WO2020034888A1 - 诊断肿瘤的dna甲基化相关的标记物及其应用 - Google Patents

诊断肿瘤的dna甲基化相关的标记物及其应用 Download PDF

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WO2020034888A1
WO2020034888A1 PCT/CN2019/099722 CN2019099722W WO2020034888A1 WO 2020034888 A1 WO2020034888 A1 WO 2020034888A1 CN 2019099722 W CN2019099722 W CN 2019099722W WO 2020034888 A1 WO2020034888 A1 WO 2020034888A1
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cancer
polynucleotide
seq
methylation
tumors
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PCT/CN2019/099722
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English (en)
French (fr)
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于文强
董世华
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上海市公共卫生临床中心
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Priority to US17/268,268 priority Critical patent/US20210340627A1/en
Priority to EP19849884.2A priority patent/EP3839070A4/en
Priority to JP2021507822A priority patent/JP7391082B2/ja
Publication of WO2020034888A1 publication Critical patent/WO2020034888A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the invention belongs to the field of diagnosis, and more particularly, the invention relates to a marker related to DNA methylation for diagnosing a tumor and an application thereof.
  • Epigenetics is a discipline that studies heritable changes in gene function without causing changes in the DNA sequence, and ultimately leads to phenotypic changes.
  • Epigenetics mainly includes biochemical processes such as DNA methylation, histone modification, and changes in microRNA levels.
  • DNA methylation refers to the process of transferring methyl groups to specific bases using S-adenosylmethionine as the methyl donor under the catalysis of DNA methyltransferase in the body.
  • DNA methylation can occur at N-6 of adenine, N-4 of cytosine, N-7 of guanine, or C-5 of cytosine.
  • DNA methylation mainly occurs on the 5'-CpG-3 'C, producing 5-methylcytosine (5mC).
  • CpG dinucleotides in the genome are scattered in repeats with transcription-dependent transposition potential.
  • these CpGs are in a state of high methylation / transcriptional silencing, while in tumor cells these CpGs undergo extensive demethylation, which results in the transcription of repeat sequences and the activation of transposons. Stability and enhanced oncogene transcription.
  • the remaining CpG which accounts for about 2% of the total, is densely distributed in smaller areas (CpG islands).
  • CpG islands exist in or near 40% -50% of the gene promoter region, suggesting that DNA methylation may be involved in the transcriptional regulation mechanism of such genes.
  • these CpG islands which were originally hypomethylated in normal cells, are hypermethylated and cause transcriptional inactivation of genes.
  • Affected genes include tumor suppressor genes such as DNA repair genes, cell cycle control genes, and anti-apoptotic genes.
  • AFP alpha-fetoprotein
  • An object of the present invention is to provide a marker related to DNA methylation for diagnosing a tumor and an application thereof.
  • an isolated polynucleotide comprising:
  • polynucleotide fragments of (a) to (d) above, and at least one of them such as 2 to 40, more specifically 3, 5, 8, 10, 15, 20, 25, 30, 35) modified CpG sites; and / or
  • the modification includes 5-methylation modification (5mC), 5-hydroxymethylation modification (5hmC), 5-aldehyde methylation modification (5-fC), or 5-carboxymethylation. (5-caC) modification.
  • an isolated polynucleotide which is transformed from the aforementioned polynucleotide, corresponding to the sequence (a) to (e) above, and a modified cytosine at the CpG site C is unchanged, and unmodified cytosine is converted to T (U).
  • the polynucleotide corresponding to (a) to (e) is transformed by bisulfite or bisulfite treatment.
  • the polynucleotide includes:
  • the tumor includes: digestive system tumors such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory system tumors such as lung cancer, pleural tumors; blood system tumors such as leukemia, Lymphoma, multiple myeloma; Gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; nervous system tumors such as glioma, neuroblastoma, meningiomas Head and neck tumors such as oral cancer, tongue cancer, throat cancer, nasopharyngeal cancer; urinary system tumors such as kidney cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer.
  • digestive system tumors such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer,
  • a method for preparing a tumor detection reagent comprising: providing a polynucleotide (e.g., one, two, three or four) of any one of the foregoing, with the polynucleotide The full length or fragment of the acid is used as a target sequence, and a detection reagent for specifically detecting the target sequence is designed; wherein, at least one of the target sequences (such as 2 to 40, more specifically, 3, 5, 8, 10, etc.) is present. , 15, 20, 25, 30, 35) modified CpG sites; preferably, the detection reagent includes (but is not limited to): primers, probes.
  • a reagent or a combined reagent which specifically detects a target sequence
  • the target sequence is the full length or fragment of any one of the foregoing polynucleotides, wherein at least one (such as 2 to 40, more specifically 3, 5, 8, 10, 15, 20, 25, 30, 35) modified CpG sites; preferably, the detection reagent includes (but is not limited to): primers , Probe.
  • the polynucleotide is a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1, and the target sequence includes nucleotides 240 to 296 in SEQ ID NO: 1 Fragment; preferably, the primers are the primers shown in SEQ ID NOs: 9-12.
  • the polynucleotide is a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 3, and the target sequence includes a nucleoside at positions 279 to 323 in SEQ ID NO: 3 Acid fragment; preferably, the primers are the primers shown in SEQ ID NOs: 12 to 15; or
  • the polynucleotide is a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 5, and the target sequence includes a nucleoside at positions 186 to 235 in SEQ ID NO: 5 Acid fragment; preferably, the primers are the primers shown in SEQ ID NOs: 12, 16-18; or
  • the polynucleotide is a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 7, and the target sequence includes a nucleoside at positions 164 to 198 in SEQ ID NO: 7 Acid fragment; preferably, the primers are the primers shown in SEQ ID NOs: 12, 19-21.
  • the use of the reagent or the combined reagent is provided to prepare a kit for detecting tumors.
  • the tumors include: digestive system tumors such as esophageal cancer, gastric cancer, colorectal Cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory tumors such as lung cancer, pleural tumor; hematological tumors such as leukemia, lymphoma, multiple myeloma; gynecological and reproductive system tumors such as breast, ovarian, and cervical cancer, Vulvar cancer, testicular cancer, prostate cancer, penile cancer; neurological tumors such as glioma, neuroblastoma, meningiomas; head and neck tumors such as oral cancer, tongue cancer, throat cancer, nasopharyngeal cancer; urinary system tumors such as Kidney cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma, liposar
  • a detection kit which includes:
  • each reagent is located in a separate container.
  • the kit further includes: bisulfite or bisulfite, DNA purification reagents, DNA extraction reagents, PCR amplification reagents and / or instruction manuals (indicating detection operation steps and results) judgement standard).
  • a method for detecting a methylation profile of a polynucleotide or a fragment thereof in a sample in vitro including:
  • step (ii) processing the sample to be tested to convert unmodified cytosine to uracil; preferably, the modification includes 5-methylation modification (5mC), 5-hydroxymethylation modification (5hmC), 5-aldehyde methylation modification or 5-carboxymethylation modification; preferably, the DNA described in step (i) is treated with bisulfite or bisulfite;
  • step (iii) Analyze the modification of the polynucleotide or fragment thereof in the genomic DNA treated in step (ii).
  • the abnormality of the methylation pattern means that C in the polynucleotide CpG is highly methylated.
  • the method of methylation profile is not for the purpose of directly obtaining the diagnosis result of the disease, or it is not a diagnostic method.
  • the analysis method includes (but is not limited to): pyrosequencing method, bisulfite conversion sequencing method, qPCR method, next-generation sequencing method, whole genome methylation sequencing Method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, or a combination thereof.
  • CTSM-4I detection results in head and neck cancer.
  • the present inventor is committed to the study of tumor markers. After extensive research and screening, the gene sequence regions with abnormal DNA methylation in the genome were identified and named as tumor markers CTSM-4F, CTSM-2BE, CTSM-3C, CTSM- 4I. Studies on clinical samples have shown that the methylation status of these sequence regions is significantly different between tumor and non-tumor tissues, and that CpG is highly methylated. Therefore, these genes are tumor markers; they can be used as the basis for designing tumor diagnostic reagents.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state.
  • sample includes a substance obtained from any individual or isolated tissue, cell, or body fluid that is suitable for detecting the state of DNA methylation.
  • high (degree) methylation refers to the presence of a high methylation, methylolation, aldehyde methylation, or carboxymethylation modification of CpG in a gene sequence.
  • MSP methylation-specific PCR
  • a PCR reaction with methylation-specific primers can obtain a positive PCR result, and the DNA (gene) region of the test can be considered to be in a hypermethylated state.
  • the determination of hypermethylation status can be analyzed for statistical differences based on the relative value of the methylation status of its control sample.
  • the tumors include but are not limited to: digestive system tumors such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory system tumors such as lung cancer, pleural tumors; blood system tumors such as leukemia Lymphoma, multiple myeloma; Gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; nervous system tumors such as glioma, neuroblastoma, meninges Tumors; head and neck tumors such as oral cancer, tongue cancer, throat cancer, nasopharyngeal cancer; urinary system tumors such as kidney cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer.
  • digestive system tumors such as esophageal cancer, gastric cancer, colorectal cancer,
  • the present invention provides an isolated polynucleotide, which is derived from the human genome and has the cores shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7
  • a nucleotide sequence generates 5-methylcytosine (5mC) at a plurality of 5'-CpG-3 'base C positions in the polynucleotide sequence.
  • the present invention also includes a fragment of a polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and at least one (such as 2 ⁇ 40, more specifically 3, 5, 8, 10, 15, 20, 25, 30, 35) methylated CpG sites.
  • the above-mentioned polynucleotides or fragments or complementary strands (antisense strands) can also be used for designing detection reagents or detection kits.
  • polynucleotides can be used as a key region for people to analyze methylation status in the genome, and their methylation status can be analyzed by various techniques known in the art. Any technique that can be used to analyze the methylation state can be applied to the present invention.
  • cytosine that has not been methylated is converted into uracil, and cytosine that has been methylated remains unchanged.
  • the present invention also provides a polynucleotide obtained by subjecting the above polynucleotide (including its complementary strand (antisense strand)) to bisulfite or bisulfite treatment, including: SEQ ID NO: 2, SEQ Polynucleotides of the nucleotide sequences shown in ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8. These polynucleotides can also be used to design detection reagents or detection kits.
  • the present invention also includes a fragment of a polynucleotide obtained by subjecting the above polynucleotide (including its complementary strand (antisense strand)) to bisulfite or bisulfite treatment, and at least one methylated CpG is present therein. Site.
  • a detection reagent designed based on the polynucleotide sequence is also provided for detecting the methylation profile of the polynucleotide in a sample in vitro. Detection methods and reagents for determining the sequence of the genome and the methylation state known in the art can be used in the present invention.
  • the present invention provides a method for preparing a tumor detection reagent, comprising: providing the polynucleotide (1, 2, 3, or 4), and using the full length or a fragment of the polynucleotide as a target sequence, Design a detection reagent that specifically detects the target sequence; wherein at least one methylated CpG site exists in the target sequence.
  • the target sequence includes a nucleotide fragment at positions 240 to 296 in SEQ ID NO: 1; or a nucleotide fragment at positions 279 to 323 in SEQ ID NO: 3; Or contains the nucleotide fragment at positions 186 to 235 in SEQ ID NO: 5; preferably, the primers are the primers shown in SEQ ID NO: 12, 16 to 18; or include SEQ ID ID NO: 7 Nucleotide fragments from 164 to 198.
  • the detection reagent includes, but is not limited to, primers, probes, and the like.
  • the reagent is, for example, a primer pair. After knowing the sequence of the polynucleotide, designing the primer is known to those skilled in the art.
  • the two primers are on both sides of the specific sequence of the target gene to be amplified (including CpG Within the sequence, it is complementary to the gene region where CpG is originally methylated, and complementary to TpG where it is the gene region originally demethylated).
  • the reagent may also be a reagent combination (primer combination), including more than one set of primers, so that the multiple polynucleotides described above can be amplified separately.
  • the primers are the primers shown in SEQ ID NO: 9 to 12; or the primers shown in SEQ ID NO: 12 to 15; or the primers shown in SEQ ID NO: 12, 16 to 18 The primers shown in the drawings; or the primers shown in SEQ ID NO: 12, 19-21.
  • the above primers are primers used for performing nested PCR. Through two rounds of PCR amplification reactions, the obtained amplified products are used for sequence identification.
  • the products obtained by the above primer amplification have a suitable length and high specificity, and have good specificity for the amplification of complex systems, and are particularly suitable for methylation-specific PCR.
  • the invention also provides a kit for detecting methylation profiles of polynucleotides in a sample in vitro.
  • the kit includes a container and the above-mentioned primer pair located in the container.
  • the kit may also include various reagents required for DNA extraction, DNA purification, PCR amplification and the like.
  • kit may further include an instruction manual, in which detection operation steps and result determination standards are marked, so as to facilitate application by those skilled in the art.
  • Determining the methylation profile of a polynucleotide can be performed by existing techniques (such as methylation-specific PCR (MSP) or real-time quantitative methylation-specific PCR, Methylight), or others are still under development and will Developed technology to proceed.
  • MSP methylation-specific PCR
  • Methylight real-time quantitative methylation-specific PCR
  • Quantitative methylation-specific PCR can also be used to detect methylation levels. This method is based on a continuous optical monitoring of fluorescent PCR, which is more sensitive than the MSP method. Its high throughput avoids analysis of its results by electrophoresis.
  • a method for detecting a methylation profile of a polynucleotide in a sample in vitro is also provided.
  • the method is based on the principle that bisulfite or bisulfite can convert unmethylated cytosine to uracil, which is converted to thymine during subsequent PCR amplification, and methylation
  • the cytosine remains unchanged; therefore, after treating the polynucleotide with bisulfite or bisulfite, the methylated site produces a polynucleotide polymorphism (SNP) similar to a C / T.
  • SNP polynucleotide polymorphism
  • the method of the present invention comprises: (a) providing a sample and extracting genomic DNA; (b) processing the genomic DNA described in step (a) with bisulfite or bisulfite, so that the genomic DNA is not Methylated cytosine is converted to uracil; (c) Analyze whether there is an abnormal methylation pattern in the genomic DNA treated in step (b).
  • the method of the present invention can be used for: (i) detecting a sample of a subject to analyze whether the subject has a tumor; and (ii) distinguishing a high-risk group of tumors.
  • the method described may also be a situation where the purpose is not to obtain a direct diagnosis of the disease.
  • DNA methylation is detected by PCR amplification and pyrosequencing.
  • PCR amplification and pyrosequencing Those skilled in the art should understand that this method is not limited to practical applications, and other DNA methylation detection methods are also possible.
  • the primers used are not limited to those provided in the examples.
  • genomic DNA is treated with bisulfite
  • unmethylated cytosine is converted to uracil and then converted to thymine in the subsequent PCR process, which will reduce the complexity of the genome sequence. Degree, making it difficult to amplify specific target fragments by PCR.
  • the inventors have discovered that using nested PCR amplification, designing two pairs of primers on the periphery and the inside, and performing two rounds of PCR amplification reactions, and using the first round of amplification products as templates for the second round of amplification can effectively improve the amplification. Increased efficiency and specificity. It should be understood that the detection methods available in the present invention are not limited to this.
  • the sequence of the CTSM-4F tumor marker is provided, as shown in SEQ ID NO: 1, where the box indicates that the base is a potential methylation CpG site, and the optimal detection area is the middle underlined area.
  • SEQ ID NO: 1 is as follows:
  • DNA extraction The genomic DNA of the test sample is extracted by conventional phenol-chloroform.
  • DNA methylation is detected by pyrosequencing.
  • the steps for DNA methylation detection are as follows:
  • SEQ ID NO: 2 After bisulfite treatment, SEQ ID NO: 2 is obtained, where Y represents C (cytosine) or T (thymine). Corresponding to SEQ ID NO: 1, the original methylated modified cytosine C is unchanged, and the original unmethylated modified cytosine C is converted to T (U), so Y is used to represent C or T.
  • SEQ ID NO: 2 is as follows:
  • the PCR amplification reagent used is the 2 ⁇ PCR Master Mix reagent produced by Lai Feng Company, the article number is PT102, but it is not limited to this reagent.
  • the first round of PCR amplification system is the first round of PCR amplification system.
  • the second round of PCR amplification is performed using the products of the first round of PCR amplification as a template.
  • the system is as follows:
  • the second round of amplification procedures is as follows:
  • CTSM-4F By analyzing sample methylation values, tumor samples can be effectively distinguished from control samples.
  • CTSM-4F In the control group samples, CTSM-4F is in a low methylation state, and in tumor samples, CTSM-4F is in a high methylation state.
  • liver cancer samples obtained from the hospital 8 cases of liver cancer samples were randomly selected, and the corresponding tissue adjacent to the cancer was used as a control sample.
  • the sequence of the CTSM-2BE tumor marker is provided, as shown in SEQ ID NO: 3, where the box indicates that the base is a potential methylated CpG site.
  • the optimal detection area is the middle underlined area.
  • SEQ ID NO: 3 is as follows:
  • SEQ ID NO: 4 is obtained, where Y represents C (cytosine) or T (thymine).
  • SEQ ID NO: 4 is as follows:
  • Example 2 Using the above primers, the same method as in Example 1 was used to extract the DNA of the test sample, and the bisulfite treatment, nested PCR amplification, agarose gel electrophoresis, pyrosequencing and analysis were used to calculate the CpG site. Methylation value.
  • Eight breast cancer samples were randomly selected from the confirmed breast cancer samples obtained from the hospital, and the corresponding adjacent tissues were selected as control samples.
  • the DNA in the above samples was obtained and subjected to bisulfite treatment, nested PCR, agarose gel electrophoresis, pyrosequencing and analysis to calculate the methylation value of the CpG site.
  • the DNA in the above samples was obtained and subjected to bisulfite treatment, nested PCR, agarose gel electrophoresis, pyrosequencing and analysis to calculate the methylation value of the CpG site.
  • the DNA in the above samples was obtained and subjected to bisulfite treatment, nested PCR, agarose gel electrophoresis, pyrosequencing and analysis to calculate the methylation value of the CpG site.
  • results are shown in FIG. 7.
  • the results show that CTSM-2BE methylation is significantly lower in the control sample than lung cancer samples, and CTSM-2BE is significantly abnormally hypermethylated in lung cancer, which can be used as a very good lung cancer marker. Further enlarged clinical samples showed the same significance.
  • pancreatic cancer samples obtained from the hospital 8 pancreatic cancer samples were randomly selected and the corresponding adjacent tissues were obtained as control samples.
  • the DNA in the above samples was obtained and subjected to bisulfite treatment, nested PCR, agarose gel electrophoresis, pyrosequencing and analysis to calculate the methylation value of the CpG site.
  • the sequence of the CTSM-3C tumor marker is provided, as shown in SEQ ID NO: 5, where the box indicates that the base is a potential methylated CpG site.
  • the optimal detection area is the middle underlined area.
  • SEQ ID NO: 5 is as follows:
  • SEQ ID NO: 6 is obtained, where Y represents C (cytosine) or T (thymine):
  • SEQ ID NO: 6 is as follows:
  • Example 2 Using the above primers, the same method as in Example 1 was used to extract the DNA of the test sample, and the bisulfite treatment, nested PCR amplification, agarose gel electrophoresis, pyrosequencing and analysis were used to calculate the CpG site. Methylation value.
  • the DNA in the above samples was obtained and subjected to bisulfite treatment, nested PCR, agarose gel electrophoresis, pyrosequencing and analysis to calculate the methylation value of the CpG site.
  • the DNA in the above samples was obtained and subjected to bisulfite treatment, nested PCR, agarose gel electrophoresis, pyrosequencing and analysis to calculate the methylation value of the CpG site.
  • the DNA in the above samples was obtained and subjected to bisulfite treatment, nested PCR, agarose gel electrophoresis, pyrosequencing and analysis to calculate the methylation value of the CpG site.
  • results are shown in FIG. 11.
  • the results show that CTSM-3C methylation is significantly lower in the control sample than in lung cancer samples, and CTSM-3C is significantly abnormally hypermethylated in lung cancer, which can be used as a very good lung cancer marker. Further enlarged clinical samples showed the same significance.
  • the sequence of the CTSM-4I tumor marker is provided, as shown in SEQ ID NO: 7, where the box indicates that the base is a potential methylated CpG site.
  • the optimal detection area is the middle underlined area.
  • SEQ ID NO: 7 is as follows:
  • SEQ ID NO: 8 is obtained, where Y represents C (cytosine) or T (thymine):
  • SEQ ID NO: 8 is as follows:
  • the DNA in the above samples was obtained and subjected to bisulfite treatment, nested PCR, agarose gel electrophoresis, pyrosequencing and analysis to calculate the methylation value of the CpG site.
  • the DNA in the above samples was obtained and subjected to bisulfite treatment, nested PCR, agarose gel electrophoresis, pyrosequencing and analysis to calculate the methylation value of the CpG site.
  • the DNA in the above samples was obtained and subjected to bisulfite treatment, nested PCR, agarose gel electrophoresis, pyrosequencing and analysis to calculate the methylation value of the CpG site.
  • the DNA in the above samples was obtained and subjected to bisulfite treatment, nested PCR, agarose gel electrophoresis, pyrosequencing and analysis to calculate the methylation value of the CpG site.

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Abstract

提供一种诊断肿瘤的DNA甲基化相关的标记物及其应用。揭示了基因组中DNA甲基化异常的基因序列区域，命名为肿瘤标志物CTSM-4F、CTSM-2BE、CTSM-3C、CTSM-4I。这些序列区域的甲基化状态在肿瘤组织和非肿瘤组织之间存在显著的差异，在肿瘤组织中其CpG发生高度甲基化。

Description

诊断肿瘤的DNA甲基化相关的标记物及其应用 技术领域
本发明属于诊断领域，更具体地，本发明涉及诊断肿瘤的DNA甲基化相关的标记物及其应用。
背景技术
随着对肿瘤这一疾病的研究的深入，本领域中越来越多的证据表明表观遗传调控的细小变化在肿瘤中具有重要的作用。
表观遗传学是研究基因在不发生DNA序列改变的情况下，基因功能发生的可遗传的变化，并最终导致了表型的变化的一门学科。表观遗传学主要包括DNA甲基化，组蛋白修饰，microRNA水平变化等生化过程。DNA甲基化是指生物体内在DNA甲基转移酶的催化下，以S-腺苷甲硫氨酸为甲基供体，将甲基转移到特定的碱基上的过程。DNA甲基化可以发生在腺嘌呤的N-6位、胞嘧啶的N-4位、鸟嘌呤的N-7位或胞嘧啶的C-5位等。但在哺乳动物中DNA甲基化主要发生在5’-CpG-3’的C上，生成5-甲基胞嘧啶(5mC)。
在基因组中98％以上的CpG二核苷酸散在的分布位于具有转录依赖性的转座潜能的重复序列中。在正常细胞中，这些CpG处于高度甲基化/转录沉默的状态，而在肿瘤细胞中这些CpG发生了广泛的去甲基化，导致重复序列的转录、转座子的活化，基因组的高度不稳定性和原癌基因转录增强。余下的占总量2％左右的CpG密集地分布于较小的区域(CpG岛)。约40％-50％的基因启动子区域或其附近存在CpG岛，暗示DNA甲基化可能参与该类基因转录调控机制。在一些肿瘤中，这些原本在正常细胞中处于低甲基化状态的CpG岛会发生高甲基化而导致基因的转录失活。受影响的基因包括DNA修复基因，细胞周期控制基因和抗凋亡基因等抑癌基因。
鉴于目前临床常用的肿瘤标记物存在很高的假阳性与假阴性，例如甲胎蛋白(AFP)，本领域亟需开发进一步的标志物。找到肿瘤细胞DNA异常甲基化谱式，是研究肿瘤生物标志物的新途径。然而，需要大量的研究以及长期对大量患者的基因鉴定、比较，才能找到真正与肿瘤相关的异常甲基化谱式。
发明内容
本发明的目的在于提供诊断肿瘤的DNA甲基化相关的标记物及其应用。
在本发明的第一方面，提供分离的多核苷酸，包括：
(a)SEQ ID NO:1所示核苷酸序列的多核苷酸；
(b)SEQ ID NO:3所示核苷酸序列的多核苷酸；
(c)SEQ ID NO:5所示核苷酸序列的多核苷酸；
(d)SEQ ID NO:7所示核苷酸序列的多核苷酸；
(e)上述(a)～(d)的多核苷酸的片段，且其中存在至少1个(如2～40个，更具体如3，5，8，10，15，20，25，30，35个)修饰的CpG位点；和/或
(f)与上述(a)～(E)的多核苷酸或片段互补的核酸(反义链)。
在一个优选例中，所述修饰包括5-甲基化修饰(5mC)、5-羟甲基化修饰(5hmC)、5-醛甲基化修饰(5-fC)或5-羧甲基化(5-caC)修饰。
在本发明的另一方面，提供分离的多核苷酸，其由前面所述的多核苷酸转变而来，对应于上述(a)～(e)的序列，其修饰的CpG位点的胞嘧啶C不变，非修饰的胞嘧啶转为T(U)。
在一个优选例中，其由对应于上述(a)～(e)的多核苷酸经过亚硫酸氢盐或重亚硫酸氢盐处理转变而来。
在另一优选例中，所述的多核苷酸包括：
(g)SEQ ID NO:2所示核苷酸序列的多核苷酸；
(h)SEQ ID NO:4所示核苷酸序列的多核苷酸；
(i)SEQ ID NO:6所示核苷酸序列的多核苷酸；
(j)SEQ ID NO:8所示核苷酸序列的多核苷酸；
(k)上述(g)～(j)的多核苷酸的片段，且其中存在至少1个(如2～40个，更具体如3，5，8，10，15，20，25，30，35个)修饰的CpG位点。
在本发明的另一方面，提供前面任一所述的多核苷酸的用途，用于制备肿瘤的检测试剂或试剂盒。
在一个优选例中，所述的肿瘤包括：消化系统肿瘤如食道癌，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
在本发明的另一方面，提供一种制备肿瘤检测试剂的方法，所述方法包括：提供前面任一所述的多核苷酸(如1，2，3或4条)，以所述多核苷酸的全长或片段作为靶序列，设计特异性检测该靶序列的检测试剂；其中，所述的靶序列中存在至少1个(如2～40个， 更具体如3，5，8，10，15，20，25，30，35个)修饰的CpG位点；较佳地，所述的检测试剂包括(但不限于)：引物，探针。
在本发明的另一方面，提供试剂或组合的试剂，其特异性检测靶序列，所述的靶序列是前面任一所述的多核苷酸的全长或片段，其中存在至少1个(如2～40个，更具体如3，5，8，10，15，20，25，30，35个)修饰的CpG位点；较佳地，所述的检测试剂包括(但不限于)：引物，探针。
在一个优选例中，所述的多核苷酸为SEQ ID NO:1所示核苷酸序列的多核苷酸，所述的靶序列包含SEQ ID NO:1中第240～296位的核苷酸片段；较佳地，所述的引物为SEQ ID NO:9～12所示的引物。
在另一优选例中，所述的多核苷酸为SEQ ID NO:3所示核苷酸序列的多核苷酸，所述的靶序列包含SEQ ID NO:3中第279～323位的核苷酸片段；较佳地，所述的引物为SEQ ID NO:12～15所示的引物；或
在另一优选例中，所述的多核苷酸为SEQ ID NO:5所示核苷酸序列的多核苷酸，所述的靶序列包含SEQ ID NO:5中第186～235位的核苷酸片段；较佳地，所述的引物为SEQ ID NO:12，16～18所示的引物；或
在另一优选例中，所述的多核苷酸为SEQ ID NO:7所示核苷酸序列的多核苷酸，所述的靶序列包含SEQ ID NO:7中第164～198位的核苷酸片段；较佳地，所述的引物为SEQ ID NO:12，19～21所示的引物。
在本发明的另一方面，提供所述的试剂或组合的试剂的用途，用于制备检测肿瘤的试剂盒；较佳地，所述的肿瘤包括：消化系统肿瘤如食道癌，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
在本发明的另一方面，提供一种检测试剂盒，其包括：
容器，以及位于容器中的所述的试剂或试剂组合；较佳地，每一种试剂位于一个独立的容器中。
在一个优选例中，所述的试剂盒中还包括：亚硫酸氢盐或重亚硫酸氢盐，DNA纯化试剂，DNA提取试剂，PCR扩增试剂和/或使用说明书(标明检测操作步骤和结果判定标准)。
在本发明的另一方面，提供一种体外检测样品中所述的多核苷酸或其片段的甲基化 谱式的方法，包括：
(i)提供样品，提取DNA；
(ii)对待测样本进行处理，使未发生修饰的胞嘧啶转化为尿嘧啶；较佳地，所述修饰包括5-甲基化修饰(5mC)、5-羟甲基化修饰(5hmC)、5-醛甲基化修饰或5-羧甲基化修饰；较佳地，利用亚硫酸氢盐或重亚硫酸氢盐处理步骤(i)所述的DNA；
(iii)分析经步骤(ii)处理的基因组DNA中所述的多核苷酸或其片段的修饰情况。
在一个优选例中，所述的甲基化谱式异常是指该多核苷酸CpG中的C发生高度甲基化。
在另一优选例中，所述的甲基化谱式的方法不以直接获得疾病的诊断结果为目的，或不是诊断性的方法。
在另一优选例中，步骤(3)中，分析的方法包括(但不限于)：焦磷酸测序法、重亚硫酸盐转化测序法、qPCR法、二代测序法、全基因组甲基化测序法、DNA富集检测法、简化亚硫酸氢盐测序技术、HPLC法、或它们的组合。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、CTSM-4F在结直肠癌中检测结果。
图2、CTSM-4F在肝癌中检测结果。
图3、CTSM-4F在头颈部肿瘤中检测结果。
图4、CTSM-4F在肺癌中检测结果。
图5、CTSM-2BE在乳腺癌中检测结果。
图6、CTSM-2BE在头颈部肿瘤中检测结果。
图7、CTSM-2BE在肺癌中检测结果。
图8、CTSM-2BE在胰腺癌中检测结果。
图9、CTSM-3C在结直肠癌中检测结果。
图10、CTSM-3C在头颈部癌中检测结果。
图11、CTSM-3C在肺癌中检测结果。
图12、CTSM-4I在乳腺癌中检测结果。
图13、CTSM-4I在结直肠癌中检测结果。
图14、CTSM-4I在头颈部癌中检测结果。
图15、CTSM-4I在肺癌中检测结果。
具体实施方式
本发明人致力于肿瘤标志物的研究，经过广泛的研究筛选，鉴定到基因组中DNA甲基化异常的基因序列区域，命名为肿瘤标志物CTSM-4F、CTSM-2BE、CTSM-3C、CTSM-4I。针对临床样本的研究显示，这些序列区域的甲基化状态在肿瘤组织和非肿瘤组织之间存在显著的差异，其CpG发生高度甲基化。因此，这些基因是肿瘤的标志物；可作为设计肿瘤诊断试剂的基础。
术语
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多核苷酸和多肽是没有分离纯化的，但同样的多核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
如本文所用，“样本”或“样品”包括从任何个体或分离的组织、细胞或体液中获得的、适合于DNA甲基化状态检测的物质。
如本文所用，“高(度)甲基化”是指在一个基因序列中CpG存在高度甲基化、羟甲基化、醛甲基化或羧甲基化修饰。例如，以甲基化特异PCR(MSP)分析手段而言，以甲基化特异性引物所进行的PCR反应可获得阳性的PCR结果即可认为该受试的DNA(基因)区处于高甲基化状态。例如，以实时定量甲基化特异性PCR而言，高甲基化状态的判定可根据其对照样品的甲基化状态的相对值分析统计学差异。
如本文所用，所述的肿瘤包括但不限于：消化系统肿瘤如食道癌，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
基因标志物
为了寻找对于诊断肿瘤有用的靶标，本发明人经过了广泛而深入的研究，最终找到了一组基因序列区域，它们是CTSM-4F、CTSM-2BE、CTSM-3C、CTSM-4I。这些基因序列区域的甲基化状态在肿瘤组织和非肿瘤组织之间存在显著的差异，只要检测到其中一个上述基因的启动子区域发生异常的甲基化状态(高度甲基化)，即可判定该受检者为肿瘤高危人员。
因此，本发明提供了分离的多核苷酸，所述的多核苷酸来自于人基因组，具有SEQ ID  NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7所示核苷酸序列，在肿瘤患者的肿瘤细胞内，该多核苷酸序列中，多处5’-CpG-3’的碱基C位置上，生成5-甲基胞嘧啶(5mC)。本发明也包含SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7所示核苷酸序列的多核苷酸的片段，且其中存在至少1个(如2～40个，更具体如3，5，8，10，15，20，25，30，35个)甲基化CpG位点。上述的多核苷酸或片段或互补链(反义链)也可以应用于设计检测试剂或检测试剂盒。
上述的多核苷酸可以作为基因组中人们分析甲基化状态的关键区域，通过各种本领域已知的技术来分析它们的甲基化状态。任何可用于分析甲基化状态的技术均可被应用于本发明。
上述的多核苷酸在经过亚硫酸氢盐或重亚硫酸氢盐处理后，其中未发生甲基化的胞嘧啶转化为尿嘧啶，而发生甲基化的胞嘧啶保持不变。
因此，本发明还提供了上述多核苷酸(包括其互补链(反义链))经过亚硫酸氢盐或重亚硫酸氢盐处理后获得的多核苷酸，包括：SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8所示核苷酸序列的多核苷酸。这些多核苷酸也可以应用于设计检测试剂或检测试剂盒。
本发明也包含上述多核苷酸(包括其互补链(反义链))经过亚硫酸氢盐或重亚硫酸氢盐处理后获得的多核苷酸的片段，且其中存在至少1个甲基化CpG位点。
检测试剂及试剂盒
基于本发明的新发现，还提供了基于所述的多核苷酸序列设计的检测试剂，用于体外检测样品中多核苷酸的甲基化谱式。本领域已知的确定基因组的序列及甲基化状态的检测方法和试剂均可被应用于本发明中。
因此，本发明提供了一种制备肿瘤检测试剂的方法，包括：提供所述的多核苷酸(1，2，3或4条)，以所述多核苷酸的全长或片段作为靶序列，设计特异性检测该靶序列的检测试剂；其中，所述的靶序列中存在至少1个甲基化CpG位点。
在本发明的优选方式中，所述的靶序列包含SEQ ID NO:1中第240～296位的核苷酸片段；或包含SEQ ID NO:3中第279～323位的核苷酸片段；或包含SEQ ID NO:5中第186～235位的核苷酸片段；较佳地，所述的引物为SEQ ID NO:12，16～18所示的引物；或包含SEQ ID NO:7中第164～198位的核苷酸片段。
所述的检测试剂包括但不限于：引物，探针，等等。
所述的试剂例如是引物对，在得知了多核苷酸的序列后，设计引物是本领域技术人员已知的，两个引物在将被扩增的目标基因特定序列的两侧(包含CpG序列在内，与其中 CpG互补为针对原为甲基化的基因区，而与其中TpG互补为针对原为去甲基化的基因区)。
所述的试剂也可以是试剂组合(引物组合)，包括多于一组的引物，从而可分别扩增上述的多条多核苷酸。
作为本发明的优选方式，所述的引物为SEQ ID NO:9～12所示的引物；或为SEQ ID NO:12～15所示的引物；或为SEQ ID NO:12，16～18所示的引物；或为SEQ ID NO:12，19～21所示的引物。上述引物为应用于进行巢式PCR的引物，通过两轮PCR扩增反应，获得的扩增产物用于序列鉴定。上述的引物扩增获得的产物具有合适的长度，且特异性高，对于复杂体系的扩增也具有良好的特异性，特别适合用于甲基化特异性PCR。
本发明还提供了体外检测样品中多核苷酸的甲基化谱式的试剂盒，该试剂盒包括：容器，以及位于容器中的上述引物对。
此外，所述的试剂盒中还可包括用于提取DNA、DNA纯化、PCR扩增等所需的各种试剂。
此外，所述的试剂盒中还可包括使用说明书，其中标明检测操作步骤和结果判定标准，以便于本领域技术人员应用。
检测方法
测定多核苷酸的甲基化谱式可通过已有的技术(如甲基化特异性PCR(MSP)或实时定量甲基化特异性PCR，Methylight)来进行，或其它仍在发展中和将被开发出来的技术来进行。
检测甲基化水平时也可使用定量甲基化特异性PCR(QMSP)的方法。这种方法是基于一种荧光PCR的持续性的光学监控，其较MSP方法更为敏感。其通量高并避免了用电泳方法对其结果进行分析。
其他可用的技术还有：焦磷酸测序法、重亚硫酸盐转化测序法、qPCR法、二代测序法、全基因组甲基化测序法、DNA富集检测法、简化亚硫酸氢盐测序技术或HPLC法以及组合基因群组检测法等该领域常规方法。应理解，在本发明的新揭示的基础上，本领域公知的这些技术以及即将发展的一些技术，均可被应用于本发明中。
作为本发明的优选方式，还提供了一种体外检测样品中多核苷酸的甲基化谱式的方法。所述的方法基于的原理是：亚硫酸氢盐或重亚硫酸氢盐可以将未甲基化的胞嘧啶转化为尿嘧啶，在后续的PCR扩增过程中转变为胸腺嘧啶，而甲基化的胞嘧啶保持不变；因而，经过亚硫酸氢盐或重亚硫酸氢盐处理多核苷酸后，甲基化的位点产生类似于一个C/T的多核苷酸多态性(SNP)。基于上述原理来鉴定检测样品中多核苷酸的甲基化谱式，可以有效区分出甲基化与非甲基化的胞嘧啶。
本发明所述的方法包括：包括：(a)提供样品，提取基因组DNA；(b)利用亚硫酸氢盐或重亚硫酸氢盐处理步骤(a)所述的基因组DNA，从而基因组DNA中未甲基化的胞嘧啶转化为尿嘧啶；(c)分析经步骤(b)处理的基因组DNA中是否存在甲基化谱式异常。
本发明的方法可用于：(i)对受试者样品进行检测，分析受试者是否患有肿瘤；(ii)区分肿瘤高危人群。所述的方法也可以是不以获得直接的疾病诊断结果为目的的情形。
在本发明的优选实施例中，通过PCR扩增及焦磷酸测序法检测DNA甲基化，本领域人员应理解，实际应用中并不限于该方法，其它DNA甲基化检测方法亦可。在进行PCR扩增中，所应用的引物也不限于是实施例中所提供的。
在本发明的优选实施例中，由于基因组DNA经过重亚硫酸盐处理后，非甲基化的胞嘧啶转变为尿嘧啶，在后续的PCR过程中又转换为胸腺嘧啶，会降低基因组的序列复杂度，使得PCR扩增出特异目标片段的难度增大。本发明人发现，采用巢式PCR扩增，设计外围与内围两对引物，进行两轮PCR扩增反应，以第一轮的扩增产物作为第二轮扩增的模板，可以有效提高扩增的效率与特异性。应理解，本发明中可用的检测方法并不限于此。
经过针对临床样本的研究验证，本发明的方法用于诊断临床肿瘤时，准确性非常高，具有很高的临床应用价值。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、肿瘤标记物CTSM-4F
一、检测序列描述
本实施例中，提供CTSM-4F肿瘤标记物的序列，如SEQ ID NO:1所示，其中方框标示碱基为潜在甲基化CpG位点，最优检测区域为中间下划线区域。
SEQ ID NO:1如下：
Figure PCTCN2019099722-appb-000001
Figure PCTCN2019099722-appb-000002
二、检测步骤
1、待测样本获取：选取临床组织样本，包括癌组织以及对应的癌旁对照组织。
2、DNA提取：通过常规的酚氯仿提取待测样本基因组DNA。
3、通过焦磷酸测序法检测DNA甲基化，DNA甲基化检测步骤如下：
(1)重亚硫酸盐处理操作：取5～200ng前述提取获得的基因组DNA进行重亚硫酸盐处理，本步骤中使用ZYMO Research公司的Methylation-Gold TM Kit试剂盒(货号D5006)，严格按照使用说明书进行处理，最终用20ul洗脱液洗脱，作为后续PCR扩增模板。
(2)经过重亚硫酸盐处理后，获得SEQ ID NO:2，其中，Y代表C(胞嘧啶)或T(胸腺嘧啶)。相应于SEQ ID NO:1，原存在甲基化修饰的胞嘧啶C不变，原非甲基化修饰的胞嘧啶转为T(U)，所以用Y代表C或T。
SEQ ID NO:2如下：
Figure PCTCN2019099722-appb-000003
Figure PCTCN2019099722-appb-000004
在SEQ ID NO:2的基础上，根据巢式PCR以及焦磷酸测序特点，提供一组扩增引物以及测序引物，如表1(对应于SEQ ID NO:2中双下划线或字体加粗序列)：
表1
Figure PCTCN2019099722-appb-000005
(3)巢式PCR扩增：使用的PCR扩增试剂为莱枫公司生产的2×PCR Master Mix试剂，货号PT102，但不限于该试剂。
第一轮PCR扩增体系：
成分 体积
2×PCR Mix 5ul
CTSM-4F-F1(10uM) 0.1ul
CTSM-4F-R1(10uM) 0.1ul
模板 1ul
双蒸水 3.8ul
第一轮PCR扩增程序：
Figure PCTCN2019099722-appb-000006
第一轮PCR扩增结束后，以第一轮PCR扩增后的产物作为模板进行第二轮PCR扩增，体系如下：
2×PCR Mix 13ul
CTSM-4F-F2(10uM) 0.5ul
CTSM-4F-R2(5’末端生物素修饰)(10uM) 0.5ul
模板 4ul
双蒸水 12ul
第二轮扩增程序如下：
Figure PCTCN2019099722-appb-000007
(4)琼脂糖凝胶电泳：为了鉴定PCR扩增产物的特异性，通过琼脂糖凝胶电泳鉴定，扩增产物长度应该为177bp。
(5)焦磷酸测序：通过QIAGEN公司生产的焦磷酸测序仪，按照具体步骤操作，检测出目标区域每个CpG位点的甲基化值。
(6)分析计算：通过本焦磷酸测序步骤共检测11个CpG位点的甲基化值，计算平均值作为样本在该区域的最终甲基化值。此处采用平均值法，其他计算方法如加和、中位数等亦可。
三、结果分析
通过分析样本甲基化值，可以有效区分肿瘤样本与对照样本，在对照组样本中，CTSM-4F处于低甲基化状态，在肿瘤样本中，CTSM-4F处于高甲基化状态。
四、临床检测和分析
1、直肠癌的检测和分析
从获自医院的已确诊的直肠癌样本中，随机选取4例结直肠癌样本，并选取对应癌旁组织作为对照样本。
应用前述步骤二～三的方法，获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图1，该结果显示，在对照样本中CTSM-4F的甲基化显著低于结直肠癌样本， CTSM-4F在结直肠癌中显著异常高甲基化，可以作为非常好的结直肠癌标记物。进一步扩大的临床样本呈现同样的显著性。
2、肝癌的检测和分析
从获自医院的已确诊的肝癌样本中，随机选取8例肝癌样本，并选取对应癌旁组织作为对照样本。
应用前述步骤二～三的方法，获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图2，该结果显示，在对照样本中CTSM-4F的甲基化显著低于肝癌样本，CTSM-4F在肝癌中显著异常高甲基化，可以作为非常好的肝癌标记物。进一步扩大的临床样本呈现同样的显著性。
3、头颈部肿瘤的检测和分析
从获自医院的已确诊的头颈部肿瘤样本中，随机选取5例头颈部肿瘤样本，并选取对应癌旁组织作为对照样本。
应用前述步骤二～三的方法，获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图3，该结果显示，在对照样本中CTSM-4F的甲基化显著低于头颈部肿瘤样本，CTSM-4F在头颈部肿瘤中显著异常高甲基化，可以作为非常好的头颈部肿瘤标记物。进一步扩大的临床样本呈现同样的显著性。
4、肺癌的检测和分析
从获自医院的已确诊的肺癌样本中，随机选取5例肺癌血浆样本作为实验组，并选取5例正常人血浆样本作为对照组。
应用前述步骤二～三的方法，获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图4，该结果显示，在对照组样本中CTSM-4F的甲基化显著低于实验组，CTSM-4F在肺癌中显著异常高甲基化，可以作为非常好的肺癌标记物。进一步扩大的临床样本呈现同样的显著性。
实施例2、肿瘤标记物CTSM-2BE
一、检测序列描述
本实施例中，提供CTSM-2BE肿瘤标记物的序列，如SEQ ID NO:3所示，其中方框标示碱基为潜在甲基化CpG位点。最优检测区域为中间下划线区域。
SEQ ID NO:3如下：
Figure PCTCN2019099722-appb-000008
经过重亚硫酸盐处理后，获得SEQ ID NO:4，其中，Y代表C(胞嘧啶)或T(胸腺嘧啶)。
SEQ ID NO:4如下：
Figure PCTCN2019099722-appb-000009
在SEQ ID NO:4的基础上，根据巢式PCR以及焦磷酸测序特点，提供一组扩增引物以及测序引物，如表2。
表2
Figure PCTCN2019099722-appb-000010
应用上述引物，采用如实施例1中相同的方法进行待测样本的DNA提取，并进行重亚硫酸盐处理、巢式PCR扩增、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲 基化值。
二、临床检测和分析
1、乳腺癌的检测和分析
从获自医院的已确诊的乳腺癌样本中，随机选取8例乳腺癌样本，并选取对应癌旁组织作为对照样本。
获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图5，该结果显示，在对照样本中CTSM-2BE的甲基化显著低于乳腺癌样本，CTSM-2BE在乳腺癌中显著异常高甲基化，可以作为非常好的乳腺癌标记物。进一步扩大的临床样本呈现同样的显著性。
2、头颈部肿瘤的检测和分析
从获自医院的已确诊的头颈部肿瘤样本中，随机选取6例头颈部肿瘤样本，并获取对应癌旁组织作为对照样本。
获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图6，该结果显示，在对照样本中CTSM-2BE的甲基化显著低于头颈部肿瘤样本，CTSM-2BE在头颈部肿瘤中显著异常高甲基化，可以作为非常好的头颈部肿瘤标记物。进一步扩大的临床样本呈现同样的显著性。
3、肺癌的检测和分析
从获自医院的已确诊的肺癌样本中，随机选取8例肺癌样本，并获取对应癌旁组织作为对照样本。
获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图7，该结果显示，在对照样本中CTSM-2BE的甲基化显著低于肺癌样本，CTSM-2BE在肺癌中显著异常高甲基化，可以作为非常好的肺癌标记物。进一步扩大的临床样本呈现同样的显著性。
4、胰腺癌的检测和分析
从获自医院的已确诊的胰腺癌样本中，随机选取8例胰腺癌样本，并获取对应癌旁 组织作为对照样本。
获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图8，该结果显示，在对照样本中CTSM-2BE的甲基化显著低于胰腺癌样本，CTSM-2BE在胰腺癌中显著异常高甲基化，可以作为非常好的胰腺癌标记物。进一步扩大的临床样本呈现同样的显著性。
实施例3、肿瘤标记物CTSM-3C
一、检测序列描述
本实施例中，提供CTSM-3C肿瘤标记物的序列，如SEQ ID NO:5所示，其中方框标示碱基为潜在甲基化CpG位点。最优检测区域为中间下划线区域。
SEQ ID NO:5如下：
Figure PCTCN2019099722-appb-000011
经过重亚硫酸盐处理后，获得SEQ ID NO:6，其中，Y代表C(胞嘧啶)或T(胸腺嘧啶)：
SEQ ID NO:6如下：
Figure PCTCN2019099722-appb-000012
在SEQ ID NO:6的基础上，根据巢式PCR以及焦磷酸测序特点，提供一组扩增引物 以及测序引物，如表3。
表3
Figure PCTCN2019099722-appb-000013
应用上述引物，采用如实施例1中相同的方法进行待测样本的DNA提取，并进行重亚硫酸盐处理、巢式PCR扩增、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
二、临床检测和分析
1、结直肠癌的检测和分析
从获自医院的已确诊的结直肠癌样本中，随机选取9例结直肠癌样本，并选取对应癌旁组织作为对照样本。
获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图9，该结果显示，在对照样本中CTSM-3C的甲基化显著低于结直肠癌样本，CTSM-3C在结直肠癌中显著异常高甲基化，可以作为非常好的结直肠癌标记物。进一步扩大的临床样本呈现同样的显著性。
2、头颈部癌的检测和分析
从获自医院的已确诊的头颈部癌样本中，随机选取5例头颈部癌样本，并选取对应癌旁组织作为对照样本。
获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图10，该结果显示，在对照样本中CTSM-3C的甲基化显著低于头颈部癌样本，CTSM-3C在头颈部癌中显著异常高甲基化，可以作为非常好的头颈部癌标记物。进 一步扩大的临床样本呈现同样的显著性。
3、肺癌的检测和分析
从获自医院的已确诊的肺癌样本中，随机选取6例肺癌样本，并选取对应癌旁组织作为对照样本。
获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图11，该结果显示，在对照样本中CTSM-3C的甲基化显著低于肺癌样本，CTSM-3C在肺癌中显著异常高甲基化，可以作为非常好的肺癌标记物。进一步扩大的临床样本呈现同样的显著性。
实施例4、肿瘤标记物CTSM-4I
一、检测序列描述
本实施例中，提供CTSM-4I肿瘤标记物的序列，如SEQ ID NO:7所示，其中方框标示碱基为潜在甲基化CpG位点。最优检测区域为中间下划线区域。
SEQ ID NO:7如下：
Figure PCTCN2019099722-appb-000014
经过重亚硫酸盐处理后，获得SEQ ID NO:8，其中，Y代表C(胞嘧啶)或T(胸腺嘧啶)：
SEQ ID NO:8如下：
Figure PCTCN2019099722-appb-000015
在SEQ ID NO:8的基础上，根据巢式PCR以及焦磷酸测序特点，提供一组扩增引物以及测序引物，如表4。
表4
Figure PCTCN2019099722-appb-000016
Figure PCTCN2019099722-appb-000017
二、临床检测和分析
1、乳腺癌的检测和分析
从获自医院的已确诊的乳腺癌样本中，随机选取4例乳腺癌样本，并选取对应癌旁组织作为对照样本。
获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图12，该结果显示，在对照样本中CTSM-4I的甲基化显著低于乳腺癌样本，CTSM-4I在肺癌中显著异常高甲基化，可以作为非常好的乳腺癌标记物。
2、结直肠癌的检测和分析
从获自医院的已确诊的结直肠癌样本中，随机选取5例结直肠癌样本，并选取对应癌旁组织作为对照样本。
获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图13，该结果显示，在对照样本中CTSM-4I的甲基化显著低于结直肠癌样本，CTSM-4I在结直肠癌中显著异常高甲基化，可以作为非常好的结直肠癌标记物。
3、头颈部癌的检测和分析
从获自医院的已确诊的头颈部癌样本中，随机选取6例头颈部癌样本，并选取对应癌旁组织作为对照样本。
获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图14，该结果显示，在对照样本中CTSM-4I的甲基化显著低于头颈部癌样本，CTSM-4I在头颈部癌中显著异常高甲基化，可以作为非常好的头颈部癌标记物。进一步 扩大的临床样本呈现同样的显著性。
4、肺癌的检测和分析
从获自医院的已确诊的肺癌样本中，随机选取7例肺癌样本，并选取对应癌旁组织作为对照样本。
获取上述样本中的DNA，并进行重亚硫酸盐处理、巢式PCR、琼脂糖凝胶电泳、焦磷酸测序及分析计算CpG位点的甲基化值。
结果如图15，该结果显示，在对照样本中CTSM-4I的甲基化显著低于肺癌样本，CTSM-4I在肺癌中显著异常高甲基化，可以作为非常好的肺癌标记物。进一步扩大的临床样本呈现同样的显著性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (13)

  1. 分离的多核苷酸，其特征在于，包括：
    (a)SEQ ID NO:1所示核苷酸序列的多核苷酸；
    (b)SEQ ID NO:3所示核苷酸序列的多核苷酸；
    (c)SEQ ID NO:5所示核苷酸序列的多核苷酸；
    (d)SEQ ID NO:7所示核苷酸序列的多核苷酸；
    (e)上述(a)～(d)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点；和/或
    (f)与上述(a)～(e)的多核苷酸或片段互补的核酸。
  2. 如权利要求1所述的分离的多核苷酸，其特征在于，所述修饰包括5-甲基化修饰、5-羟甲基化修饰、5-醛甲基化修饰或5-羧甲基化修饰。
  3. 分离的多核苷酸，其特征在于，其由权利要求1或2所述的多核苷酸转变而来，对应于权利要求1的序列，其修饰的CpG位点的胞嘧啶C不变，非修饰的胞嘧啶转为T。
  4. 如权利要求3所述的多核苷酸，其特征在于，包括：
    (g)SEQ ID NO:2所示核苷酸序列的多核苷酸；
    (h)SEQ ID NO:4所示核苷酸序列的多核苷酸；
    (i)SEQ ID NO:6所示核苷酸序列的多核苷酸；
    (j)SEQ ID NO:8所示核苷酸序列的多核苷酸；
    (k)上述(g)～(j)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点。
  5. 权利要求1～4任一所述的多核苷酸的用途，其特征在于，用于制备肿瘤的检测试剂或试剂盒。
  6. 如权利要求5所述的用途，其特征在于，所述的肿瘤包括：消化系统肿瘤如食道癌，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
  7. 一种制备肿瘤检测试剂的方法，其特征在于，所述方法包括：提供权利要求1～4任一所述的多核苷酸，以所述多核苷酸的全长或片段作为靶序列，设计特异性检测该靶序列的检测试剂；其中，所述的靶序列中存在至少1个修饰的CpG位点；较佳地，所述的检测试剂包括：引物，探针。
  8. 试剂或组合的试剂，其特征在于，其特异性检测靶序列，所述的靶序列是权利要求1～4任一所述的多核苷酸的全长或片段，其中存在至少1个修饰的CpG位点；较佳地，所述的检测试剂包括：引物，探针。
  9. 如权利要求8所述的试剂或组合的试剂，其特征在于，所述的多核苷酸为SEQ ID NO:1所示核苷酸序列的多核苷酸，所述的靶序列包含SEQ ID NO:1中第240～296位的核苷酸片段；较佳地，所述的引物为SEQ ID NO:9～12所示的引物；或
    所述的多核苷酸为SEQ ID NO:3所示核苷酸序列的多核苷酸，所述的靶序列包含SEQ ID NO:3中第279～323位的核苷酸片段；较佳地，所述的引物为SEQ ID NO:12～15所示的引物；或
    所述的多核苷酸为SEQ ID NO:5所示核苷酸序列的多核苷酸，所述的靶序列包含SEQ ID NO:5中第186～235位的核苷酸片段；较佳地，所述的引物为SEQ ID NO:12，16～18所示的引物；或
    所述的多核苷酸为SEQ ID NO:7所示核苷酸序列的多核苷酸，所述的靶序列包含SEQ ID NO:7中第164～198位的核苷酸片段；较佳地，所述的引物为SEQ ID NO:12，19～21所示的引物。
  10. 权利要求8或9所述的试剂或组合的试剂的用途，用于制备检测肿瘤的试剂盒；较佳地，所述的肿瘤包括：消化系统肿瘤如食道癌，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；呼吸系统肿瘤如肺癌，胸膜瘤；血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；妇科及生殖系统肿瘤如乳腺癌，卵巢癌，宫颈癌，外阴癌，睾丸癌，前列腺癌，阴茎癌；神经系统肿瘤如胶质瘤，神经母细胞瘤，脑膜瘤；头颈部肿瘤如口腔癌，舌癌，喉癌，鼻咽癌；泌尿系统肿瘤如肾癌，膀胱癌，皮肤及其他系统如皮肤癌、黑色素瘤、骨肉瘤，脂肪肉瘤，甲状腺癌。
  11. 一种检测试剂盒，其特征在于，其包括：
    容器，以及位于容器中的权利要求8或9所述的试剂或试剂组合。
  12. 一种体外检测样品中权利要求1所述的多核苷酸或其片段的甲基化谱式的方法，其特征在于，包括：
    (i)提供样品，提取DNA；
    (ii)对待测样本进行处理，使未发生修饰的胞嘧啶转化为尿嘧啶；较佳地，所述修饰包括5-甲基化修饰、5-羟甲基化修饰、5-醛甲基化修饰或5-羧甲基化修饰；较佳地，利用亚硫酸氢盐或重亚硫酸氢盐处理步骤(i)所述的DNA；
    (iii)分析经步骤(ii)处理的基因组DNA中权利要求2或3所述的多核苷酸或其片段的修饰情况。
  13. 如权利要求12所述的方法，其特征在于，步骤(3)中，分析的方法包括：焦磷酸测序法、重亚硫酸盐转化测序法、qPCR法、二代测序法、全基因组甲基化测序法、DNA富集检测法、简化亚硫酸氢盐测序技术、HPLC法、或它们的组合。
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