JP2005185143A - Prostatic cancer, method for determining its risk or its early contraction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a medicine for diagnosing the contraction risk of prostatic cancer by finding a DNA methylated domain having high specificity for prostatic cancer patients and low specificity for prostatic hypertrophy patients and then measuring the methylation frequency of the DNA. <P>SOLUTION: The subject method for determining the prostatic cancer, its risk or its early contraction comprises measuring a methylation frequency in one or more base sequences selected from base sequences represented by sequence number 1, sequence number 2, and sequence number 3 in the gene of a specimen separated from a living body, or an index value having a relative relation to the methylation frequency. The medicine for diagnosing the prostatic cancer, its risk or its early contraction contains a primer capable of examining a methylation frequency in one or more base sequences selected from base sequences represented by the sequence number 1, the sequence number 2, and the sequence number 3. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for determining prostate cancer, its risk, or its early morbidity, and a diagnostic agent.

  Prostate cancer is a cancer that is sensitive to androgen, a male hormone, and is generally older and has a relatively mild growth rate. On the other hand, if the discovery is delayed, it can also metastasize to the whole body including bone marrow metastasis, resulting in death of the patient. Elderly people also deteriorate their quality of life (QOL) in terms of physical and psychology, such as urinary excretion after treatment.

  In Europe and the United States, prostate cancer accounts for the first to third ranks of male malignancies in terms of both incidence and mortality, and is the most diagnosed cancer among American men. It also accounts for about 20% of cancer mortality in the West. On the other hand, the mortality rate of prostate cancer in Japan has been relatively low, about 3.5%. However, in recent years, the incidence of prostate cancer has been rapidly increasing against the background of the westernization of eating habits and an aging society.

  Three types of diagnostic methods are used to diagnose prostate cancer: digital rectal examination, transrectal ultrasonography, and measurement of prostate specific antibody (PSA) in the blood. After that, a method has been used in which a definitive diagnosis is made by pathological diagnosis of tissue collected by biopsy.

  In addition, since prostate cancer is a hormone-dependent cancer, the most basic treatments are castration for the removal of androgen (bilateral orchiectomy) and sustained release of agonists against luteinizing hormone-releasing hormone from the pituitary gland. Administration of an agent (androgen removal method). At present, in addition to these androgen removal methods, maximum androgen blockade (MAB) therapy using an antiandrogen agent for the purpose of removing residual androgen from the adrenal gland is being actively performed. However, these hormonal therapies have a heavy burden on patients such as loss of male function and side effects due to androgen removal. In addition, since surgical operations such as castration require 5 to 6 hours for general anesthesia, patients require considerable physical strength and are burdensome for the elderly. In addition, if cancer is confined to the prostate, surgical treatment and local treatment such as radiotherapy are also performed.

  Furthermore, with the malignant transformation of cancer cells, androgen is no longer necessary for their growth, which has the detrimental effect that hormone therapy is not effective. In such cases, hormones such as ethylestradiol, ifosamide (IFO; alkylating agent), tegafur uracil (UFT; antimetabolite), peplomycin sulfate (PEP; anticancer antibiotic) Chemotherapy using cisplatin (CDP; platinum antineoplastic agent), etc. is performed, but not only the side effects are serious, but the duration of the effect is short and the response cannot be expected. ing. Therefore, early diagnosis and early detection of prostate carcinogenesis is important in mass screening and the like.

  However, the diagnosis of benign prostatic hyperplasia, which is a disease in the same tissue, is also performed by a combination of confirmation of subjective symptoms by inquiry, measurement of mean urine flow rate, palpation, or measurement of prostate size by ultrasound, etc. In recent years, prostatic hypertrophy has been differentiated using a value obtained by dividing the amount of PSA by the size of the prostate and the PSA density as an index. Therefore, the conventional method for diagnosing prostate cancer has a problem that prostatic hypertrophy is also judged positive. In addition, the occurrence of false-positive judgments is unavoidable, and when there is a suspicion of developing prostate cancer, the final diagnosis was made by pathological diagnosis of the tissue collected by biopsy, but there is also a problem that the burden on the patient is heavy. It was. Furthermore, it has been pointed out that these diagnostic methods may also miss early cancers without tissue hypertrophy.

In this way, patients with prostate cancer and benign prostatic hyperplasia are forced to undergo treatment that places a heavy burden on the body, such as loss of male function, and in order to improve the burden, detection as early as possible is necessary. It was. However, in any of the conventional methods, it is impossible to make a definitive diagnosis unless the prostate tissue becomes large to some extent, and diagnosis in the initial stage is impossible.
The inventors have already reported a method for diagnosing morbidity risk by identifying a polymorphism of the HPC2 / ELAC2 gene as a diagnostic method for detecting morbidity at an early stage or diagnosing the morbidity risk (patent application). 2002-334941), the diagnostic method is also not sufficient as a method for specifically diagnosing prostate cancer because it positively determines prostatic hypertrophy. Therefore, there has been a demand for a diagnostic method that can be carried out by mass screening, has high specificity for prostate cancer, and is useful for early detection.

  As a result of intensive studies to solve the above problems, the present inventors have found a DNA methylation region that is highly specific for prostate cancer patients and low in specificity for prostatic hypertrophy patients. Further, the present inventors have found that the risk of prostate cancer can be diagnosed before the onset of disease by measuring the methylation frequency of the DNA, thereby completing the present invention.

That is, the present invention relates to SEQ ID NO: 1 (hereinafter sometimes referred to as “MFPC3”), SEQ ID NO: 2 (hereinafter sometimes referred to as MFPC68), and SEQ ID NO: 3 (hereinafter also referred to as “MFPC68”) in a gene isolated from a living body. (Hereinafter, also referred to as MFPC138). A prostate that measures a methylation frequency or an index value correlated with the methylation frequency in one or more base sequences selected from the base sequences represented by A method for determining cancer, its risk, or its initial morbidity is provided.
The present invention also includes a primer capable of examining the methylation frequency in one or more base sequences selected from the base sequences represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. It provides a diagnostic agent for prostate cancer, its risk or its early morbidity.

  Since the methylation of SEQ ID NOs: 1 to 3 of the present invention has not been found in cancer, and the methylation state is completely different from that of prostatic hypertrophy, it is a genetic modification specific to prostate cancer. Therefore, by using the methylation analysis of SEQ ID NOs: 1 to 3 of the present invention, it is possible to easily diagnose prostate cancer that does not include prostatic hypertrophy, its risk of morbidity, and its initial morbidity that could not be diagnosed by a known method. I can do it.

  In mammals, there is a phenomenon in which only cytosine is methylated out of four types of bases constituting a gene (genomic DNA). The DNA methylation modification is performed in a base sequence represented by 5′-CG-3 ′ (C represents cytosine and G represents guanine. Hereinafter, the base sequence may be referred to as CpG). Limited to cytosine. The site that is methylated in cytosine is at position 5. During DNA replication prior to cell division, only cytosine in CpG of the template strand is methylated immediately after replication, but cytosine in CpG of the nascent strand is also methylated immediately by the action of methyltransferase. . Therefore, the DNA methylation state is inherited as it is by two new sets of DNA even after DNA replication.

  When the methylation frequency in the base sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 is high in the gene in the sample used in the present invention, the subject having the gene has a risk of developing prostate cancer patients It can be determined that the patient or an early affected patient. High expression of the methylation frequency is not observed in patients with benign prostatic hyperplasia.

  The determination method of the present invention can be performed using the methylation frequency of cytosine in one or more base sequences represented by 5′-CG-3 ′ among these base sequences as an index, and in particular, methylated cytosine. It is preferable to use as an index the cytosine methylation frequency in a region where Cd is dense (CpG island). Such a CpG island is represented by nucleotide numbers 3173 to 3844 in SEQ ID NO: 1, nucleotide numbers 36768 to 37234 in SEQ ID NO: 2, and nucleotide numbers 109344 to 109768 in SEQ ID NO: 3.

  The methylation frequency is represented by the ratio of haploids in which the cytosine is methylated when the presence or absence of methylation of cytosine in CpG to be investigated is examined for a plurality of haploids. The index value correlated with the methylation frequency is, for example, the amount of the gene expression product of the nucleotide sequence represented by SEQ ID NO: 1 to 3, more specifically, the amount of the transcription product of the gene, Examples include the amount of translation product of the gene. In the case of the amount of such an expression product, there is a negative correlation that decreases as the methylation frequency increases.

  The sample used in the present invention is a sample separated from a living body and may be a sample containing DNA. For example, cancer cells such as prostate cancer cells or tissues containing the same, and cancer cells such as prostate cancer cells Cells that may contain DNA derived from them, tissues containing them (the tissues here have a broad meaning including body fluids such as blood, plasma, serum, and lymph, lymph nodes, etc.) or bodies. Biological samples such as secretions (urine, milk, etc.) can be mentioned. These biological samples may be used as specimens as they are, or DNA-containing fractions prepared by various operations such as separation, fractionation, and immobilization from such biological samples may be used as samples. When the sample is blood, it can be expected to use the determination method of the present invention in periodic health examinations and simple tests. In this case, in order to use it effectively while keeping the false determination rate low, it is under 55 years old. Human blood is preferable.

  DNA is extracted from these samples using, for example, a commercially available DNA extraction kit. Incidentally, when blood is used as a specimen, plasma or serum is prepared from blood according to a normal method, and the prepared plasma or serum is used as a specimen for free DNA (DNA derived from prostate cancer cells). Analysis), DNA derived from prostate cancer cells can be analyzed while avoiding DNA derived from blood cells, cancer cells, tissues containing the same can be detected, and sensitivity can be improved. Then, after the extracted DNA is contacted with a reagent that modifies unmethylated cytosine, one or more CpGs present in the promoter sequence of the base sequence represented by SEQ ID NOs: 1 to 3 or the base sequence of the coding region Amplification of DNA containing cytosine in the base sequence shown by the polymerase chain reaction (hereinafter referred to as PCR) using primers capable of discriminating the presence or absence of cytosine methylation to be analyzed. Check the amount. Here, as the base sequence represented by one or more CpGs present in the base sequence of the promoter sequence or coding region of the base sequence represented by SEQ ID NO: 1 to 3, for example, the base represented by SEQ ID NO: 1 to 3 The base sequence of the genomic DNA containing the exon region of the sequence and the promoter region located 5 ′ upstream thereof can be mentioned. In particular, base numbers 3173 to 3844 in SEQ ID NO: 1 and base numbers in SEQ ID NO: 2 36768 to 37234, and base numbers 109344 to 109768 in SEQ ID NO: 3 can be mentioned. Cytosine in CpG present in a region where these CpGs are densely present exhibits a high methylation frequency (ie, hypermethylation) in cancer cells.

  As a reagent for modifying unmethylated cytosine, for example, bisulfite such as sodium bisulfite can be used.

  In order to contact the extracted DNA with a reagent that modifies unmethylated cytosine, for example, the DNA is first bisulphite such as sodium bisulfite (concentration in the solution) in an alkaline solution (pH 9 to 14). : For example, the final concentration is 3 M) and the like, and the treatment is performed at 55 ° C. for about 10 to 16 hours. In this case, unmethylated cytosine is converted to uracil, while methylated cytosine is not converted to uracil and remains cytosine. Next, when DNA treated with bisulfite or the like is used as a template and methylated cytosine is included, the base sequence [cytosine at the methylated position (cytosine in CpG) remains cytosine, PCR using a pair of methylation-specific primers each selected from a non-methylated cytosine (a cytosine not included in CpG is a uracil base sequence) and a base sequence complementary to the base sequence , And may also be referred to as methylation-specific PCR.) And nucleotide sequences when DNA treated with bisulfite or the like is used as a template and cytosine is not methylated (all cytosines became uracil) PCR using a pair of unmethylated specific primers each selected from a base sequence) and a base sequence complementary to such base sequence (hereinafter also referred to as unmethylated specific PCR) Succoth is also there.) And perform. In the above PCR, in the case of PCR using methylation specific primers (the former), DNA in which cytosine to be analyzed is methylated is amplified, while in the case of PCR using unmethylation specific primers ( In the latter case, DNA in which cytosine to be analyzed is not methylated is amplified. By comparing the amounts of these amplified products, the presence or absence of methylation of the target cytosine is examined. In this way, the methylation frequency can be measured.

  Here, the methylation-specific primer is a cytosine that has undergone methylation in consideration that cytosine that has not been methylated is converted to uracil, and that cytosine that has undergone methylation is not converted to uracil. Design PCR primers (methylation specific primers) specific to nucleotide sequences containing, and PCR primers (non-methylation specific primers) specific to nucleotide sequences containing unmethylated cytosine To do. Since it is designed based on DNA strands that have been chemically converted by bisulfite treatment and are no longer complementary, based on each strand of DNA that was originally double-stranded, a methylation-specific primer and Unmethylated specific primers can also be made. Such a primer is preferably designed to contain cytosine in CpG in the vicinity of the 3 ′ end of the primer in order to increase the specificity of methyl and non-methyl. Further, in order to facilitate analysis, one of the primers may be labeled.

  Among such primers, examples of unmethylated specific primers are shown in Table 1.

  As an example of a methylation specific primer, the primer in case Y is C or R is G in the primer of Table 3 mentioned later is mentioned.

As a reaction solution in methylation-specific PCR, for example, 20 ng of DNA as a template, 1 μl of each primer solution of 30 pmol / μl, 3 μl of 2 mM dNTP, 10 × buffer (100 mM Tris-HCl pH 8. 3, 500 mM KCl, 20 mM MgCl 2) and 3 μl of heat-resistant DNA polymerase 5 U / μl are mixed with 0.2 μl, and sterilized ultrapure water is added thereto to make the reaction volume 30 μl. As the reaction conditions, for example, the above reaction solution is kept at 94 ° C. for 10 minutes, then at 94 ° C. for 30 seconds, then at 55 to 65 ° C. for 60 seconds, and further at 72 ° C. for 45 seconds for one cycle. The conditions for carrying out 40 cycles of heat insulation are mentioned. After performing such PCR, the amount of amplification product obtained is compared. For example, an analytical method (denaturing polyacrylamide gel electrophoresis or agarose gel) that can compare the amount of each amplification product obtained by PCR using a methylation specific primer and PCR using an unmethylated specific primer. In the case of electrophoresis, the gel after electrophoresis is stained with DNA to detect the band of the amplification product, and the concentration of the detected band is compared. Here, instead of DNA staining, a pre-labeled primer can be used to compare the band concentrations using the label as an index.
Such a method is generally called methylation-specific PCR, and is a method reported by Herman et al. (Herman et al., Proc. Natl. Acad. Sci USA, 93, 9821-9826, 1996). Thus, this method utilizes the difference in chemical properties between cytosine and 5-methylcytosine.

  On the other hand, regardless of whether it is abnormal or normal, first amplification is performed by PCR, then the amplified sequence is cleaved with a restriction enzyme, and diagnosis can be made based on whether the bands obtained by electrophoresis remain one or two. it can. When there is methylated cytosine, it is divided into two. In the case of this method, PCR may be performed by mixing an unmethylated specific primer and a methylated specific primer and / or a primer having Y in T and R in A in Table 3. After amplifying all the genes, the amplified product may be cleaved with a restriction enzyme to confirm the position of the band.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.

Example 1 Preparation of DNA
As a DNA extraction source, prostate cells, biopsy tissue, or surgically appropriate tissue removed from serum or prostate tissue and mixed in urine are used. That is, proteinase K was added to the collected cells or tissue pieces to a final concentration of 500 μg / ml and sodium dodecyl sulfate (SDS) to 0.5%, and the mixture was left stirring at 54 ° C. The same amount of proteinase K was added every 24 hours, and digestion was carried out until the tissue pieces were completely digested and the turbidity of the liquid disappeared. An equal amount of phenol was added to the obtained digestive juice and stirred, followed by centrifugation at 15,000 rpm for 5 minutes. After centrifugation, the uppermost layer separated into three layers was removed, and an equivalent amount of a phenol / chloroform (1: 1) mixed solution was added to the remaining digested solution, and the same operation was performed. After centrifugation, the top layer was similarly removed, and the same operation was performed by adding an equal amount of chloroform to the remaining digestive juice. After centrifugation, the uppermost layer was similarly removed, and then 2.2 times 100% ethanol and 0.1 times 3M sodium acetate (pH 5.2) were added to the remaining digested solution. The mixture was gently stirred and allowed to stand at −20 ° C. for 2 hours or longer, and then centrifuged at 4 ° C. and 15,000 rpm for 20 minutes. After centrifugation, the supernatant was discarded and air-dried while removing residual ethanol to obtain DNA.

Example 2 Identification of methylated CpG site The DNA used in the test was prepared in Example 1, and the CpG site in the DNA was identified using MCA / RDA (methylated CpG island amplification-representational difference analysis) method. did. The MCA-RDA method was performed according to a previous report (Toyota M, et al. Cancer Res., 59. 2307 (1999)). That is, 5 μg of DNA was digested in the order of 100 units of SmaI and 20 units of XmaI (each restriction enzyme was purchased from New England Biolabs). After treatment, the methylated DNA site becomes a sticky end. An RMCA adapter (universal adapter) was bound to the restriction enzyme-treated DNA fragment using T4 DNA ligase (New England Biolabs), and PCR was performed with primers corresponding to the adapter (PCR condition: 3 μL RMCA adapter) 100 μL of reaction mixture (10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl ₂ , 50 mM KCl, 0.5 M betaine, 2% DMSO, 200 μM each dNTP mixture, 100 pmol RMCA, 24 mer primer , 15 units Taq polymerase) (PCR cycle: 72 ° C, 5 minutes, then 95 ° C, 3 minutes, then 95 ° C, 1 minute, 95 ° C, 1 minute, then 77 ° C) , 3 minutes 25 cycles, and finally the reaction at 77 ° C for 10 minutes)). Table 2 shows MCA amplification sequences derived from prostate cancer cell line DU145, RDA (analysis of expressed differences) as testers, and MCA (methylated CpG island amplification products) total PCR products derived from 3 independent cases of prostate hypertrophy The result of MCA-RDA analysis when using amplicon (called amplicon) as an RDA driver is shown.

  As described in Table 2, a total of 8 clones were isolated and identified. Criterion for deterministic determination of CpG islands (concentrated CpG sites) (length,> 200bp; GC content,> 50%; CpG score;> 0.6, Gradiner GM, and Frommer MJ Mol. Biol., 196, 261-282 (1987) ), The regions in MFPC3, Flamingo1, MFPC68, and MFPC138 among the isolated CpG sites were identified as CpG islands. Using these MCA amplicons, RDA was performed three more times in the order of JMCA, NMCA, and JMCA adapter. The amplified RDA product was cloned into the pBluescriptII plasmid vector (Stratagene). The cloned DNA fragment was sequenced by a conventional method, and the position on the genome was identified using the BLAST database (FIG. 1). In FIG. 1, the CpG sequences of MFPC3, MFPC68, and MFPC138 identified in Table 2 are indicated by vertical bars, and the regions isolated by MCA-RDA analysis and the regions analyzed by subsequent analysis are indicated by arrows. .

Example 3 Analysis of DNA methylation status in prostate tissue of identified CpG island (1: Bisulfite sequence method)
When single-stranded DNA is treated with bisulfite (sodium bisulfite, bisulfite), sulfonation and hydroamination reactions occur. Subsequent desulfonation converts cytosine to uracil. On the other hand, in methylated cytosine, the reaction rate of sulfonation is so slow that uracil is not converted and remains methylated cytosine, but unmethylated cytosine is replaced by thymine (Frommer M, et al, Proc. Natl. Acad. Sci. USA, 891827-1831, (1992). Clark SJ, et al. Nucleic Acids Res., 22, 2990 (1994)). Using this sequence difference (C and T), the methylation state can be analyzed, and this method is called a bisulfite treatment method. More specific experimental methods using bisulfite treatment include the bisulfite sequencing method (Clark SJ, et al. Nucleic Acids Res., 22, 2990 (1994)) and the combined bisulfite restriction analysis (COBRA) method (Xiong Z & Laird PW. COBRA: Nucleic Acids Res., 25, 2532 (1997)) for the first time, there are several experimental methods, and it is possible to select an experimental method suitable for the purpose of analysis.

  Bisulfite treatment is performed as follows. That is, 2 μg of chromosomal DNA is denatured in 0.3 M NaOH at 37 ° C. for 15 minutes. Subsequently, sodium bisulfite is added to a final concentration of 3.1 M, and hydroquinone is added to a final concentration of 0.5 mM. React at 55 ° C for 16 hours. The reaction solution is desalted using Wizard DNA purification (Promega, Medison, Wisconsin). The bisulfite treatment is completed by reaction in 0.3 M NaOH at 37 ° C for 15 minutes. The modified DNA is dried after ethanol precipitation and subsequent washing with 70% ethanol, and finally dissolved in 50 μL of distilled water.

  Table 3 shows the experimental conditions when the methylation states of MFPC3, Flamingo1, MFPC68, and MFPC138 were analyzed by the bisulfite sequence method. That is, 2 μL of bisulfite-treated DNA derived from three types of prostate cancer cell lines (LNCaP, DU145, PC-3) and bisulfite-treated from the three independent prostate hypertrophy tissues used in Table 2 2 μL of the obtained DNA was subjected to PCR amplification under the conditions shown in Table 3. PCR products were subcloned into TOPO TA creening vectors (Invitrogen, San Diego, California), and 10 clones for each product were sequenced by the Big Dye terminator method. The average level of methylation at each CpG site was categorized into 5 levels: 0-19%, 20-39%, 40-59%, 60-79%, 80-100% (Figures 2 and 3 (Figure 3 Is a continuation of FIG. 2)).

  2 and 3, two CpG islands were identified in MFPC3, and a total of five CpG islands were analyzed. MFPC3 was hypermethylated in all prostate cancer cell lines on both CpG islands and hypomethylated in benign prostatic hyperplasia tissue. MFPC68 was methylated in all prostate cancer cell lines and was not methylated in benign prostatic hyperplasia tissue. MFPC138 was only methylated in the DU145 cell line and not methylated in other prostate cancer cell lines and benign prostatic hyperplasia. Flamingo1 was methylated in all prostate cancer cell lines and all benign prostatic hyperplasia tissues. MFPC3, MFPC68, and MFPC138 were considered to be methylated specifically in cancer tissues. Flamingo1 may be methylated not only in prostate cancer but also in prostate disease. Hereinafter, MFPC3, MFPC68, and MFPC138 were used as target regions for cancer-specific DNA modification.

Example 4 Analysis of DNA methylation status in prostate cancer tissue of identified CpG island (2: COBRA method)
Analysis of the methylation status of MFPC3, MFPC68, and MFPC138 in prostate cancer tissues was analyzed using the COBRA method, which can more easily detect DNA methylation. In addition, the methylation status of the same region was compared with prostate cancer cell lines and benign prostatic hyperplasia tissue. That is, DNA subjected to bisulfite treatment was PCR amplified using the same primers and PCR conditions as in the bisulfite sequencing method. The PCR products were digested with the following restriction enzymes (New England Biolabs.) Under the reaction conditions: MFPC3 and MFPC68 were TaqI at 65 ° C. for 4 hours, and MFPC138 was RsaI at 37 ° C. for 4 hours. The digested DNA fragment was electrophoresed on 4% agarose gel (Seakem GTG agarose gels. BMA, Rockland, ME) and then stained with ethidium bromide. If digestion of the DNA fragment is detected, it can be determined that the DNA has been methylated (FIG. 4). From FIG. 4, it was found that the presence or absence of DNA methylation can be easily visualized and can be easily and clearly determined without false positives.

  The results of FIG. 4 are summarized in Table 4. As a result, MFPC3, MFPC68, and MFPC138 were specifically methylated in cancer tissues. In benign prostatic tissue, methylation was not detected in MFPC68 and MFPC138, and methylation was detected in MFPC3 only in one case. In prostate cancer cell lines, methylation was detected in all of MFPC3 and MFPC68, and methylation was detected in one cell line in MFPC138. It was shown that detection by the combination of the three regions increases the sensitivity.

Example 5 Confirmation of re-expression of MFPC3 gene not expressed by methylation by demethylation treatment
We examined whether silencing (inactivation or non-expression) of the MFPC3 gene depends on methylation in the upstream region of the gene by demethylation treatment for prostate cancer cell lines not expressing the MFPC3 gene . That is, the recovery of MFPC3 gene expression by demethylation treatment was confirmed. Prostate cancer cell lines (LNCaP, DU145, PC-3) were treated with 5-aza-2′-deoxycytidine (Sigma Chemical Co., St. Louis, Missouri) alone or in combination with trichostatin A (Sigma Chemical Co.). Total RNA was extracted with isogen (Nippon Gene, Tokyo, Japan). CDNA was prepared from 1 μg of total RNA using Superscript Reverse transcriptase (GIBCO BRL, Gaithersburg, Maryland). The MFPC3 gene was amplified by RT-PCR. The control was GAPDH gene, and PCR conditions were as follows: forward primer; AGT CCA GAC ACT CAG GAT TTG TAC, reverse primer, CTC CGT GTG ATT GGT AAC ATG. Amplification condition: 95 ° C 30 seconds, 64 ° C 30 seconds, 30 cycles of 72 ° C for 1 minute. PCR products were electrophoresed on a 2.0% agarose gel and stained with ethidium bromide to confirm the presence or absence of amplification (FIG. 5). As a result, re-expression of the MFPC3 gene in LNCaP and DU145 was confirmed by the demethylation treatment, and the degree thereof was enhanced depending on the increase in 5-aza-2′-deoxycytidine concentration. The expression level of MFPC3 gene in PC-3 was not changed by demethylation treatment.

FIG. 3 is a diagram showing a map of CpG islands on RDA products. It is a figure which shows the result of a bisulfite sequence. It is a figure which shows the result of a bisulfite sequence (continuation of FIG. 2). It is a figure which shows the analysis result (agarose gel electrophoresis image) of COBRA method. It is a figure which shows the confirmation result of the reexpression by the demethylation process of MFPC3 gene.

Claims (4)

  There is a correlation with the methylation frequency or the methylation frequency in one or more base sequences selected from the base sequences represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in genes in a sample isolated from a living body A method for determining prostate cancer, its risk, or its initial morbidity, characterized by measuring a certain index value.   The determination method according to claim 1, wherein the methylation frequency is a methylation frequency of cytosine in a region (CpG island) where methylated cytosines are densely packed.   The CpG island of SEQ ID NO: 1 is the region of base numbers 3173-3844, the CpG island of SEQ ID NO: 2 is the region of SEQ ID NOs: 36768-37234, and the CpG island of SEQ ID NO: 3 is the region of SEQ ID NOs: 109344-109768 The determination method according to claim 2.   A prostate cancer comprising a primer capable of detecting a methylation frequency in one or more base sequences selected from the base sequences represented by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, A diagnostic for its initial morbidity.

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