KR101384326B1 - Composition for diagnosing metastatic characteristics of ovarian cancer using cpg methylation status of ntn4 gene and uses thereof - Google Patents
Composition for diagnosing metastatic characteristics of ovarian cancer using cpg methylation status of ntn4 gene and uses thereof Download PDFInfo
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Abstract
The present invention relates to compositions, kits and methods for diagnosing ovarian cancer metastasis or predicting metastasis risk by detecting the methylation level of the CpG region of the NTN4 (netrin 4) gene.
Description
The present invention relates to compositions, kits and methods for diagnosing ovarian cancer metastasis or predicting metastasis risk by detecting the methylation level of the CpG region of the NTN4 (netrin 4) gene.
Ovarian cancer is an incurable cancer disease with the highest mortality among female cancers. The frequency of ovarian cancer has been increasing steadily as western lifestyle, westernized hormone replacement therapy, and elderly population increase. In the case of ovarian cancer, there is no clear symptom in the early stage, so about 70% or more patients are first detected as advanced stage ovarian cancer with at least 3 stages.
The method of treating ovarian cancer is determined according to the type and stage of the cancer, and there are surgical treatment, radiation therapy, and combination therapy, but it is not very effective in treating metastatic cancer caused by relapse. This is because the cancer metastasis involves the induction of the formation of new blood vessels and the movement of cells, and thus the phenomenon differs from that of cancer itself. In order to prevent metastasis of cancer, induction of new blood vessels and cell migration must be inhibited. The effect and the anticancer effect are different from each other. Therefore, although the diagnosis of cancer is important, the development of a biomarker that can determine the recurrence and metastasis after the treatment of ovarian cancer is expected to contribute greatly to increase the survival rate and treatment efficiency. In addition, since cancer recurrence and metastasis are fundamentally different from the onset of cancer, it is necessary to develop biomarkers different from biomarkers for diagnosing cancer in order to predict cancer recurrence and metastasis.
More specifically, metastatic cancers have been reported to differ in biological properties from early cancers produced at the primary site. This is because gene expression of metastatic cancer differs from that of cancer in the primary site. For example, various growth hormones are required for tumor cells to grow, and surviving the effects of anticancer drugs, ultimately metastatic cancer cells must induce changes in gene expression to favor survival. This pattern of expression appears to play a very important role in determining cancer metastasis. Therefore, it is difficult to say that metastasis is caused by increased expression of one of the genes involved in tumor cells (primary site).
In this aspect, the present invention compared gene expression patterns between primary cancer cells and metastasized tissues, and it was confirmed that the methylation change of the CpG region among the genes showing changes in gene expression in metastasized tissues affected gene expression. Genes were finally screened. Furthermore, specific CpG sites that influenced the expression of the gene were identified, and the present invention was completed by confirming that the risk of metastatic ovarian cancer can be predicted by measuring the degree of methylation occurring at a specific CpG position of the gene.
On the other hand, domestic patent registration No. 1169127, Japanese Patent Publication No. 2010-178650, US Patent Publication No. 2010-0279301 discloses that the genes selected in the present invention can be used as a marker for diagnosing the development of various cancers. Compared to the above documents, the present invention has a significant difference in that it diagnoses or predicts the recurrence and metastasis of ovarian cancer using specific hypermethylation of a specific CpG region of the gene.
One object of the present invention is an agent for measuring the methylation level of CpG sites of at least one gene selected from the group consisting of ADAM12 (a disintegrin and metalloproteinase 12), NTN4 (netrin 4) and PTGS2 (prostaglandin-endoperoxide synthase 2) It includes, to provide a composition for diagnosing or predicting metastasis risk of ovarian cancer.
Another object of the present invention to provide a kit for diagnosing or predicting metastasis risk of ovarian cancer comprising the composition.
It is another object of the present invention to provide a method of diagnosing metastasis of ovarian cancer or predicting the risk of metastasis by measuring the methylation level of the CpG region of the gene.
Another object of the present invention is to provide information for diagnosing metastasis of ovarian cancer or predicting the risk of metastasis, comprising measuring the methylation level at the CpG site of the gene from a biological sample of a patient suspected of metastatic ovarian cancer. It is to provide a way.
The present invention is based on the discovery that specific CpG sites of the ADAM12, NTN4 and PTGS2 genes are specifically hypermethylated in ovarian cancer metastatic tissues. Provide techniques to predict the risk of metastasis.
Since the CpG of the ADAM12, NTN4, and PTGS2 genes are each specifically hypermethylated in ovarian cancer metastatic tissues, each of them is used as a sole biomarker for diagnosing ovarian cancer metastasis or predicting metastasis risk, or two of them. More than one species may be used as the composite biomarker.
Therefore, in one aspect, the present invention comprises a composition for measuring the methylation level of the CpG region of one or more genes selected from the group consisting of ADAM12, NTN4 and PTGS2, the composition for diagnosing or predicting metastasis risk of ovarian cancer It relates to a kit comprising the same.
In one preferred embodiment, the present invention relates to a composition for diagnosing or predicting metastasis risk of ovarian cancer, and a kit comprising the same, comprising an agent for measuring the methylation level of the CpG region of the ADAM12 gene.
In this case, more preferably, the composition and kit may further comprise an agent for measuring the methylation level of the CpG site of at least one gene selected from the group consisting of NTN4 and PTGS2.
As another preferred embodiment, the present invention relates to a composition for diagnosing or predicting metastasis risk of ovarian cancer, comprising a preparation for measuring the methylation level of the CpG region of the NTN4 gene, and a kit comprising the same.
In this case, more preferably, the composition and kit may further comprise an agent for measuring the methylation level of the CpG site of at least one gene selected from the group consisting of ADAM12 and PTGS2.
As another preferred embodiment, the present invention relates to a composition for diagnosing or predicting metastasis risk of ovarian cancer, comprising a preparation for measuring the methylation level of the CpG region of the PTGS2 gene, and a kit comprising the same.
In this case, more preferably, the composition and kit may further comprise an agent for measuring the methylation level of the CpG site of at least one gene selected from the group consisting of ADAM12 and NTN4.
In the present invention, mRNAs of the ADAM12, NTN4 and PTGS2 genes can be confirmed by their sequence information in a known gene database. For example, the nucleic acid sequence of the human ADAM12 gene can be found in Genbank accession number NM_003474, the nucleic acid sequence of the human NTN4 gene can be found in Genbank accession number NM_021229, and the nucleic acid sequence of the human PTGS2 gene can be found in Genbank accession number NM_000963. have.
As used herein, the term "methylation" refers to the attachment of a methyl group to the base constituting the DNA. Preferably, whether or not methylation in the present invention means whether or not methylation occurs in the cytosine of a specific CpG region of a specific gene. When methylation occurs, the binding of transcription factors is disturbed thereby inhibiting the expression of specific genes. In contrast, when demethylation or hypomethylation occurs, expression of specific genes increases.
In genomic DNA of mammalian cells, in addition to A, C, G and T, there is a fifth base called 5-methylcytosine (5-mC) with a methyl group attached to the fifth carbon of the cytosine ring. do. Methylation of 5-methylcytosine occurs only in C of CG dinucleotides (5'-mCG-3 ') called CpG, and methylation of CpG inhibits the expression of alu or transposons and genome repeats. In addition, CpG is a site where most epigenetic changes occur frequently in mammalian cells because 5-mC of CpG is naturally deaminoated to easily become thymine (T).
As used herein, the term "measurement of methylation level" refers to measuring the methylation level of the CpG region of the ADAM12, NTN4 and / or PTGS2 genes, and is characterized by methylation specific PCR, for example methylation-specific polymerase chain reaction. , MSP), real time methylation-specific polymerase chain reaction (PCR), PCR using methylated DNA-specific binding proteins, or quantitative PCR. Or, it may be measured by a method such as automatic base analysis such as pyro sequencing and bisulfite sequencing, but is not limited thereto.
Preferably, the CpG site of the ADAM12, NTN4 and / or PTGS2 gene refers to the CpG site present on the DNA of the gene. The DNA of the gene is a concept including all a series of structural units necessary for the gene to express and operably linked to each other, for example, a promoter region, an open reading frame (ORF) and a terminator It includes an area. Thus, the CpG region of the ADAM12, NTN4 and / or PTGS2 genes may be present in the promoter region, open reading frame (ORF) or terminator region of the gene.
Preferably, measuring the methylation level of the CpG region of the ADAM12 gene in the present invention may mean measuring the methylation level of the cytosine of the CpG region appearing in the 127779782 to 127779903 bases of
More preferably, measuring the methylation level of the CpG region of the ADAM12 gene in the present invention may mean measuring the methylation level of cytosine located at 127779842th (SEQ ID NO: 1) of
Also preferably, in the present invention, measuring the methylation level of the CpG region of the NTN4 gene may mean measuring the methylation level of the cytosine of the CpG region appearing in the 96184755 to 96184876 bases of chromosome 12. In the present invention, the 96184755 to 96184876 base of the chromosome 12 is shown in SEQ ID NO: 2.
More preferably, measuring the methylation level of the CpG region of the NTN4 gene in the present invention may mean measuring the methylation level of cytosine located at 96184815th (SEQ ID NO: 2) of chromosome 12.
Also preferably, in the present invention, measuring the methylation level of the CpG region of the PTGS2 gene may mean measuring the methylation level of the cytosine of the CpG region appearing in the 186650381 to 186650502 bases of
More preferably, measuring the methylation level of the CpG region of the PTGS2 gene in the present invention may mean measuring the methylation level of cytosine located at the 186650441 th (SEQ ID NO: 3) of
In the present invention, the nucleotide sequence of the human genomic chromosome region was expressed according to the latest version The February 2009 Human reference sequence (GRCh 37), The specific sequence of the human genomic chromosome region may have a slightly changed expression as the results of genomic sequence studies are updated, and the expression of the human genomic chromosome region of the present invention may be different. Therefore, the human genome chromosome region expressed according to The February 2009 Human reference sequence (GRCh 37) of the present invention has been updated after the filing date of the present invention so that the expression of the human genome chromosome region is now available. Even if it is changed differently from the above, it will be apparent that the scope of the present invention extends to the modified human genomic chromosome region. Such modifications are readily apparent to those skilled in the art to which the present invention pertains.
In light of the fact that the gene expression of early cancer and metastasized cancer sites in the primary site of cancer is different, the present inventors compared gene expression patterns between primary cancer cell lines and metastasized tissues, and compared the changes in gene expression in metastasized tissues. Of the genes shown, the genes identified as having a change in methylation of CpG affected gene expression were finally selected. Furthermore, specific CpG sites that influenced the expression of genes were identified, and it was confirmed that metastasis and risk of metastasis of ovarian cancer could be predicted by measuring the degree of methylation occurring at specific CpG positions of the genes.
More specifically, the present inventors implanted the primary ovarian cancer cell line SK-OV-3 into 10 nude mouse abdominal cavity to establish an ovarian cancer metastasis animal model, and extracted genomic DNA and RNA of tumor tissue obtained from this animal model , DNA methylation microarray using Illumina Human Methylation 450 Bead Chip and gene expression microarray analysis using Affymetrix Human Gene 1.0 ST were performed, and the results of these analyzes were integrated into the CpG methylation of each gene through integration analysis. The genes were estimated to have altered the gene expression.
Among the selected genes, ADAM12 decreased gene expression by 11-19 times compared with primary cancer cell line in ovarian cancer metastasis model mice, and increased DNA methylation by 2.2-2.5 times. NTN4 decreased the expression of genes by 1.8-6.9 times compared with the primary cancer cell line in ovarian cancer metastasis model mice, and at the same time increased DNA methylation of specific CpG by 2.7-4.0 times. PTGS2 had a 1.5-17.4-fold decrease in gene expression in ovarian cancer metastasis model mice, and at the same time 1.6-fold increased DNA methylation of specific CpG.
In addition, when the primary cell line SKOV-3 was treated with 5-aza-2'-deoxycytidin, a de-DNA methylation agent, the expression of the ADAM12, NTN4 and PTGS2 genes increased by about 2-fold, respectively. Means controlled by methylation.
Therefore, hypermethylation of DNA methylation at the CpG positions of ADAM12, NTN4 and / or PTGS2 can be used as a biomarker to diagnose metastasis of ovarian cancer or predict the risk of metastasis.
In the present invention, "metastasis diagnosis" refers to identifying a state in which ovarian cancer has metastasized to tissues other than the ovary. Since ovarian cancer generally metastasizes to various organ tissues through the abdominal cavity, tissues other than the ovary may be, for example, various intraperitoneal organ tissues including a large intestine, a small intestine, and a liver periphery. More preferably, the metastasis diagnosis in the present invention may mean confirming the metastatic state of ovarian cancer by distinguishing between the primary ovarian cancer sample and the metastatic patient's sample that have not been metastasized.
In the present invention, "transition risk prediction" means to determine in advance the possibility of ovarian cancer metastasis to tissues other than the ovary. More preferably, in the present invention, metastasis risk prediction means that the ovarian cancer metastasis patient undergoes ovarian cancer treatment using surgical therapy, radiation therapy, or chemotherapy, and then, in advance, the possibility of recurrence and metastasis of ovarian cancer in the treated tissue. It can mean judging. In another aspect, the prediction of metastasis risk in the present invention may mean determining a metastasis potential of an ovarian cancer patient by diagnosing a primary ovarian cancer sample that has not metastasized and a sample of a patient at risk of metastasis. .
In addition, abnormal methylation changes in cancer tissue show significant similarity to changes in methylation of genomic DNA obtained from biological samples such as cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine. When used for the diagnosis of metastasis of ovarian cancer or the prediction of metastasis risk, there is an advantage that can be easily diagnosed through blood or body fluids.
In the present invention, an agent for determining the methylation level of a CpG site is a compound that modifies an unmethylated cytosine base or a primer specific for the methylated allele sequence of the methylation sensitive restriction enzyme, ADAM12, NTN4 and / or PTGS2 genes, and non Primers specific for the methylated allele sequence may be included.
The compound that modifies the unmethylated cytosine base may be, but is not limited to, bisulfite or a salt thereof, preferably sodium bisulfite. The use of such bisulfites to modify unmethylated cytosine residues to detect the methylation of CpG sites is well known in the art (WO01 / 26536; US2003 / 0148326A1).
In addition, the methylation sensitive restriction enzyme may be a restriction enzyme containing CG as a recognition site of the restriction enzyme as a restriction enzyme capable of specifically detecting the methylation of the CpG region. For example, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I and the like are not limited thereto. Depending on the methylation or demethylation of the restriction enzyme recognition site, the cleavage by restriction enzymes is different and can be detected by PCR or Southern blot analysis. Methylation sensitive restriction enzymes other than the restriction enzymes are well known in the art.
As a representative method for measuring the methylation level at the CpG region of the ADAM12, NTN4 and PTGS2 genes of a patient suspected of ovarian cancer metastasis, genomic DNA is obtained from a biological sample of the patient and unmethylated cytosine bases are obtained from the obtained DNA. After treatment with the compound to be modified or methylation sensitive restriction enzyme, the treated DNA can be measured by amplification by PCR using a primer and confirming the presence of the amplified result.
Thus, the formulations of the present invention may comprise primers specific for the methylated allele sequences of the ADAM12, NTN4 and PTGS2 genes and primers specific for the unmethylated allele sequences. In the present invention, the term “primer” refers to a short nucleic acid sequence that can form base pairs with complementary templates with nucleic acid sequences having short free 3-terminal hydroxyl groups and that serves as a starting point for template strand copying. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In addition, primers, as sense and antisense nucleic acids having 7 to 50 nucleotide sequences, may incorporate additional features that do not change the basic properties of the primers serving as the starting point for DNA synthesis.
Primers of the present invention may be preferably designed according to the sequence of a specific CpG site to be analyzed for methylation, primer pairs that can specifically amplify cytosine that is methylated and not modified by bisulfite, And primer pairs that are not methylated to specifically amplify cytosine modified by bisulfite.
In addition to the formulation, the composition and kit may further include a polymerase agarose, a buffer solution for electrophoresis, and the like.
As another aspect, the present invention relates to a method for diagnosing ovarian cancer metastasis or predicting metastasis risk by measuring the methylation level at the CpG site of at least one gene selected from the group consisting of ADAM12, NTN4 and PTGS2.
As another aspect, the invention provides information for diagnosing metastasis or predicting metastasis risk of ovarian cancer, comprising measuring the methylation level at the CpG site of the gene from a biological sample of a patient suspected of metastatic ovarian cancer. It is about how to provide.
In one preferred embodiment, the present invention comprises the steps of measuring the methylation level of the CpG region of the ADAM12 gene from a biological sample of a patient suspected of ovarian cancer metastasis; And comparing the methylation level with the methylation level of the CpG of the corresponding gene of the primary ovarian cancer sample, to provide information for diagnosing or predicting metastasis risk of ovarian cancer.
In this case, more preferably, the method further comprises measuring the methylation level of the CpG region of at least one gene selected from the group consisting of NTN4 and PTGS2 from biological samples of patients suspected of having ovarian cancer metastasis; And comparing the methylation level with the methylation level of CpG of the gene of the primary ovarian cancer sample.
In another preferred embodiment, the present invention provides a method for treating ovarian cancer, comprising measuring the methylation level of the CpG region of the NTN4 gene from a biological sample of a patient suspected of having ovarian cancer metastasis; And comparing the methylation level with the methylation level of the CpG of the corresponding gene of the primary ovarian cancer sample, to provide information for diagnosing or predicting metastasis risk of ovarian cancer.
In this case, more preferably, the method further comprises measuring the methylation level of the CpG region of at least one gene selected from the group consisting of ADAM12 and PTGS2 from biological samples of patients suspected of having ovarian cancer metastasis; And comparing the methylation level with the methylation level of CpG of the gene of the primary ovarian cancer sample.
In another preferred embodiment, the present invention provides a method for treating ovarian cancer, comprising: measuring the methylation level of the CpG region of the PTGS2 gene from a biological sample of a patient suspected of having ovarian cancer metastasis; And comparing the methylation level with the methylation level of the CpG of the corresponding gene of the primary ovarian cancer sample, to provide information for diagnosing or predicting metastasis risk of ovarian cancer.
In this case, more preferably, the method further comprises measuring the methylation level of the CpG region of at least one gene selected from the group consisting of ADAM12 and NTN4 from biological samples of patients suspected of having ovarian cancer metastasis; And comparing the methylation level with the methylation level of CpG of the gene of the primary ovarian cancer sample.
As used herein, the term "biological sample" refers to samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine that differ in methylation levels of the ADAM12, NTN4 and / or PTGS2 genes due to ovarian cancer metastasis. Including but not limited to.
First, in order to obtain genomic DNA from patients suspected of ovarian cancer metastasis and to measure methylation levels, the obtaining of genomic DNA is performed by phenol / chloroform extraction, SDS extraction (Tai et al., Plant Mol. Biol.Reporter, 8: 297-303, 1990), CTAB isolation (Cetyl Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) or commercially available DNA extraction kits. Can be.
Determining the methylation level of the CpG region of the gene, more specifically, (a) treating genomic DNA in the obtained sample with a compound or methylation sensitive restriction enzyme that modifies the unmethylated cytosine base; And (b) amplifying the treated DNA by PCR using a primer capable of amplifying a specific CpG site of the gene.
The compound that modifies the unmethylated cytosine base in step (a) may be bisulfite, preferably sodium bisulfite. The use of such bisulfites to modify unmethylated cytosine residues to detect the methylation of specific CpG sites is well known in the art.
In addition, the methylation-sensitive restriction enzyme in step (a) may be a restriction enzyme containing CG as a recognition site of the restriction enzyme as a restriction enzyme that can specifically detect methylation of a specific CpG site, as described above. For example, Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I and the like, but is not limited thereto.
Amplification in step (b) may be performed by a conventional PCR method. In this case, the primers used may be preferably designed according to the sequence of a specific CpG region to be analyzed for methylation, as described above, and specifically amplify cytosine that has been methylated and not modified by bisulfite. Possible primer pairs and unmethylated primer pairs that can specifically amplify cytosine modified by bisulfite.
Measuring the methylation level of the CpG region of the gene may further comprise (c) confirming the presence of the result amplified in step (b). The presence of the result amplified in step (c) may be performed according to a method known in the art, for example, electrophoresis, according to whether a band of a desired position is detected. For example, in the case of using a compound that modifies an unmethylated cytosine residue, primers capable of specifically amplifying two kinds of primer pairs used in step (a), namely, cytosine that is methylated and not modified by bisulfite, are used. The degree of methylation can be determined according to the presence or absence of PCR results amplified by pairs and primer pairs that can specifically amplify cytosine modified by bisulfite without methylation. Preferably, whether or not methylation can be determined using a bisulfite genome sequencing method in which the sample genomic DNA is treated with bisulfite, the CpG site of the gene is amplified by PCR, and the nucleotide sequence of the amplified site is analyzed. .
In addition, even when restriction enzymes are used, it is determined that CpG sites are methylated when PCR products are present in the DNA treated with restriction enzymes, for example, when PCR products are displayed in mock DNA. If there is no PCR result in the DNA treated with restriction enzymes, the methylation can be determined according to the determination that the CpG site is unmethylated, which is obvious to those skilled in the art. The mock DNA in the above means the sample DNA which is separated from the sample and is not treated at all.
According to this method, when a specific CpG region of the ADAM12, NTN4 and / or PTGS2 genes in a patient sample is measured in a high methylation state, ovarian cancer can be predicted to be metastatic or at risk of metastasis.
Therefore, according to the present invention, it is possible to effectively determine whether the methylation of the CpG of the ADAM12, NTN4 and / or PTGS2 gene methylation can easily determine the risk of metastasis or metastasis of ovarian cancer.
According to the present invention, the degree of methylation of a CpG region of a specific gene of genomic DNA collected from a biological sample of a patient is measured by a method such as MSP (methylation-specific PCR) based on PCR technique, and thus the risk of ovarian cancer metastasis within several hours. This enables the development of highly accurate and convenient diagnostic kits.
1 is a schematic diagram showing a process of integrating mRNA and CpG methylation data.
2 is a photograph showing that the SK-OV-3 cell line was injected into the abdominal cavity of nude mice to construct an animal model of ovarian cancer metastasis.
Figure 3 shows the overall DNA methylation distribution trend in the tumor tissue (n = 7; 1C ~ 8C) of the primary ovarian cancer cell line (SK-OV-3) and seven ovarian cancer metastases.
Figure 4 is a Heatmap result for the genes showing significant changes in expression in metastatic tumor tissue compared with the primary ovarian cancer cell line.
Figure 5 shows the results of changes in DNA methylation and gene expression in metastatic ovarian cancer animal model.
Figure 6 shows the results of verifying the expression changes of the ADAM12 gene in qRT-PCR in tumor tissue of metastatic ovarian cancer animal model (n = 7; 1C ~ 8C).
Figure 7 shows the results of verifying the changes in the expression of NTN4 gene in qRT-PCR in tumor tissue of metastatic ovarian cancer animal model (n = 7; 1C ~ 8C).
Figure 8 shows the results of verifying the change in the expression of PTGS2 gene in the tumor tissue of the metastatic ovarian cancer animal model (n = 7; 1C ~ 8C) by qRT-PCR.
9 shows the results of DNA methylation microarray analysis of changes in DNA methylation at the CpG region of the ADAM12 gene in tumor tissues of metastatic ovarian cancer animal models (n = 7; 1C-8C).
10 shows the results of DNA methylation microarray analysis of changes in DNA methylation at the CpG region of the NTN4 gene in tumor tissues of metastatic ovarian cancer animal models (n = 7; 1C ~ 8C).
Figure 11 shows the results of DNA methylation microarray analysis of changes in DNA methylation at the CpG region of PTGS2 gene in tumor tissue of metastatic ovarian cancer animal model (n = 7; 1C ~ 8C).
12 shows the results of confirming the change in ADAM12 gene expression after 5-aza-2'-deoxycytidin treatment in SK-OV-3 cell line.
Figure 13 shows the results of confirming the change in NTN4 gene expression after 5-aza-2'-deoxycytidin treatment to SK-OV-3 cell line.
Figure 14 shows the result of confirming the change in PTGS2 gene expression after 5-aza-2'-deoxycytidin treatment to SK-OV-3 cell line.
Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.
Example 1. Cell Line and Ovarian Cancer Metastasis Model Mice
Human ovarian cancer cell line SK-OV-3 was purchased from American type culture collection (ATCC no.HTB-77) and contained 10% FBS (fetal bovine serum), 100 U / mL penicillin and 100 μg / mL streptomycin. Incubated in McCoy's 5a medium.
To create an ovarian cancer metastasis model rat, 2 x 10 6 SK-OV-3 cells were suspended in cell culture medium and injected into the abdominal cavity of 10 4-6 week old female BALB / c nude mice. After 4 weeks, the cell lines moved along the abdominal cavity, and the tumor-forming tumor tissues (intestinal organs including the large intestine, the small intestine, and the liver periphery) were removed and stored in liquid nitrogen.
Example 2. Total RNA extraction
Total RNA was extracted from SK-OV-3 cell line and tumor tissue using RNeasy mini kit (Qiagen), respectively. Extraction method was performed according to the manufacturer's manual. The extracted total RNA was quantified using a spectrophotometer, and RNA status was confirmed by degradation by electrophoresis on 1% agarose gel.
Example 3. Quantitative Real Time ( Quantitative real - time ) PCR ( qRT - PCR )
Superscript II reverse transcriptase (Invitrogen) was used for cDNA synthesis. 1 μg of total RNA and 50 ng oligo dT were denatured at 70 ° C for 10 minutes, followed by 4 μl of 5X RT buffer, 2 μl of 0.1 mM DTT, 4 μl of 2.5 mM dNTP mixture, Superscript II reverse transcriptase 200 units and
Example 4. 5- aza -2'- deoxycytidine (5- aza - dC ) process
SK-OV-3 cell line was treated with 5-aza-2'-deoxycytidine (Sigma-Aldrich), a methylation inhibitor, at 5, 10, and 20 μM concentrations for 3 days and then the qRT- expression of ADAM12, NTN4 and PTGS2 genes It was measured using PCP.
Example 5. mRNA Microarray
MRNA microarrays were performed using GeneChip Human Gene 1.0 ST arrays.
The expression value of each gene obtained after scanning is determined by background correction, RMA normalization (Biostatistics. 2003 Apr; 4 (2): 249-64.Exploration, normalization, and summaries of high density. Oligonucleotide array probe level data) and log2 transformation were used for the final statistical analysis. Bayesian t-test (Limma: Linear Models for Microarray Data. Gordon K. Smyth.) Was used to identify differentially expressed genes (DEGs) in both groups. Finally, DEG was selected as the gene with p value <0.05 and the absolute value of log2 (fold change) greater than 0.585.
Example 6. DNA Methylation Microarray
A DNA methylation microarray was performed using the Infinium (®) Human Methylation 450K BeadChip. The degree of DNA methylation is expressed as a β value with a value of 0 to 1, and a β value of 0 means that the CpG site is completely unmethylated, and 1 means fully methylated.
Bayesian t-test was used to identify differentially methylated genes (DMGs) in both groups. Finally, the CpG site with p value <0.05 and the absolute value β difference ≥0.3 was selected as the differentially methylated CpG site and the gene with the changed degree of methylation at the CpG site was selected as DMG.
Example 7. DEG And DMG Integrated analysis of data
Following the procedure of FIG. 1, the final determined DEG and DMG data were combined.
Experiment result
One. Building an Animal Model for Ovarian Cancer Metastasis
The SK-OV-3 ovarian cancer cell line was injected into the abdominal cavity of 10 female nude mice, respectively, to construct an ovarian cancer metastatic animal model (FIG. 2).
2. Ovarian Cancer Metastasis of Animal Models Epigenetics Change analysis
DNA methylation microarray using Illumina Human Methylation 450 BeadChip by extracting genomic DNA from tumor tissues (intestinal peritoneal organs including colon, small intestine, and liver periphery) and SK-OV-3 ovarian cancer cell lines obtained from metastatic animal models CpG sites that showed significant changes in DNA methylation in metastatic tumor tissues were analyzed in comparison with the primary ovarian cancer cell line. As a result, it was observed that global hypomethylation showed a lower overall distribution of DNA methylation in metastatic tumor tissue compared to the primary ovarian cancer cell line (FIG. 3).
3. Analysis of Gene Expression Changes in Animal Models of Metastatic Ovarian Cancer
RNA was extracted from the tumor tissues and SK-OV-3 ovarian cancer cell lines obtained from metastatic animal models and subjected to expression microarray using Affymetrix Human Gene 1.0 ST and compared with primary ovarian cancer cell lines. Genes showing changes in expression were analyzed (FIG. 4). As a result, the expression of genes associated with cell adhesion, cell cycles, wound healing, and coagulation has been increased, while transcription, transcription regulation, and apoptosis have been increased. And the expression of genes involved in the regulation of apoptosis was significantly reduced (Table 2).
(GOTERM_BP_FAT)
Cell-to-cell adhesion
Protein localization
4. Metastatic Ovarian Cancer Animal Model Epigenetics Integrated Analysis of Changes and Gene Expression
Compared to the primary ovarian cancer cell line, the genes showing differences in DNA methylation and genes showing differences in gene expression in metastatic tumor tissues were selected. Genes presumed to have affected gene expression were selected (FIG. 5).
In addition, as a result of integrating mRNA expression and CpG methylation data, 277 genes whose gene expression was increased by hypomethylation of specific CpG were selected from the transition group, and gene expression was reduced by hypermethylation of CpG. 120 reduced genes were selected.
5. CpG Methylation Metastasis Diagnosis of Ovarian Cancer Marker selection
Through integration analysis, we selected genes whose changes in methylation of CpG in each gene had an effect on gene expression, and then among them, genes whose function was reported in relation to cancer metastasis were selected. The expression changes of these genes were verified using quantitative real-time PCR to select candidate genes for transition specific molecular targets with significant differences. In addition, the primary cell line SK-OV-3 was treated with 5-aza-2'-deoxycytidin, a de- DNA methylation agent, to express three genes that were regulated by DNA methylation. ADAM12, NTN4 and PTGS2) were finally selected as diagnostic markers for ovarian cancer metastasis using specific CpG methylation changes.
(fold change)
6. Selection of Genes from Tumor Tissue in Animal Model of Metastatic Ovarian Cancer. CpG Methylation Changes and changes in gene expression
Expression of all three genes (ADAM12, NTN4, and PTGS2) in tumor tissues of metastatic ovarian cancer animal model was decreased by the results of expression microarrays, and qRT-PCR confirmed similar expression trends. It could be confirmed (FIGS. 6-8).
As a result of DNA methylation microarray analysis, all three kinds of genes (ADAM12, NTN4, and PTGS2) showed significantly higher DNA methylation at specific CpG sites (FIGS. 9 to 11).
In addition, DNA methyl methylation was observed after treatment of the primary DNA cell SK-OV-3 with 5-aza-2'-deoxycytidin, a de- DNA methylation agent, for 3 days and then the expression of three genes (ADAM12, NTN4 and PTGS2). When the reduction was reduced, the expression of the genes was confirmed to increase. This means that the expression of these three genes is regulated by DNA methylation (FIGS. 12-14).
These results indicate that the rapid decrease of the three genes (ADAM12, NTN4 and PTGS2) in the ovarian cancer metastasis model is regulated by hypermethylation of specific CpG region of each gene and is specific to the ovarian cancer metastasis model. Shows.
Attach an electronic file to a sequence list
Claims (13)
Compounds or methylation sensitive restriction enzymes that modify unmethylated cytosine bases;
Primers specific for the methylated sequence of the CpG region of the NTN4 gene; And
A composition comprising primers specific for unmethylated sequences.
A method of providing information for diagnosing or predicting the risk of metastasis of ovarian cancer.
(a) treating genomic DNA in the obtained sample with a compound or methylation sensitive restriction enzyme that modifies an unmethylated cytosine base; And
(b) amplifying the treated DNA by PCR using primers capable of amplifying the CpG region of NTN4 gene.
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| US14/433,397 US20160265061A1 (en) | 2013-09-06 | 2014-09-04 | Composition for diagnosing ovarian cancer metastasis by using cpg methylation in gene, and use thereof |
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