methylation region associated with cancer was correlated with the clinicopathologic characteristics of the cancer patient and that the level of methylation of the HBD-2 gene could be accurately diagnosed.

In a specific example of the present invention, expression patterns of HBD-2 gene in prostate epithelial cell line and prostate cancer tissue were examined by RT-PCT and quantitative real-time PCR (qPCR) method, respectively (Example 2). As a result, when the DNA methylation inhibitor 2'-deoxy-5-azacytidine (DAC) was treated with the prostate cancer cell line DU145 and PC-3, which inhibited the expression of HBD-2 mRNA, the expression of HBD-2 was induced Observation revealed that methylation of HBD-2 gene is a cause of inhibition of HBD-2 expression (Example 2-1).

In addition, the inventors of the present invention performed bisulfite genomic sequencing using gDNA extracted from a prostate cancer cell line to identify the position of a promoter CpG site important for transcriptional regulation of HBD-2 gene. As a result, as shown in FIG. 2, it was found that the 5'-terminal region of HBD-2 was a low CpG-content promoter (LCP) lacking a typical CpG island and the 1 Kb promoter region was replaced with LCP1 (L1) and LCP2 The methylation trends of the CpG dinucleotides contained in the respective fractions were determined by the bisulfite genomic sequencing method (FIG. 2B). Thus, it was confirmed that CpG 4 and CpG 5 in the L1 region were important positions for HBD-2 transcriptional regulation in prostate cancer cell lines (highlighted by the red boxes in FIGS. 2B and 2C).

In another embodiment of the present invention, quantitative analysis of the methylation of the CpG 5 and CpG 5 dinucleotides in the HBD-2 LCP site in prostate cancer patient tissue samples was performed to compare the results obtained from samples of prostate cancer patients classified by gleason score T stage compared to the results obtained from patient samples (Example 4). As a result, the difference in methylation tendency between normal tissues and tumors was statistically significant at the histopathological stage in both CpG 4 and CpG 5 of HBD-2 LCP region. That is, the methylation of CpG 4 and CpG 5 in the HBD-2 LCP region was significantly reduced in the tumor compared to the normal tissue. In particular, the CpG 4 position was found to be a good biomarker for diagnostic use in distinguishing between Gleason score ≤6 and 7 tumors (P = 0.003), and Gleason score ≤6 and ≥8 tumors (P = 0.003) 3A). In addition, the results of the T stage classified as prostate cancer patients showed a significant difference between normal T3a and T3b (CpG 4, P = 0.021, CpG 5, P = 0.02) , And field effect biomarkers can be expected.

That is, when CpG methylation of the HBD-2 gene promoter was detected in a clinical sample according to the method of the present invention, it was found that when CpG of the HBD-2 gene promoter was hypomethylated, The tissue can be diagnosed as a tumor.

More particularly, the present invention relates to a method for detecting a CpG methylation of an HBD-2 gene promoter from (a) obtaining a clinical sample from an individual, (b) detecting the CpG methylation of the HBD-2 gene promoter from the clinical sample, and (c) And comparing the result with the results of the normal group to provide information for cancer diagnosis.

In the present invention, a clinical sample refers to a sample obtained from a mammal suspected of having a cancer, and preferably refers to a sample obtained from a human. The sample may be a tissue, a cell, a spleen, a feces, a urine, a cell membrane, a cerebrospinal fluid, a amniotic fluid, an eye, an organ or a blood of a carcinoma to be diagnosed, and may be different depending on a carcinoma to be diagnosed.

The cancer to be diagnosed according to the present invention includes, but is not limited to, cervical cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, colon cancer, bone cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, Cancer of the endometrium, cancer of the endocrine system, cancer of the endocrine system, cancer of the endocrine system, cancer of the thyroid, parathyroid cancer, soft tissue sarcoma, urethral cancer, penile cancer, Prostate cancer, bladder cancer, kidney, or urothelial cancer, and is preferably a prostate cancer.

SEQ ID NO: 1 is a promoter region from -433 bp to -334 bp from the transcription initiation site (TSS, +1) of the HBD-2 gene, and a total of 100 nucleotide sequences are as follows.

5'-ATG GAT AGA ATT AGA TGG AAA GAG TTT TTA ATT TGG TTT GAG ATT GTT TTT AGA TAT TTA GGA AAA ATA GGA YGT YGT ATA GAG TGG GTA GTA GGT GAG T-3 '

The CpG 4 position indicated by Y in each of the above sequences is a pyrimidine base located at -361 bp from the transcription start point (TSS, +1) of the HBD-2 gene and at -358 bp at the CpG 5 position, If the unmethylated protein is methylated, then it can be classified as cytosine.

The present invention also relates to a method of detecting methylation by PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA-specific binding protein, and methylation detection using methylation-sensitive restriction enzyme , Quantitative PCR, DNA chip, pyrosequencing, and bisulfite sequencing. The present invention is not limited to the method.

Specifically, the method of methylation-specific PCR is a method of designing primers to be subjected to PCR after treating the sample DNA with bisulfite, and designing different kinds of primers depending on whether methylation of the CpG dinucleotide is performed. When the primer binding site has been methylated, the PCR is carried out by the methylated primer. If the methylation is not carried out, the PCR proceeds by the normal primer. In other words, after treating the sample DNA with bisulfite, PCR is performed using two kinds of primers at the same time, and the results are compared.

Real-time methylation-specific PCR is the conversion of methylation-specific PCR method to real-time measurement method. It is designed by designing PCR primer corresponding to the methylation of bisphosphate after processing genomic DNA, PCR. 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 is prepared using an in vitro methylated DNA sample, and a gene having no 5'-CpG-3 'sequence is amplified together with a negative control group in the nucleotide sequence for standardization to quantitatively analyze the degree of methylation. .

In the method of measuring methylation using a methylation-sensitive restriction enzyme, a methylation-sensitive restriction enzyme has a CpG dinucleotide as a site of action and does not act as an enzyme when this site is methylated. Therefore, when the sample DNA is treated with a methylation-sensitive restriction enzyme and then amplified by PCR to include an enzyme target site, the methylated region is cleaved by a restriction enzyme, Since it is not amplified, it is possible to measure methylation of a specific DNA site. The methylation-sensitive restriction enzyme is a restriction enzyme capable of specifically detecting the methylation of CpG islands, and is preferably a restriction enzyme containing CG as a recognition site of a restriction enzyme. For example, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI, and the like. The methylation or non-methylation of the restriction enzyme recognition site at C may or may not be cleaved by restriction enzymes and may be detected by PCR or Southern blot analysis. Other methylation sensitive restriction enzymes than the above restriction enzymes are well known in the art.

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 . After mixing genomic DNA with methylated DNA-specific binding protein, only methylated DNA is selectively isolated. These separated DNAs are amplified using a PCR primer corresponding to the intron region and then subjected to agarose electrophoresis to determine whether or not they are methylated. 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 . Here, the methylated DNA-specific binding protein is not limited to MBD2bt.

In addition, bisulfite pyrosequencing of DNA with bisulfite treatment is based on the following principle. When methylation occurs at the CpG dinucleotide site, 5-methylcytosine (5-mC) is formed, which changes to uracil upon treatment with bisulfite. When the DNA extracted from the sample is treated with bisulfite, the CpG dinucleotide is converted to uracil if it is methylated and preserved as cytosine if it is not methylated. Sequence analysis of the bisulfite treated DNA can be preferably performed using a pyrosequencing method. A detailed description of pyrosequencing is known in the prior art [Ronaghi et al, Science 1998 Jul 17, 281 (5375), 363-365; Ronaghi et al, Analytical Biochemistry 1996 Nov 1, 242 (1), 84-9; Ronaghi et al. Analytical Biochemistry 2000 Nov 15, 286 (2): 282-288; Nyr, P. Methods Mol Biology 2007, 373, 114].

Therefore, by using the method of detecting methylation of the present invention, it is possible to effectively confirm the methylation of the HBD-2 gene promoter. Whether or not the methylation of the gene is correlated with cancer, preferably prostate cancer, . ≪ / RTI >

Preferably, the method for detecting methylation is a) obtaining a sample from an individual, b) obtaining genomic DNA from the sample, c) reacting the obtained genomic DNA with a compound that transforms the non-methylated cytosine base D) obtaining the PCR product by amplifying the treated DNA by PCR using a primer for pyrosequencing capable of amplifying the HBD-2 gene promoter, and e) amplifying the PCR product with the primer set forth in SEQ ID NO: 2 And measuring the degree of methylation by pyrosequencing using a sequencing primer having a gene sequence.

The genomic DNA of step b) can be obtained by the phenol / chloroform extraction method, the SDS extraction method (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990), the CTAB separation method Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) or commercially available DNA extraction kits.

The compound capable of modifying the non-methylated cytosine residue in step c) may be bisulfite. The method of detecting whether the methylation of the promoter is modified by modifying the non-methylated cytosine residue using such a bisulfite is widely known in the art (Herman JG et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 9821-9826; WO01 / 26536; US2003 / 0148326A1).

In the step d), amplification can be performed by a conventional PCR method, but is not limited thereto. At this time, the primer pair used may preferably be the primer pair of SEQ ID NOS: 3 and 4.

The present invention further provides a method for detecting whether CpG methylation of the HBV-1 (human beta defensin-1) gene promoter is additionally detected from the clinical sample.

The promoter of the HBD-1 gene may have the DNA sequence of SEQ ID NO: 5, and the 83rd and 87th cytosines of the sequence may be methylated (hereinafter referred to as CpG 5 and CpG 6 Quot;). The method of detecting the methylation of CpG 5 and CpG 6 in the promoter of the HBD-1 gene can be performed by the same method as the method of detecting the methylation of the HBD-2 promoter. For more details, refer to Korean Patent Application No. 10- RTI ID = 0.0 > 2015-0049580. ≪ / RTI >

The present invention also provides a method for diagnosing cancer, comprising: (a) detecting whether the HBD-1 and HBD-2 promoters are methylated and (b) comparing the methylation rate of HBD-1 with the HBD-2 promoter The method comprising:

Preferably, a method is provided for detecting the methylation frequency of the HBD-1 and HBD-2 promoters and comparing the methylation ratios of the HBD-1 and HBD-2 promoters to provide information for cancer diagnosis.

In a specific example of the present invention, the inventors investigated the possibility of biomarkers for molecular diagnosis of prostate cancer patients using the ratio of CpG 5, CpG 6 position of the HBD-1 promoter and CpG 4 and CpG 5 positions of the HBD-2 promoter . (CpG 4 and CpG 5) of the proto-oncogene HBD-2 promoter were measured as the denominator of the methylation frequency of the two CpG positions (CpG 5 and CpG 6) of the tumor suppressor gene HBD-1 promoter. The HBD-2 / HBD-1 ratio was calculated from the total of four types of HBD-2 / HBD-1 and HBD-2 / HBD-1 ratios.

As a result, the ratio of HBD-2 CpG 4 / HBD-1 CpG 5 and the ratio of HBD-2 CpG 5 / HBD-1 CpG 5 showed statistical significance between normal tissue samples and tumor tissue samples of prostate cancer patients The proportion of HBD-2 CpG 4 / HBD-1 CpG 5 was tumor specific, and the Gleason score 3 + 3 tissue sample was found to have a Gleason score of 7 (3 + 4 & 4 + 3) (P = 0.004), respectively. On the other hand, the ratio of HBD-2 CpG 5 / HBD-1 CpG 5 was found to be significantly different between the Gleason score 3 + 3 and 4 + 3 and the Gleason score 3 + 4 and 4 + 3 in the normal tissues of prostate cancer patients (P = 0.004), confirming the possibility as a field effect biomarker (Example 5).

In other words, when the methylation ratio of the HBD-2 / HBD-1 promoter is used, it is possible to obtain a result that the distinction between normal tissue samples and tumor tissues of prostate cancer patients at all histopathological stages becomes more definite. Thus, the results of the HBV-2 / HBD-1 ratio, which is a combination of HBV-1 CpG 5 and CpG 6 methylation frequency measurements and HBD-2 CpG 4 and CpG 5 methylation frequency measurements, Markers and field effects can be expected to greatly increase the utility value of biomarkers.

In the above step (b), the methylation ratios of the HBD-1 and HBD-2 promoters were compared with the methylation frequencies of the two CpG positions (CpG 5 and CpG 6) of the HBD-1 promoter as denominators. The proto-oncogene HBD The pathological state of the clinical sample can be predicted through the calculated value of the methylation frequency of two CpG positions (CpG 4 and CpG 5) of the -2 promoter as a numerator.

The promoter and methylation of the HBD-1 gene is very similar to the mechanism of loss of function in the carcinogenesis process of well-known typical tumor suppressor genes. In addition, HBD-2, which is considered to be one of the causes of many cancers, and thus chronic inflammation, facilitates early tumor formation through the function of inducing inflammation-promoting factors, so that promoter methylation is reduced Prostate epithelial cells with increased expression of HBD-2 may be expected to provide a microenvironment (i. E., A tumor-prone microenvironment) that is more likely to be carcinogenic in inflammation-mediated carcinogenesis. Thus, the contrary promoter methylation pattern of HBD-1, a tumor suppressor gene and the proto-oncogene HBD-2, (ie, the former is tumor-specific hypermethylation versus normal, the latter is tumor-specific hypomethylation) , We found that the first combination of methylation of specific CpG dinucleotides found in the present study could reveal a more meaningful or obscure clinical relevance than the molecular diagnosis and prognosis of individual genes alone I expected. In fact, the mean values of% methylation values measured by 3 repetitions of CpG 5 and CpG 6 sites of HBD-1 and CpG 4 and CpG 5 sites of HBD-2 were combined into four combinations, and classified into Gleason score and T stage Statistical analysis was performed on samples of prostate cancer patients, and Table 3 and Table 4 of the patent plan were obtained. In the results of A and B in Fig. 3, the percentage of methylation of CpG 4 and CpG 5 sites of the HBD-2 promoter in GS 3 + 3 patients is not statistically significant (NT) and tumor (T) But not statistically significant, as shown in Table 3, the p value was 0.001 or 0.002 in all four cases in combination with the percent methylation of CpG 5 and CpG 6 sites of the HBD-1 promoter. Most importantly, when the percent methylation of the HBD-1 and HBD-2 promoter CpG sites were used, a specific distinction was made only between the tumor samples in the prostate cancer samples classified by the Gleason score (I. E., No significant p value between NT samples was obtained), HBD-2 as in Table 3; CpG 4 / HBD-1; CpG 5 and HBD-2; CpG 5 / HBD-1; For CpG 5, it was possible to distinguish between statistically significant NT samples (p = 0.009 and p = 0.004, respectively). In particular, HBD-2; CpG 5 / HBD-1; In CpG 5 combination, the classification of 3 + 4 and 4 + 3 patients with GS 7, which is very important in predicting the aggressiveness of prostate cancer, was found to be present between NT samples. Therefore, histopathologically, It is particularly noteworthy that it provided a basis for judging whether a potential genetic alteration of the prostate site is visible. As shown in Table 4, the results of the statistical analysis of the NT samples in the vicinity of the tumor that have not yet been prostate-carcinoma-like are again shown in the combination of HBD-2 / HBD-1 promoter CpG sites using T stage-divided prostate cancer patient samples As in Table 3, HBD-2; CpG 4 / HBD-1; CpG 5 and HBD-2; CpG 5 / HBD-1; CpG 5 combinations can provide information on the more significant distinctions between NT samples at p = 0.002 and p = 0.003, respectively. This is more statistically significant than the p values shown in FIGS. 4A and B, and is more useful in that it shows not only the NT samples of the T3a and T3b patients but also the T2 and T3b samples, Feature. In conclusion, we calculated the percentage of the methylation% of the promoters CpG 5 and CpG 6 sites of HBD-1 gene and the percentage of methylation% of CpG 4 and CpG 5 sites of HBD-2 gene promoter, When applied to the diagnosis and prognosis of clinical specimens, "field effect" (gradual accumulation of epigenetic abnormalities during the course of normal epithelial cell metastatic cancer, histologic abnormalities are not evident but are not already normal The effects of the primary carcinoma surrounding epithelium were found to be new potential as biomarkers.

These biomarkers can be used for the purpose of investigating and preventing prophylactic changes in prostate epithelial cells inherited before the onset of prostate cancer among health holders, such as apparently healthy, or prostate or prostatic hyperplasia And it is necessary to emphasize the importance of biomarkers with these characteristics in the present situation.

The present invention also provides a cancer diagnostic composition comprising a substance capable of detecting whether or not the HBD-2 gene promoter CpG is methylated.

The present invention also provides a composition for cancer diagnosis, which further comprises a substance capable of detecting the methylation of the HBD-1 gene promoter CpG in the cancer diagnostic composition.

The substance capable of detecting methylation of the CpG is selected from the group consisting of a primer pair capable of amplifying a fragment containing methylated CpG, a probe capable of hybridizing with the methylated CpG, a methylation specificity capable of binding to the methylated CpG An antigen-binding protein, an anti-methylation-specific binding antibody, a sequencing, a sequencing biosynthesis, and a sequencing bi-ligation primer.

The present invention also provides a cancer diagnostic kit comprising a pair of primers for amplifying a fragment comprising the methylated CpG of the HBD-2 gene promoter.

The present invention also provides a cancer diagnostic kit further comprising a pair of primers for amplifying a fragment containing the HBD-1 gene promoter CpG in the cancer diagnostic kit.

 The cancer diagnostic kit of the present invention may be a gene amplification kit. The gene amplification kit of the present invention includes a primer that is a polynucleotide complementary to the HBD-1 and HBD-2 gene promoter sequences. The primer may have one or more mismatch nucleotide sequences and does not need to have a sequence completely complementary to a part of the template. The primer may be hybridized with the template to give sufficient complementarity Is sufficient.

The kit for cancer diagnosis of the present invention includes reagents necessary for gene amplification reaction such as buffer, DNA polymerase, DNA polymerase joinder (for example, Mg ²⁺ ) and dNTPs (ATP, GTP, CTP, TTP) can do. The kit of the present invention can be made with a number of separate packaging or compartments containing the reagent components described above. The nucleic acid sample to be analyzed can be extracted from the tissues of the patients. The cancer diagnosis kit of the present invention can be used to diagnose cancer by analyzing the methylation PCR results of a nucleic acid sample of a healthy control group and a cancer patient.

Preferably, in the present invention, the pair of primers for pyrosequencing for amplifying the HBD-2 gene promoter fragment is a sequence represented by SEQ ID NOS: 3 and 4, wherein the HBD-1 gene promoter fragment is The primer pair for amplification may be a sequence represented by SEQ ID NOs: 6 and 7, but is not limited thereto.

The present invention also provides a cancer diagnostic chip comprising a probe capable of hybridization with a fragment containing a methylation site of the HBD-2 gene promoter.

Preferably, the cancer diagnostic chip is characterized in that the sequence represented by SEQ ID NOS: 8 to 11 is immobilized as a probe.

The present invention also provides a cancer diagnostic chip further comprising a probe capable of hybridization with a fragment containing a methylation site of the HBD-1 gene promoter.

The cancer diagnostic chip may be characterized in that the sequence represented by SEQ ID NOS: 12 to 15 is additionally fixed as a probe.

The cancer diagnostic chip may be provided in the form of a DNA chip, which may include essential elements necessary for performing DNA chip analysis. The DNA chip may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and reagents, preparations, enzymes, and the like for producing a fluorescent-labeled probe. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.

In addition, the cancer diagnostic chip may be provided in the form of a protein chip, which may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, etc. for immunological detection of the antibody have. The substrate is not particularly limited, but a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, and a glass slide glass can be used, and the coloring enzyme is not particularly limited The fluorescent substance may be FITC, RITC or the like, and the coloring substrate liquid is not particularly limited, but ABTS (2, 3, 4, 5, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) or OPD (o-phenylenediamine), TMB (tetramethylbenzidine).

In the present invention, the presence or absence of cancer in a clinical sample can be diagnosed by detecting the methylation of the HBD-2 gene using a kit or a chip.

As described above, the methylation of CpG in the HBD-2 gene promoter affects the expression of HBD-2 in cancer cells, and the methylation of HBD-2 gene is specifically expressed in cancer cells, particularly prostate cancer. The method or composition of the present invention can be used to diagnose cancer by detecting the methylation of the HBD-2 gene.

FIG. 1A shows the expression levels of HBD-2 mRNA in normal prostate cancer cell lines (HPEpiC) and prostate cancer cell lines (LNCaP, DU145 and PC-3).
FIG. 1B shows the changes in the expression level of HBD-2 mRNA after treatment of DNA methylation inhibitor with prostate cancer cell lines DU145 and PC-3.
2A is a schematic diagram of a CpG island (CGI) plot at the 5'-terminal region of the HBD-2 gene.
FIG. 2B is a schematic representation of bisulfite genomic sequencing results for CpG sites in prostate cancer cell lines LNCaP, DU145, PC-3, and DU145 and PC-3 cell lines treated with DNA methylation inhibitor DAC.
FIG. 2C shows the ratio (Obs _CpG / Exp _CpG , O / E) of the total amount (GC%) of the guanine plus cytosine base and the observed / predicted ratio of the CpG dinucleotide frequency at the 5 'end of the HBD-2 gene.
FIG. 3A shows the correlation between the methylation frequency of CpG 4 position in HBD-2 LCP site and the prostate cancer tissue classified by Gleason score (NT: normal tissue, T: tumor tissue).
FIG. 3B shows the correlation between the methylation frequency of the CpG 5 site in the HBD-2 LCP region and the prostate cancer tissue classified by the Gleason score (NT: normal tissue, T: tumor tissue).
FIG. 4A shows the correlation between the methylation frequency of CpG 4 position in HBP-2 LCP site and T stage-differentiated prostate cancer tissue (NT: normal tissue, T: tumor tissue).
FIG. 4B shows the correlation between the methylation frequency of CpG 5 position of HBP-2 LCP site and T stage-differentiated prostate cancer tissues (NT: normal tissue, T: tumor tissue).

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Prostate cancer sample tissue and cell culture

<1-1> Preparation of Clinical Samples for Prostate Cancer

 In the present invention, a total of 60 prostate cancer patient tissues were obtained (Korea Prostate Bank, Catholic University Seoul St. Mary's Hospital). Hematoxylin and eosin (H & E) were used to stain frozen sections of each patient's tissues obtained through radical prostatectomy (RP), and micro- -dissection). GDNA was extracted from the matched samples (tumor vs. normal tissue) of each patient by proteinase K treatment. All sample collections and analyzes were approved by the Clinical Trials Board of Chung-Ang University Hospital and Seoul St. Mary's Hospital and were approved by each patient.

<1-2> Cell culture

 Human prostate cancer cell lines LNCaP, DU145 and PC-3 were purchased from the American Type Culture Collection (Manassas, USA) and the human normal prostate cell line HPEpiC from ScienCell Research Laboratories (Carlsbad, USA). 10% fetal bovine serum (WelGENE, Korea) was added to F-12K medium (WelGENE, Korea) for PC-3 cell line and RPMI1640 (WelGENE, Korea) for LNCaP cell line, Eagle's Minimum Essential Medium And HPEpiC cell lines were cultured in Prostate Epithelial Cell Medium (ScienCell Research Laboratories, USA). All cell lines were maintained at 37 ° C in a 5% carbon dioxide (CO2) condition.

&Lt; Example 2 >

Expression Pattern of HBD-2 Gene in Prostate Cancer Epithelial Cell Line and Prostate Cancer Tissue

PCR was performed to determine whether the expression of HBD-2 mRNA was inhibited in the prostate cancer cell line. To determine whether the expression inhibition was due to DNA methylation, DNA methylation inhibitor was treated to express HBD-2 mRNA And the change in the shape of the nose.

DNA methyltransferase (DNMT) inhibitor 2'-deoxy-5-azacytidine (DAC) (Sigma-Aldrich, St. Louis, USA) was treated for 72 hours with prostate cancer cell line DU145 and PC-3.

Total RNA was extracted from prostate cancer cell line using RNeasy Mini Kit (Qiagen), and then reverse transcription polymerase chain reaction (RT-PCR) and qPCR (quantitative real-time PCR) were performed. First, for the RT-PCR, 1 μg of total RNA was synthesized using ImProm-II ™ Reverse Transcription System (Promega). The primer sequence for RT-PCR for HBD-2 gene was as follows; Forward primer, 5'-TGC CTC TTC CAG GTG TTT TTG GT-3 ', Reverse primer, 5'-ATG GCT TTT TGC AGC ATT TTG TTCCA-3'. Amplification conditions were 94 ° C for 3 minutes, followed by 33-35 cycles of 94 ° C for 30 seconds, 65 ° C for 30 seconds, and 72 ° C for 20 seconds, and finally 72 ° C for 7 minutes. All PCR reactions were performed using AccuPower PCR PreMix ). As a control, β-actin gene was amplified and used. For qPCR, cDNA was synthesized from 1 μg of total RNA using QuantiTect Reverse Transcription Kit (Qiagen), and 1 μl of the cDNA was used for amplification reaction using Rotor-Gene SYBR Green PCR Kit (Qiagen). PCR and quantitative analysis were performed using a Rotor-Gene Q 5 complex HRM system (Qiagen). The amplification conditions were 95 ° C for 5 minutes, 95 ° C for 5 seconds, and 60 ° C for 10 seconds. Were calibrated using the QuantiTect Primer Assay (Qiagen) of the β-actin gene.

The results are shown in Fig.

As shown in FIG. 1, it was confirmed that the expression of HBD-2 mRNA was inhibited in prostate cancer cell lines LNCaP, DU145 and PC-3 (FIG. 1A), and the expression of HBD-2 mRNA was inhibited in prostate cancer cell line We observed that HBD-2 expression was induced after treatment with DNA methyltransferase (DNMT) inhibitor 2'-deoxy-5-azacytidine (DAC) in DU145 and PC-3. (Fig. 1B). &Lt; tb &gt; &lt; TABLE &gt;

&Lt; Example 3 >

Identification of Promoter CpG Affecting Transcriptional Regulation of HBD-1 Gene

After confirming that the expression of mRNA and protein of HBD-2 was inhibited in the prostate cancer cell line and the prostate cancer clinical sample as in the case of Example 2, it was confirmed that CpG that is methylated in the promoter affecting the transcriptional regulation of HBD- Respectively.

Genomic DNA was extracted using DNeasy Tissue Kit (Qiagen), followed by bisulfite treatment with EZ DNA Methylation-Lightning ™ Kit (Zymo Research) for each 500 ng of prostate cancer cell line and 200 ng of prostate cancer patient tissue sample Respectively. The promoter region of the HBD-2 gene was amplified by dividing it into LCP1 (L1) (-646 to -235 bp from the transcription initiation site) and LCP2 (L2 (-1,158 to -692 bp from the transcription initiation site) The sequence is as follows; Reverse primer, 5'-TCC TAT CTT ACA CCA TTT AAA ACC C-3 ', GGA TAG ATT TGG GAG AGG AAG TGG, HBD-2 L2, forward primer, 5'-AGA TAT GGA TAT TGG GGT TTG TTT G-3 ', reverse primer, 5'-CAA AAT TCA CCC AAA CCT AAA CCAA-3'. The amplification conditions were 94 ° C for 10 minutes, followed by 45 to 50 cycles of 94 ° C for 30 seconds, 52 ° C for 30 seconds, and 72 ° C for 30 seconds, and finally, 72 ° C for 10 minutes. The PCR products were purified and then ligated into pGEM- T Easy Vector (Promega) for sequencing reaction. In the case of pyrosequencing analysis, bisulfite-treated prostate cancer patient tissue gDNA samples were PCR-amplified with the primer pairs of SEQ ID NO: 3 and SEQ ID NO: 4 and prepared as pyrosequencing reagents using PyroMark Gold Q96 Reagents (Qiagen) . Sequencing primers of SEQ ID NO: 2 on the PyroMark Q96 ID (Qiagen) platform were used to quantitatively analyze the% methylation of CpG 4 and CpG 5 sites in the HBD-2 L1 region for a total of 3 repetitions.

The results are shown in Fig.

As shown in FIG. 2A, after finding that the 5 'terminal region of HBD-2 is a low CpG-content promoter (LCP) without a typical CpG island, the 1 Kb region of the promoter is divided into LCP1 (L1) and LCP2 The methylation of the contained CpG dinucleotide was determined by the bisulfite genomic sequencing method (FIG. 2B). As a result, it was confirmed that CpG 4 and CpG 5 in the L1 region are important positions for HBD-2 transcriptional regulation in prostate cancer cell lines (highlighted by the red boxes in FIGS. 2B and 2C).

More specifically, FIG. 2A shows the CpG of the region corresponding to the 5 'terminal region of the HBD-2 gene, that is, the promoter and the 5'-untranslated region (5'-UTR) island (CGI) plot. The positions of LCP 1 (L1) and LCP 2 (L2) were amplified to amplify the promoter-1 kb region downstream of the CGI plot showing typical low CpG-content promoter (LCP)

As shown in Fig. 2B, prostate cancer cell lines LNCaP, DU145, PC-3, and gDNA extracted from PC14 and DM14 cells treated with the DNA methylation inhibitor DAC were used to express 6 and 10 Each of the boxes represents an individual CpG site, of which the white box is unmethylated and the black box is the methylated CpG representation of bisulfite genomic sequencing results for individual CpG sites. In addition, the number of boxes on the vertical axis was the number of clones used in the analysis, and approximately 10 to 12 clones were determined for methylation. In particular, in the results of the L1 region, the red line highlighted the CpG 4 and CpG 5 sites, which are in good agreement with the results of HBD-2 expression in the prostate cancer cell line identified in FIG.

FIG. 2C is a graph showing the ratio (Obs _CpG / Exp _CpG , O / E) of the total amount of the guanine plus cytosine base (GC%) and the observed / estimated ratio of the CpG dinucleotide frequency at the 5 'end of the HBD- As a result, it is concluded that this region lacks typical CpG island features. In the lower part, the sequence of 412 bp of L1 region examined by bisulfite sequencing analysis and the position of CpG 4 and CpG 5 sites emphasized by red box are shown.

<Example 4>

Assessment of methylation of HBD-2 gene promoter in prostate cancer patient tissue

<4-1> Clinical sample analysis of prostate cancer patients classified by Gleason score

The most important predictor of the status of prostate cancer inheritance is the Gleason-score, now named by the American pathologist Dr. Donald Gleason. With this score, a specially trained pathologist evaluates the tumor's aggressiveness or proliferation potential under a microscope.

 Gleason-score refers to the sum of the scores obtained by grading 1-5 grades (GP1-5, Gleason pattern1-5) in two places where the most part of the cancer is removed from the removed prostate. A grade of 1 indicates a near-normal differentiation, and the malignancy is low. However, if the score is 5, malignancy that does not show the shape of normal cells is the most severe cancer. Theoretically, the Gleason score (GS) combined with the two Gleason grades (GS) is 2-10, but recent trends are below 6 points (low malignancy), 7 points (intermediate malignancy), 8-10 points Malignancy) are classified into three broad categories.

In the present invention, the correlation between the methylation tendency of the HBsAg-2 gene promoter CpG and the clinical sample of the patient classified by Gleason-score was confirmed, and as a biomarker for diagnostic use of CpG 4 and CpG 5 in HBD-2 gene promoter Respectively.

The results are shown in Fig.

As shown in Fig. 3, the percent methylation differences between normal tissues and tumors were statistically significant at all histopathological stages of both CpG4 and CpG5 positions. Particularly, in the case of CpG 4 position, there was a good possibility of distinguishing between Gleason score ≤6 and 7 tumors (P = 0.003) and ≤6 and ≥8 tumors (P = 0.003) as diagnostic biomarkers 3A).

<4-2> Clinical sample analysis of patients with prostate cancer classified as T stage

The steady-state of the cancer allows you to see how specific a cancer is spread. Knowing what stage of cancer is at present is very important in determining the direction of treatment in the future.

Cancer can be assessed for its stage with the TNM stage system, where T evaluates the tumor itself, N for lymph nodes, and M for metastases. Among these, T stage in prostate cancer is divided into T1 to T4 stages. Specifically, T1 stage is a very small stage that can not be recognized by the patient and can not be confirmed by scanning. The T2 stage is a stage in which the cancer is completely contained within the prostate, and the T3 stage is a state in which the cancer is split into capsules that cover the prostate gland but have not yet metastasized to other organs. The T4 stage represents the spread of cancer to the tissues surrounding the prostate.

The inventors of the present invention have revealed the correlation between the status of T stage-classified prostate cancer and the methylation tendency of CpG 4 and CpG 5 in the HBD-2 promoter of clinical samples, and as a biomarker for diagnostic use of CpG 4 and CpG 5 Respectively.

The results are shown in Fig.

As shown in Fig. 4, the% methylation difference between the normal tissue and the tumor of the patient corresponding to the specific T stage at both the CpG 4 and CpG 5 positions was statistically significant, confirming a great possibility as a biomarker for diagnostic use . In addition, the results of T stage-classified prostate cancer patients showed a significant difference in non-tumor specificity between T3a and T3b (CpG 4, P = 0.021, CpG 5, P = 0.02) It can be expected that it can be used not only as a diagnostic biomarker but also as a field effect biomarker.

&Lt; Example 5 >

Comparison of methylation ratios of HBD-1 and HBD-2 promoters

The possibility of biomarkers for molecular diagnosis of prostate cancer patients was evaluated by using the combination of CpG position of HBD-1 promoter and CpG position of HBD-2 promoter.

(%) Of the two CpG positions (CpG 4 and CpG 5) of the proto-oncogene HBD-2 promoter, denominator% of the methylation of the two CpG positions (CpG 5 and CpG 6) of the HBD- The methylation rates of the four HBD-2 / HBD-1 promoters obtained by combining the measured values of the methylation values with the numerator were determined, and statistical analysis was performed to predict the combination of the most significant biomarkers.

As can be seen from the following Table 1, the ratio of HBD-2 CpG 4 / HBD-1 CpG 5 and the ratio of HBD-2 CpG 5 / HBD-1 CpG 5 were determined as NT (non-tumor) tissue samples The proportion of HBD-2 CpG 4 / HBD-1 CpG 5 was tumor-specific, and the Gleason score 3 + 3 tissue specimen showed a Gleason score of 7 (3) +4 & 4 + 3) and the Gleason score ≥8 tissue samples (p = 0.004). On the other hand, the ratio of HBD-2 CpG 5 / HBD-1 CpG 5 was normal (NT) -specific and Gleason score 3 + 3 vs. HBD- 4 + 3 and Gleason score 3 + 4 vs. (P = 0.004), the possibility of field effect biomarkers could be confirmed.

As shown in Table 2, the most statistically significant ratio is the ratio of HBD-2 CpG 4 / HBD-1 CpG 5 "and the ratio of HBD-2 CpG 5 / HBD-1 CpG 5 ratio. In particular, the ratio of T2 and T3b and T3a and T3b were significantly different between the two groups (HBD-2 CpG 4 / HBD-1 CpG 5, p = 0.002; HBD-2 CpG 5 / HBD -1 CpG 5, p = 0.003). Thus, the possibility of field effect biomarkers in patients with prostate cancer classified as T stage was confirmed again.

In conclusion, there have been many reports of high CpG-content promoters (HCPs), ie, GC-rich and CpG islands. However, the CpG 4 and CpG 5 positions in the LCP1 region of the HBD-2 gene, which we first identified, are specific methylation variable positions (MVPs) in the CpG-poor and non-CpG island promoters, It is a genetic counterpart. Therefore, the results of the% methylation of CpG 4 and CpG 5 in the HBV-2 promoter region, a new proto-oncogene, were added to the list of tumor suppressor genes for molecular diagnostics when PSA test and biopsy analysis were performed in patients who had been performed mainly in urology. Which is expected to complement the accuracy of tumor detection of prostate epithelial cells.

The methylation of CpG in the HBD-2 gene promoter affects the expression of HBD-2 in cancer cells and the methylation of HBD-2 gene is specifically expressed in cancer cells, particularly prostate cancer, And the detection of methylation of the HBD-2 gene can be used for diagnosis of cancer, which is highly likely to be used in industry.

<110> CHUNG ANG University industry Academic Cooperation Foundation <120> Method for dececting methylation of HBD-2 promoter and          composition for diagnosing cancer using therof <130> NP15-0019 <160> 15 <170> Kopatentin 2.0 <210> 1 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> HBD-2 promoter sequence <400> 1 atggatagaa ttagatggaa agagttttta atttggtttg agattgtttt tagatattta 60 ggaaaaatag gaygtygtat agagtgggta gtaggtgagt 100 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 2 cctactaccc actctatac 19 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> pyrosequencing primer forward <400> 3 atggatagaa ttagatggaa agagtt 26 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> pyrosequencing primer reverse <400> 4 actcacctac tacccactct at 22 <210> 5 <211> 154 <212> DNA <213> Artificial Sequence <220> <223> HBD-1 promoter sequence <400> 5 tttttgtaag ggaagagggt gaagtttgag tttgttttgt aggaagataa ttaaattaaa 60 gaggttaata ttagtttaga gtygagyggt tttttgttta gagttttttt gtggtttttt 120 tttatgtgat ttagaaggag ggattttagt gtga 154 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HBD-1 promoter pyrosequencing primer forward <400> 6 tttttgtaag ggaagagggt gaag 24 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> HBD-1 promoter pyrosequencing primer reverse [biotin] <400> 7 tcacactaaa atccctcctt ctaaatcac 29 <210> 8 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> HBD-2 Unmethylated CpG 4 and unmethylated CpG 5 <400> 8 tgttg 5 <210> 9 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> HBD-2 Methylated CpG 4 and unmethylated CpG 5 <400> 9 cGTTG 5 <210> 10 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> HBD-2 Unmethylated CpG 4 & methylated CpG 5 <400> 10 tgtcg 5 <210> 11 <211> 5 <212> DNA <213> Artificial Sequence <220> <223> HBD-2 Methylated CpG 4 & methylated CpG 5 <400> 11 cgtcg 5 <210> 12 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> HBD-1 Unmethylated CpG 5 and unmethylated CpG 5 <400> 12 tgagtg 6 <210> 13 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> HBD-1 Methylated CpG 5 and unmethylated CpG 6 <400> 13 cgagtg 6 <210> 14 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> HBD-1 Unmethylated CpG 5 & methylated CpG 6 <400> 14 tgagcg 6 <210> 15 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> HBD-1 Methylated CpG 5 & methylated CpG 6 <400> 15 cgagcg 6

Claims (12)

Among the CpG bases consisting of cytosine at -361 bp and cytosine at -358 bp from the transcription initiation site (TSS, +1) of HBD-2 (human beta defensin-2) gene from clinical samples for the diagnosis of prostate cancer, Methylation of one or more selected cytosine positions.
2. The method according to claim 1, wherein the HBD-2 (human beta defensin-2) gene promoter has the DNA sequence of SEQ ID NO: 1.
The method according to claim 1, wherein the methylation is selected from the group consisting of PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, methylation using methylation sensitive restriction enzyme A DNA chip, a pyrosequencing, and a bisulfite sequencing.
The method according to claim 1, further comprising detecting whether the human beta defensin-1 (HBD-1) gene promoter is CpG methylated from the clinical sample.
(a) the cytosine at the -361 bp position and the cytosine at the -358 bp position from the transcription initiation site (TSS, +1) of HBD-2 (human beta defensin-2) gene and the CpG methylation of the HBD-1 promoter Detecting the methylation of one or more cytosine sites selected from CpG bases; And
(b) comparing the methylation of CpG of the HBD-1 promoter with the methylation ratio of cytosine in the HBD-2 promoter.
Methylation of one or more cytosine sites selected from CpG bases consisting of cytosine at -361 bp position and cytosine at -358 bp position from the transcription initiation site (TSS, +1) of HBD-2 (human beta defensin- A composition for diagnosing prostate cancer, comprising a detectable substance.
The composition for diagnosing prostate cancer according to claim 6, further comprising a substance capable of detecting whether or not methylation of the HBD-1 gene promoter CpG is detected.
Methylated cytosine at one or more cytosine positions selected from CpG bases consisting of cytosine at -361 bp position and cytosine at -358 bp position from the transcription initiation point (TSS, +1) of HBD-2 (human beta defensin- And a primer pair for amplifying a fragment comprising the primer.
9. The kit for diagnosing prostate cancer according to claim 8, wherein the primer pair is a sequence represented by SEQ ID NOS: 3 and 4.
9. The kit for diagnosing prostate cancer according to claim 8, further comprising a pair of primers for amplifying a fragment containing methylated CpG of the HBD-1 gene promoter.
Methylated cytosine at one or more cytosine positions selected from CpG bases consisting of cytosine at -361 bp position and cytosine at -358 bp position from the transcription initiation point (TSS, +1) of HBD-2 (human beta defensin- And a probe capable of hybridizing with the fragment containing the probe.
12. The prostate cancer diagnostic chip according to claim 11, further comprising a probe capable of hybridization with a fragment containing the methylation site of the HBD-1 gene promoter.

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