WO2020135861A1 - 基于甲基化修饰的肿瘤标记物stamp-ep4 - Google Patents

基于甲基化修饰的肿瘤标记物stamp-ep4 Download PDF

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WO2020135861A1
WO2020135861A1 PCT/CN2019/129827 CN2019129827W WO2020135861A1 WO 2020135861 A1 WO2020135861 A1 WO 2020135861A1 CN 2019129827 W CN2019129827 W CN 2019129827W WO 2020135861 A1 WO2020135861 A1 WO 2020135861A1
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cancer
samples
seq
methylation
polynucleotide
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PCT/CN2019/129827
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English (en)
French (fr)
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李振艳
罗怀兵
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上海奕谱生物科技有限公司
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Priority to US17/309,901 priority Critical patent/US20220177972A1/en
Priority to JP2021538269A priority patent/JP7399169B2/ja
Priority to EP19901853.2A priority patent/EP3950945A4/en
Publication of WO2020135861A1 publication Critical patent/WO2020135861A1/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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the invention belongs to the field of disease diagnosis markers. More specifically, the invention relates to a tumor marker STAMP (Specific Tumor Aligned Methylation of Pan-cancer) based on methylation modification.
  • STAMP Specific Tumor Aligned Methylation of Pan-cancer
  • tumor markers were mainly cell secretions such as hormones, enzymes, and proteins.
  • CCA carcinoembryonic antigen
  • AFP alpha-fetoprotein
  • CA125 can be used as a marker for cervical cancer
  • PSA prostate specific antigen
  • DNA methylation is one of the epigenetic mechanisms, which refers to S-adenosylmethionine (SAM) as the methyl group in the organism catalyzed by DNA methyltransferase (DMT) The process of transferring methyl groups to specific bases.
  • SAM S-adenosylmethionine
  • DMT DNA methyltransferase
  • Liquid biopsy technology is a technique for diagnosing and predicting tumors by using circulating tumor cells or circulating tumor DNA in blood as detection targets.
  • the sensitivity and specificity are not high enough, the tumor itself is very heterogeneous, and contains multiple subtypes of cell populations, and clinical samples, especially blood samples, have a very small proportion of tumor DNA .
  • the existing tumor markers are difficult to meet the sensitivity of clinical requirements, and are likely to cause misdiagnosis in the clinic; secondly, one marker only has a good effect on one or a few tumors, and the source of DNA in the blood is very complicated. Therefore, existing tumor markers cannot cope with complex tumor sources, metastasis and other problems. Due to the existence of these complex situations, it is difficult for many DNA methylated tumor markers to have a uniform use standard when applied to the clinic, which seriously affects the sensitivity and accuracy of the markers.
  • the object of the present invention is to provide a method for detecting tumors by using DNA methylation modification as a tumor marker and utilizing abnormal hypermethylation at specific sites in the tumor.
  • an isolated polynucleotide including: (a) a polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1; (b) a nucleotide sequence shown in SEQ ID NO: 2 Polynucleotide; (c) a fragment of the polynucleotide of (a) to (b) above, and at least one modified CpG site (such as 2 to 54 or 2 to 204, more specifically 3 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 80, 100, 120, 140, 160, 180, 200); (d) and (a) ⁇ (c ) Polynucleotides or fragments complementary nucleic acids (such as SEQ ID NO: 5 or SEQ ID NO: 6 nucleotide sequence shown in polynucleotide).
  • the modification includes 5-methylation modification, 5-hydroxymethylation modification, 5-aldehyde methylation modification or 5-carboxymethylation modification.
  • an isolated polynucleotide which is converted from the polynucleotide, corresponding to the sequence of the aforementioned first aspect, the cytosine C of the modified CpG site is unchanged, non The modified cytosine is converted to T or U.
  • the polynucleotide includes: (e) a polynucleotide having a nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 7; (f) SEQ ID NO: 4 or SEQ ID NO: polynucleotide of the nucleotide sequence shown in 8; (g) the fragment of the polynucleotide of (e) to (f) above, and there are at least one modified CpG site (such as 2 to 54) Or 2 to 204, more specifically 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 80, 100, 120, 140, 160, 180, 200).
  • modified CpG site such as 2 to 54
  • 2 to 204 more specifically 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 80, 100, 120, 140, 160, 180, 200.
  • a third aspect of the present invention there is provided the use of the polynucleotide according to the aforementioned first or second aspect, for preparing a tumor detection reagent or kit.
  • the tumors include (but are not limited to): hematological tumors such as leukemia, lymphoma, multiple myeloma; digestive system tumors such as esophageal cancer (esophageal cancer), gastric cancer, colorectal cancer, liver cancer, pancreas Cancer, bile duct and gallbladder cancer; respiratory system tumors such as lung cancer, pleural tumor; nervous system tumors such as glioma, neuroblastoma, meningioma; head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer; Gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; urinary system tumors such as kidney cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma , Liposarcoma, thyroid cancer.
  • hematological tumors such as le
  • the tumor samples include: tissue samples, paraffin embedded samples, blood samples, pleural effusion samples and alveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, fecal samples, urine Fluid samples, saliva samples, sputum samples, cerebrospinal fluid samples, cell smear samples, cervical scraping or brush samples, tissue and cell biopsy samples.
  • a method for preparing a tumor detection reagent comprising: providing the polynucleotide according to the first aspect or the second aspect, with the full-length or fragment of the polynucleotide As the target sequence, design a detection reagent that specifically detects the modification of the CpG site of the target sequence; wherein, there are at least one of the target sequence (such as 2 to 54 or 2 to 204, more specifically, 3, 5,10,15,20,25,30,35,40,45,50,60,80,100,120,140,160,180,200) modified CpG sites; preferably, the Detection reagents include (but are not limited to): primers, probes.
  • a reagent or a combined reagent which specifically detects the modification of the CpG site of the target sequence, and the target sequence is the polynucleotide according to any one of the foregoing first aspect or second aspect
  • the target sequence is the polynucleotide according to any one of the foregoing first aspect or second aspect
  • the reagent or the combined reagent is directed to a gene sequence (designed based on the gene sequence) including the target sequence, and the gene sequence includes a gene panel or a gene group.
  • the detection reagents include: primers and probes.
  • the primers are selected from the group consisting of primers shown in SEQ ID NO: 9 and 10; primers shown in SEQ ID NO: 11 and 12; primers shown in SEQ ID NO: 13 and 14. Primers; primers shown in SEQ ID NO: 15 and 16; primers shown in SEQ ID NO: 17 and 18.
  • the use of the reagent or the combined reagent according to the fifth aspect of the present invention is provided for preparing a kit for detecting tumors; preferably, the tumors include (but are not limited to): Digestive system tumors such as esophageal cancer (esophageal cancer), stomach cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory system tumors such as lung cancer, pleural tumor; hematological 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, meningioma; head and neck tumors such as oral cancer , Tongue cancer, laryngeal cancer, nasopharyngeal cancer; urinary system tumors such as esophageal cancer
  • a detection kit comprising: a container, and the aforementioned reagent or reagent combination located in the container; preferably, 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 instructions for use (indicating the detection operation steps and The result judgment standard).
  • a method for detecting the methylation pattern of a sample in vitro includes: (i) providing a sample and extracting a nucleic acid; (ii) detecting the CpG site of the target sequence in the nucleic acid of (i)
  • the target sequence is the polynucleotide described in the first aspect or the polynucleotide described in the second aspect derived therefrom.
  • the analysis methods include: pyrophosphate sequencing method, bisulfite conversion sequencing method, methylation chip method, qPCR method, digital PCR method, second generation sequencing method, third generation sequencing Method, whole-genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, MassArray, methylation-specific PCR, or a combination thereof and part or all of the sequence shown in the invention Combined gene group in vitro detection method and in vivo tracer detection method for basification sites.
  • other other methylation detection methods and newly-developed methylation detection methods in the future can also be applied to the present invention.
  • step (ii) includes: (1) processing the product of (i) to convert unmodified cytosine into uracil; preferably, the modification includes 5-methyl Modification, 5-hydroxymethylation modification, 5-aldehyde methylation modification or 5-carboxymethylation modification; preferably, the treatment described in step (i) using bisulfite or bisulfite Nucleic acid; (2) analysis of the modification of the CpG site of the target sequence in the nucleic acid treated by (1).
  • the abnormal methylation pattern means that the C in the polynucleotide CpG is highly methylated.
  • the method of methylation spectrum does not aim to directly obtain the diagnosis result of the disease, or it is not a diagnostic method.
  • a tumor diagnostic kit which includes a primer pair designed using the sequence shown in the first aspect or the second aspect of the present invention and a gene Panel or gene group containing the sequence.
  • the basification state detection obtains the characteristics of normal cells and tumor cells.
  • Figure 1 The difference in the degree of methylation of 001-054 methylation sites in SEQ ID NO: 1 in normal liver cells and liver cancer cells.
  • Figure 2 The difference in the degree of methylation between 001-044 methylation sites in SEQ ID NO: 2 in normal liver cells and liver cancer cells.
  • Figure 3 The difference in the degree of methylation of 045-125 methylation sites in SEQ ID NO: 2 in normal liver cells and liver cancer cells.
  • Figure 4 The difference in the degree of methylation between 126-204 methylation sites in SEQ ID NO: 2 in normal liver cells and liver cancer cells.
  • the inventors are devoted to the study of tumor markers. After extensive research and screening, they provide a universal DNA methylated tumor marker STAMP (SpecificTumorAlignedMethylationofPan-cancer), which is low in normal tissues. Methylation status, where STAMP is highly methylated in tumor tissues, can be used for clinical tumor detection and as a basis for designing tumor diagnostic reagents.
  • STAMP SpecificTumorAlignedMethylationofPan-cancer
  • 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 the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state.
  • sample or “sample” includes a substance obtained from any individual or isolated tissue, cell, or body fluid (such as plasma) that is suitable for the detection of DNA methylation status.
  • high (degree) methylation refers to the presence of highly methylated, hydroxymethylated, aldehyde methylated, or carboxymethylated modifications of CpG in a gene sequence.
  • MSP methylation specific PCR
  • a PCR reaction performed with methylation-specific primers can obtain a positive PCR result, and the DNA (gene) region of the test is considered to be in a hypermethylated state .
  • the determination of high methylation status can be analyzed 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 (esophageal cancer), gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; gynecological and reproductive system tumors such as breast cancer, ovarian cancer Cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; hematological tumors such as leukemia, lymphoma, multiple myeloma; respiratory system tumors such as lung cancer, pleural tumor; nervous system tumors such as glioma, neuroma Cell tumors, meningiomas; head and neck tumors such as oral cancer, tongue cancer, laryngeal 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
  • the present inventors have conducted extensive and in-depth research to determine the target of STAMP-EP4.
  • the methylation status of the STAMP-EP4 gene sequence region is significantly different between tumor tissues and non-tumor tissues, as long as an abnormal methylation status (highly methylated) is detected in the promoter region of one of the above genes, It can be determined that the subject is a high-risk person.
  • the significant differences between STAMP-EP4 and tumor tissues exist in various types of tumors, including solid tumors and non-solid tumors.
  • the present invention provides an isolated polynucleotide from the human genome, having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 (which is the reverse of SEQ ID NO: 1 To the complementary sequence) or SEQ ID NO: 6 (which is the reverse complementary sequence of SEQ ID NO: 2), in the tumor cell of the tumor patient, there are multiple 5's in the polynucleotide sequence -At the base C position of CpG-3', 5-methylcytosine (5mC) is generated.
  • SEQ ID NO: 1 SEQ ID NO: 2
  • SEQ ID NO: 5 which is the reverse of SEQ ID NO: 1 To the complementary sequence
  • SEQ ID NO: 6 which is the reverse complementary sequence of SEQ ID NO: 2
  • the present invention also includes a fragment of the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 6, and at least one of them (such as 2 ⁇ 54 or 2 to 204, more specifically 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 80, 100, 120, 140, 160, 180, 200) Methylated CpG sites.
  • the above-mentioned polynucleotides or fragments can also be used to design detection reagents or detection kits.
  • the polynucleotide fragment is, for example, a fragment including the 132th to 164th bases in SEQ ID NO: 2 (including CpG Nos. 010 to 015 in SEQ ID NO: 2 Site); contains the fragment that starts at the first position and ends at the 500th to 529th bases in SEQ ID NO: 2 (contains the CpG positions 1 to 44 in SEQ ID NO: 2); contains the SEQ ID NO : 2 fragments starting at 501-530 positions and ending at 1213-1228 bases (containing SEQ ID NO: 2 CpG sites 45-125); including SEQ ID NO: 2 starting at Fragments from positions 1214 to 1229, terminating at base 1848 (containing CpG sites 126 to 204 in SEQ ID NO: 2). Antisense strands of the above fragments are also available. Moreover, these fragments are examples of preferred embodiments of the present invention, and other fragments may be selected based on the information provided by the present invention.
  • a gene panel or gene group including the nucleotide sequence or sequence fragment shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 6 is also included in the present invention.
  • the characteristics of normal cells and tumor cells can also be obtained by detecting the DNA methylation status.
  • polynucleotides can be used as key regions for analyzing 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 status can be used in the present invention.
  • the present invention also provides polynucleotides obtained after the above polynucleotides have been treated with bisulfite or bisulfite, including: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or The polynucleotide of nucleotide sequence shown in 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 the polynucleotide obtained after the above-mentioned polynucleotide or its antisense strand is treated with bisulfite or bisulfite, and at least one methylated CpG site (such as 2 ⁇ 54 or 2 to 204, more specifically 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 80, 100, 120, 140, 160, 180, 200) .
  • the number of each CpG site in the antisense strand corresponding to the sense strand is easily obtained according to the content provided by the present invention.
  • a detection reagent designed based on the polynucleotide sequence is also provided for in vitro detection of the methylation pattern of the polynucleotide in the sample.
  • the detection methods and reagents known in the art for determining the sequence and methylation status of the genome can be applied to the present invention.
  • the present invention provides a method for preparing a tumor detection reagent, comprising: providing the polynucleotide, using the full-length or fragment of the polynucleotide as a target sequence, and designing a detection reagent that specifically detects the target sequence ; Wherein, there is at least one methylated CpG site in the target sequence.
  • the detection reagents of the present invention include but are not limited to: primers, probes, and so on.
  • the reagent is, for example, a primer pair.
  • the design of 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 Including the sequence, it is complementary to CpG for the gene region that was originally methylated, and TpG is complementary to the gene region that was originally demethylated). It should be understood that, according to the new findings of the present invention, for a CpG site at different positions on the target sequence or a combination thereof, a person skilled in the art can design a variety of primers or probes or other types of detection reagents. It is included in the technical solution of the present invention.
  • the primers are selected from the group consisting of primers shown in SEQ ID NO: 9 and 10; primers shown in SEQ ID NO: 11 and 12; and primers shown in SEQ ID NO: 13 and 14. Primers shown; SEQ ID NO: 15 and 16 primers; SEQ ID NO: 17 and 18 primers.
  • the reagent may also be a reagent combination (primer combination), including more than one set of primers, so that the above-mentioned multiple polynucleotides can be amplified separately.
  • the present invention also provides a kit for in vitro detection of the methylation profile of polynucleotides in a sample.
  • the kit includes a container and the above primer pair located in the container.
  • the kit can also include various reagents required for DNA extraction, DNA purification, PCR amplification, etc.
  • kit may also include instructions for use, which indicate the detection operation steps and result judgment standards, so that those skilled in the art can use them.
  • Determination of the methylation profile of polynucleotides can be carried out 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 Carried out by the developed technology.
  • 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 throughput is high and the analysis of its results by electrophoresis is avoided.
  • a method for in vitro detection of the methylation profile of a polynucleotide in a sample 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 in the subsequent PCR amplification process, while methylation The cytosine remains unchanged; therefore, after processing 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 includes: (a) providing a sample and extracting genomic DNA; (b) treating 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) Analysis of the genomic DNA treated in step (b) for abnormal methylation patterns.
  • the method of the present invention can be used to: (i) test a sample of a subject and analyze whether the subject has a tumor; (ii) distinguish between high-risk groups of tumors.
  • the method described may also be a situation where the goal is not to obtain a direct disease diagnosis result.
  • DNA methylation is detected by PCR amplification and pyrosequencing.
  • PCR amplification and pyrosequencing Those skilled in the art should understand that the actual application is not limited to this method, and other DNA methylation detection methods are also possible.
  • the primers used are not limited to those provided in the examples.
  • telomeres Since the genomic DNA undergoes bisulfite treatment, unmethylated cytosine is converted to uracil, which is converted to thymine in the subsequent PCR process, which will reduce the sequence complexity of the genome and make the PCR amplify a specific target The difficulty of the clip increases. Therefore, preferably nested PCR amplification can be used, two pairs of primers on the outer and inner sides are designed, two rounds of PCR amplification reactions are performed, and the amplification products of the first round are used as templates for the second round of amplification, which can effectively improve the amplification Efficiency and specificity.
  • the detection methods available in the present invention are not limited thereto.
  • the method of the present invention is very accurate when used to diagnose clinical tumors.
  • the invention can be applied to the fields of tumor auxiliary diagnosis, curative effect judgment, prognosis monitoring and the like, and has high clinical application value.
  • SEQ ID NO: 3 (where Y represents C or U):
  • SEQ ID NO: 4 (where Y represents C or U):
  • SEQ ID NO: 5 The reverse complementary sequence of the above nucleotide sequence shown in SEQ ID NO: 1 is as follows SEQ ID NO: 5:
  • SEQ ID NO: 6 The reverse complementary sequence of the nucleotide sequence shown in SEQ ID NO: 2 above is as follows SEQ ID NO: 6:
  • SEQ ID NO: 5 after bisulfite treatment (where Y represents C or U) is as follows SEQ ID NO: 7 (where Y represents C or U):
  • SEQ ID NO: 6 after bisulfite treatment (where Y represents C or U) is as follows SEQ ID NO: 8 (where Y represents C or U):
  • Example 2 Verification of methylation differences between STAMP-EP4CpG sites in tumor cells and non-tumor cells——Sequencing after bisulfite treatment (BSP-Bisulfite Sequencing PCR)
  • DNA extraction extract the DNA of the experimental group and the control group separately; this experiment uses phenol chloroform extraction method, but not limited to this method;
  • Primer design According to the characteristics of STAMP-EP4 sequence SEQ ID NO: 2, design PCR amplification primers and pyrosequencing primers to detect the methylation value of CpG sites 10-15 in SEQ ID NO: 2, as STAMP -Representative of EP4 methylation value, PCR primer amplification sequence, pyrophosphate sequencing primer sequence, pyrophosphate sequencing on-line detection sequence and detection sites are shown in SEQ ID NO 17-20 (Table 2);
  • PCR amplification and agarose gel electrophoresis using bisulfite-treated samples as PCR products for PCR amplification, the amplified products are identified by agarose gel electrophoresis for specificity of PCR amplification;
  • STAMP-EP4 methylation value calculation Pyrosequencing can independently detect the methylation of a single CpG site in the target area, calculate the average of all CpG site methylation as STAMP-EP4 in this sample Methylation value;
  • pancreatic cancer 4 samples were obtained as a control group, 4 samples of pancreatic cancer were obtained as an experimental group, and the methylation level of STAMP-EP4 was analyzed according to the pyrophosphate test procedure of Example 3 above.
  • Example 10 STAMP-EP4: verification of clinical samples of esophageal cancer-pyrosequencing method

Abstract

本发明提供了一种甲基化肿瘤标志物STAMP-EP4及其应用，属于疾病诊断标记物领域。本发明提供了甲基化肿瘤标志物STAMP-EP4在制备肿瘤诊断试剂中的应用。

Description

基于甲基化修饰的肿瘤标记物STAMP-EP4 技术领域
本发明属于疾病诊断标记物领域，更具体地，本发明涉及基于甲基化修饰的肿瘤标记物STAMP(Specific Tumor Aligned Methylation of Pan-cancer)。
背景技术
肿瘤被认为是一种遗传性疾病的观念在本领域持续几十年。人类几次系统的大规模测序证实了癌组织中体细胞的突变数量明显少于预期，这些结果暗示了癌症并非简单的遗传性疾病。
为了实现肿瘤的诊断，近年来许多新型的肿瘤标记物被发现并用于临床诊断。1980年之前，肿瘤标记物主要是一些激素、酶类、蛋白质等细胞分泌物，例如癌胚抗原(CEA)、甲胎抗原(AFP)等可以作为胃癌和肝癌等多种肿瘤的标记物，糖类抗原125(CA125)可以作为宫颈癌的标记物，前列腺特异性抗原(PSA)可作为前列腺癌标记物，目前这一类肿瘤标记物虽然临床仍在用，但其敏感性与准确性已难以满足临床需求。
越来越多的证据表明，表观遗传调控的细小变化在肿瘤中的重要作用。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(DNA methylation)，组蛋白修饰(Histone modification)，microRNA水平变化等生化过程。DNA甲基化是表观遗传学机制之一，其是指生物体内在DNA甲基转移酶(DNA methyltransferase，DMT)的催化下，以S-腺苷甲硫氨酸(SAM)为甲基供体，将甲基转移到特定的碱基上的过程。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)。
液体活检技术是以血中循环肿瘤细胞或循环肿瘤DNA为检测靶标对肿瘤进行诊断和预测的技术。该技术存在诸多不足：首先，敏感性与特异性不够高，肿瘤本身有很大的异质性，包含多种亚型的细胞群，而临床样本尤其是血液样本，肿瘤DNA所占比例非常小，现有的肿瘤标记物难以满足临床要求的敏感性，在临床容易造成误诊；其次，一种标记物只针对一种或少数几种肿瘤有较好效果，而血液中的DNA来源非常复杂，因此现有的肿瘤标记物无法应对复杂的肿瘤来源、转移等问题。由于这些复杂情况的存在，使得很多DNA甲基化肿瘤标记物在应用于临床时难以有统一的使用标准，严重影响标记物的敏感度以及准确性。
综上，在肿瘤诊断领域中，亟待发现和研究更多的新型肿瘤标志物，从而为肿瘤的诊断提供更多的途径。
发明内容
本发明的目的在于提供以DNA甲基化修饰作为肿瘤标记物，利用肿瘤中特异性位点异常高甲基化现象来检测肿瘤的方法。
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:2所示核苷酸序列的多核苷酸；(c)上述(a)～(b)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点(如2～54个或2～204个，更具体如3，5，10，15，20，25，30，35，40，45，50，60，80，100，120，140，160，180，200个)；(d)与上述(a)～(c)的多核苷酸或片段互补的核酸(如SEQ ID NO:5或SEQ ID NO:6所示核苷酸序列的多核苷酸)。
在一个优选例中，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。
在本发明的第二方面，提供分离的多核苷酸，其由所述的多核苷酸转变而来，对应于前述第一方面的序列，其修饰的CpG位点的胞嘧啶C不变，非修饰的胞嘧啶转为T或U。
在一个优选例中，其由对应于前述第一方面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：(e)SEQ ID NO:3或SEQ ID NO:7所示核苷酸序列的多核苷酸；(f)SEQ ID NO:4或SEQ ID NO:8所示核苷酸序列的多核苷酸；(g)上述(e)～(f)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点(如2～54个或2～204个，更具体如3，5，10，15，20，25，30，35，40，45，50，60，80，100，120，140，160，180，200个)。
在本发明的第三方面，提供前述第一方面或第二方面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，所述肿瘤包括(但不限于)：血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；消化系统肿瘤如食道癌(食管癌)，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；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；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；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。
在另一优选例中，所述肿瘤的样本包括：组织样本、石蜡包埋样本、血液样本、胸腔积液样本以及肺泡灌洗液样本、腹水及灌洗液样本、胆汁样本、粪便样本、尿液样本、唾液样本、痰液样本、脑脊液样本、细胞涂片样本、宫颈刮片或刷片样本、组织及细胞活检样本。
在本发明的第四方面，提供一种制备肿瘤检测试剂的方法，所述方法包括：提供前述第一方面或第二方面所述的多核苷酸，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CpG位点修饰情况的检测试剂；其中，所述的靶序列中存在至少1个(如2～54个或2～204个，更具体如3，5，10，15，20，25，30，35，40，45，50，60，80，100，120，140，160，180，200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。
在本发明的第五方面，提供试剂或组合的试剂，其特异性检测靶序列的CpG位点修饰情况，所述的靶序列是前述第一方面或第二方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2～54个或2～204个，更具体如3，5，10，15，20，25，30，35，40，45，50，60，80，100，120，140，160，180，200个)修饰的CpG位点。
在一个优选例中，所述的试剂或组合的试剂针对包含所述靶序列的基因序列(基于所述基因序列而设计)，所述的基因序列包括基因Panel或基因群组。
在另一优选例中，所述的检测试剂包括：引物，探针。
在另一优选例中，所述的引物选自下组：SEQ ID NO:9和10所示的引物；SEQ ID NO:11和12所示的引物；SEQ ID NO:13和14所示的引物；SEQ ID NO:15和16所示的引物；SEQ ID NO:17和18所示的引物。
在本发明的第六方面，提供本发明的第五方面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(但不限于)：消化系统肿瘤如食道癌(食管癌)，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。
在本发明的第七方面，提供一种检测试剂盒，其包括：容器，以及位于容器中的前面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。
在另一优选例中，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)。
在本发明的第八方面，提供一种体外检测样品的甲基化谱式的方法，包括：(i)提供样品，提取核酸；(ii)检测(i)的核酸中靶序列的CpG位点修饰情况，所述的靶序列是前述第一方面所述的多核苷酸或由其转变而来的前述第二方面所述的多核苷酸。
在一个优选例中，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、甲基化芯片法、qPCR法、数字PCR法、二代测序法、三代测序法、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、MassArray、甲基化特异PCR、或它们的组合以及本发明所示序列中部分或全部甲基化位点的组合基因群组体外检测方法及体内示踪检测方法。并且，其它其他甲基化检测方法及未来新开发的甲基化检测方法也可被应用于本发明中。
在另一优选例中，步骤(ii)包括：(1)对(i)的产物进行处理，使其中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)所述的核酸；(2)分析经(1)处理的核酸中所述的靶序列的CpG位点的修饰情况。
在另一优选例中，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。
在本发明的第九方面，提供一种肿瘤诊断试剂盒，包括利用本发明的第一方面或第二方面所示序列设计的引物对以及包含该序列的基因Panel或基因群组，通过DNA甲基化状态检测获取正常细胞和肿瘤细胞的特征。
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、SEQ ID NO:1中001-054甲基化位点在正常肝细胞与肝癌细胞中甲基化程度差异。
图2、SEQ ID NO:2中001-044甲基化位点在正常肝细胞与肝癌细胞中甲基化程度差异。
图3、SEQ ID NO:2中045-125甲基化位点在正常肝细胞与肝癌细胞中甲基化程度差异。
图4、SEQ ID NO:2中126-204甲基化位点在正常肝细胞与肝癌细胞中甲基化程度差异。
图5、在乳腺癌临床样本中，STAMP-EP4在实验组中甲基化值显著高于癌旁组织；其中，p<0.001。
图6、在白血病临床样本中，STAMP-EP4在实验组中甲基化值显著高于非癌组织；其中，p<0.01。
图7、在结直肠癌临床样本中，STAMP-EP4在实验组中甲基化值显著高于癌旁组织；其中，p<0.001。
图8、在肝癌临床样本中，STAMP-EP4在实验组中甲基化值显著高于癌旁组织；其中，p<0.01。
图9、在肺癌临床样本中，STAMP-EP4在实验组中甲基化值显著高于癌旁组织；其中，p<0.01。
图10、在胰腺癌临床样本中，STAMP-EP4在实验组中甲基化值显著高于癌旁组织；其中，p<0.01。
图11、在食管癌临床样本中，STAMP-EP4在实验组中甲基化值显著高于癌旁组织；其中，p<0.01。
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，提供一种通用型的DNA甲基化肿瘤标志物STAMP(Specific Tumor Aligned Methylation of Pan-cancer)，在正常的组织中STAMP处于低甲基化状态，在肿瘤组织中STAMP呈高甲基化状态，可用于临床肿瘤的检测，以及用于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(如血浆)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(食管癌)，胃癌，结直肠癌，肝癌，胰腺癌，胆管及胆囊癌；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；血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；呼吸系统肿瘤如肺癌，胸膜瘤；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，确定了STAMP-EP4这一靶标。STAMP-EP4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。并且，STAMP-EP4呈现的这种在在肿瘤组织和非肿瘤组织之间存的显著差异广谱地存在于各种种类的肿瘤中，包括实体瘤以及非实体瘤。
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:2、SEQ ID NO:5(为SEQ ID NO:1的反向互补序列)或SEQ ID NO:6(为SEQ ID NO:2的反向互补序列)所示的核苷酸序列，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:2、SEQ ID NO:5或SEQ ID NO:6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2～54个或2～204个，更具体如3，5，10，15，20，25，30，35，40，45，50，60，80，100，120，140，160，180，200个)甲基化CpG位点。上述的多核苷酸或片段也可以应用于设计检测试剂或检测试剂盒。
在本发明的一些具体实施例中，所述的多核苷酸的片段例如是：包含SEQ ID NO:2中第132～164位碱基的片段(包含SEQ ID NO：2中010～015号CpG位点)；包含SEQ ID  NO:2中起始于第1位、终止于第500～529位碱基的片段(含有SEQ ID NO：2中1～44号CpG位点)；包含SEQ ID NO:2中起始于第501～530位、终止于第1213～1228位碱基的片段(含有SEQ ID NO：2中45～125号CpG位点)；包含SEQ ID NO:2中起始于第1214～1229位、终止于第1848位碱基的片段(含有SEQ ID NO：2中126～204号CpG位点)。上述片段的反义链也是可用的。并且，这些片段是本发明的较佳实施方式的举例，根据本发明提供的信息，也可以选择其它的片段。
此外，包含SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:5或SEQ ID NO:6所示核苷酸序列或序列片段的基因Panel或基因群组也被包含在本发明中。针对所述的基因Panel或基因群组，也可以通过DNA甲基化状态检测获取正常细胞和肿瘤细胞的特征。
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中。
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:3、SEQ ID NO:4、SEQ ID NO:7或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或其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(如2～54个或2～204个，更具体如3，5，10，15，20，25，30，35，40，45，50，60，80，100，120，140，160，180，200个)。反义链中各个CpG位点相应于正义链的编号是根据本发明所提供的内容易于获得的。
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，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。
本发明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)。应理解，根据本发明的新发现，针对所述靶序列上的不同位置的CpG位点或其组合，本 领域技术人员可以设计出多种引物或探针或其它类型的检测试剂，这些均应被包含在本发明的技术方案中。在本发明的优选实施例中，所述的引物选自下组：SEQ ID NO:9和10所示的引物；SEQ ID NO:11和12所示的引物；SEQ ID NO:13和14所示的引物；SEQ ID NO:15和16所示的引物；SEQ ID NO:17和18所示的引物。
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。
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。
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。因此优选地可采用巢式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。然而应理解，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。本发明可应用于肿瘤辅助诊断、疗效判定、预后监测等等领域，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、针对STAMP-EP4检测的核酸序列
提供STAMP-EP4肿瘤标记物的序列1，如下SEQ ID NO:1(chr6:391206-391694/hg19)所示，其中下划线标示碱基为甲基化CpG位点，下划线下面的数字表示该位点的编号。
Figure PCTCN2019129827-appb-000001
提供STAMP-EP4肿瘤标记物的序列2，如下SEQ ID NO:2(chr6:391780-393627/hg19)所示，其中下划线标示碱基为甲基化CpG位点，下划线下面的数字表示该位点的编号。
Figure PCTCN2019129827-appb-000002
Figure PCTCN2019129827-appb-000003
上述SEQ ID NO:1序列经重亚硫酸盐处理后序列如下SEQ ID NO:3(其中Y代表C或U)：
Figure PCTCN2019129827-appb-000004
上述SEQ ID NO:2序列经重亚硫酸盐处理后序列如下SEQ ID NO:4(其中Y代表C或U)：
Figure PCTCN2019129827-appb-000005
Figure PCTCN2019129827-appb-000006
上述SEQ ID NO:1所示核苷酸序列的反向互补序列如下SEQ ID NO:5：
Figure PCTCN2019129827-appb-000007
上述SEQ ID NO:2所示核苷酸序列的反向互补序列如下SEQ ID NO:6：
Figure PCTCN2019129827-appb-000008
Figure PCTCN2019129827-appb-000009
上述SEQ ID NO:5序列经重亚硫酸盐处理后序列(其中Y代表C或U)如下SEQ ID NO:7(其中Y代表C或U)：
Figure PCTCN2019129827-appb-000010
上述SEQ ID NO:6序列经重亚硫酸盐处理后序列(其中Y代表C或U)如下SEQ ID NO:8(其中Y代表C或U)：
Figure PCTCN2019129827-appb-000011
Figure PCTCN2019129827-appb-000012
实施例2、STAMP-EP4CpG位点在肿瘤细胞与非肿瘤细胞的甲基化差异验证——亚硫酸氢盐处理后测序法(BSP-Bisulfite Sequencing PCR)
1.提取肝癌细胞系HepG2与正常肝细胞系基因组DNA；
2.分别用重亚硫酸盐处理提取的HepG2与正常肝细胞系基因组DNA，作为后续PCR扩增的模板；本实验中用ZYMO Research公司的EZ DNA Methylation-Gold Kit，货号D5006；但本发明不限于该试剂盒；
3.分别根据SEQ ID NO:1或2的序列设计扩增引物(SEQ ID NO:9～16；表1)，针对不同序列区域，常规方法进行扩增。
4.PCR扩增之后，2％琼脂糖凝胶电泳检测PCR片段特异性，切胶回收目的片段，连接插入T载体，转化感受态大肠杆菌，涂菌板，第二天挑克隆测序，每个片段挑取10个克隆进行Sanger测序。
表1、BSP引物
Figure PCTCN2019129827-appb-000013
SEQ ID NO:1中001-054甲基化位点在正常肝细胞与肝癌细胞中甲基化程度差异的测定结果如图1。
SEQ ID NO:2中001-044甲基化位点在正常肝细胞与肝癌细胞中甲基化程度差异的测定结果如图2。
SEQ ID NO:2中045-125甲基化位点在正常肝细胞与肝癌细胞中甲基化程度差异的测定结果如图3。
SEQ ID NO:2中126-204甲基化位点在正常肝细胞与肝癌细胞中甲基化程度差异的测定结果如图4。
上述结果显示，SEQ ID NO:1，SEQ ID NO:2区域肝癌细胞系甲基化水平显著 高于肝正常细胞系。
实施例3、STAMP-EP4在肿瘤与非肿瘤临床组织样本中的甲基化差异验证—焦磷酸测序法
1.获取临床样本：从临床获取癌旁/非癌-癌样本，癌旁样本作为对照组，肿瘤样本作为肿瘤检测实验组；
2.DNA提取：分别提取实验组和对照组DNA；本实验用酚氯仿抽提法，但不限于该方法；
3.重亚硫酸盐处理：以重亚硫酸盐处理提取的DNA样本，严格按照步骤操作；本实验中用ZYMO Research公司的EZ DNA Methylation-Gold Kit，货号D5006，但不限于该试剂盒；
4.引物设计：根据STAMP-EP4序列SEQ ID NO：2特点，设计PCR扩增引物与焦磷酸测序引物，检测SEQ ID NO：2中10-15号CpG位点的甲基化值，作为STAMP-EP4甲基化值的代表，PCR引物扩增序列、焦磷酸测序引物序列、焦磷酸测序上机检测序列以及检测位点如SEQ ID NO 17-20所示(表2)；
5.PCR扩增及琼脂糖凝胶电泳：以重亚硫酸盐处理后的样本作为PCR的产物，进行PCR扩增，扩增后的产物通过琼脂糖凝胶电泳鉴定PCR扩增的特异性；
6.焦磷酸测序：通过QIAGEN公司的Pyro Mark Q96ID焦磷酸测序仪进行检测，严格按照说明书步骤进行操作；
7.STAMP-EP4甲基化值计算：焦磷酸测序可以独立检测出目标区域内单个CpG位点的甲基化情况，计算所有CpG位点甲基化平均值作为STAMP-EP4在该样本中的甲基化值；
8.结果分析：比较癌旁/非癌对照组与肿瘤实验组STAMP-EP4甲基化值。
表2、Pyro引物
Figure PCTCN2019129827-appb-000014
实施例4、STAMP-EP4：乳腺癌临床样本验证-焦磷酸测序法
临床获取5例乳腺癌癌旁样本作为对照组，获取5例乳腺癌组织样本作为实验组，按照以上实施例3的焦磷酸检验步骤，比较对照组与实验组STAMP-EP4甲基化水平。
结果如图5所示，在乳腺癌临床样本中，STAMP-EP4在实验组中甲基化值显著高于癌旁组织。
实施例5、STAMP-EP4：白血病临床样本验证-焦磷酸测序法
从临床获取8例白血病骨髓涂片样本作为实验组，另获取8例非白血病骨髓涂片样本作为对照组，按照以上实施例3的焦磷酸检验步骤，比较对照组与实验组STAMP-EP4甲基化水平。
结果如图6所示，在白血病临床样本中，STAMP-EP4在实验组中甲基化值显著高于非癌组织。
实施例6、STAMP-EP4：结直肠癌临床样本验证-焦磷酸测序法
临床获取8例结直肠癌癌旁样本作为对照组，获取8例结直肠癌样本作为实验组，按照以上实施例3的焦磷酸检验步骤，分析STAMP-EP4甲基化水平。
结果如图7所示，在结直肠癌临床样本中，STAMP-EP4在实验组中甲基化值显著高于癌旁组织。
实施例7、STAMP-EP4：肝癌临床样本验证-焦磷酸测序法
临床获取8例肝癌癌旁样本作为对照组，获取8例肝癌样本作为实验组，按照以上实施例3的焦磷酸检验步骤，分析STAMP-EP4甲基化水平。
结果如图8所示，在肝癌临床样本中，STAMP-EP4在实验组中甲基化值显著高于癌旁组织。
实施例8、STAMP-EP4：肺癌临床样本验证-焦磷酸测序法
临床获取4例肺癌癌旁样本作为对照组，获取4例肺癌样本作为实验组，按照以上实施例3的焦磷酸检验步骤，分析STAMP-EP4甲基化水平。
结果如图9所示，在肺癌临床样本中，STAMP-EP4在实验组中甲基化值显著高于癌旁组织。
实施例9、STAMP-EP4：胰腺癌临床样本验证-焦磷酸测序法
临床获取4例胰腺癌癌旁样本作为对照组，获取4例胰腺癌样本作为实验组，按照以上实施例3的焦磷酸检验步骤，分析STAMP-EP4甲基化水平。
结果如图10所示，在胰腺癌临床样本中，STAMP-EP4在实验组中甲基化值显著高于癌旁组织。
实施例10、STAMP-EP4：食管癌临床样本验证-焦磷酸测序法
临床获取10例食管癌癌旁样本作为对照组，获取10例食管癌样本作为实验组，按照以上实施例3的焦磷酸检验步骤，分析STAMP-EP4甲基化水平。
结果如图11所示，在食管癌临床样本中，STAMP-EP4在实验组中甲基化值显著高于癌旁组织。
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 (16)

  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:2所示核苷酸序列的多核苷酸；
    (c)上述(a)～(b)的多核苷酸的片段，且其中存在至少1个修饰的CpG位点；
    (d)与上述(a)～(c)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或U。
  4. 如权利要求3所述的多核苷酸，其特征在于，包括：
    (e)SEQ ID NO:3或SEQ ID NO:7所示核苷酸序列的多核苷酸；
    (f)SEQ ID NO:4或SEQ ID NO:8所示核苷酸序列的多核苷酸；
    (g)上述(e)～(f)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；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；血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；呼吸系统肿瘤如肺癌，胸膜瘤；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. 如权利要求5或6所述的用途，其特征在于，所述肿瘤的样本包括：组织样本、石蜡包埋样本、血液样本、胸腔积液样本以及肺泡灌洗液样本、腹水及灌洗液样本、胆汁样本、粪便样本、尿液样本、唾液样本、痰液样本、脑脊液样本、细胞涂片样本、宫颈刮片或刷片样本、组织及细胞活检样本。
  8. 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CpG位点修饰情况的检测试剂；其中，所述的靶序列中存在至少1个修饰的CpG位点。
  9. 试剂或组合的试剂，其特征在于，其特异性检测靶序列的CpG位点修饰情况，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。
  10. 如权利要求9所述的试剂或组合的试剂，其特征在于，所述的试剂或组合的试 剂针对包含所述靶序列的基因序列，所述的基因序列包括基因Panel或基因群组。
  11. 如权利要求9所述的试剂或组合的试剂，其特征在于，所述的试剂或组合的试剂包括：引物或探针；较佳地，所述的引物选自下组：SEQ ID NO:9和10所示的引物；SEQ ID NO:11和12所示的引物；SEQ ID NO:13和14所示的引物；SEQ ID NO:15和16所示的引物；SEQ ID NO:17和18所示的引物。
  12. 权利要求9～11任一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；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；血液系统肿瘤如白血病，淋巴瘤，多发性骨髓瘤；呼吸系统肿瘤如肺癌，胸膜瘤；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。
  13. 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9～11任一所述的试剂或试剂组合。
  14. 一种体外检测样品甲基化谱式的方法，其特征在于，包括：
    (i)提供样品，提取核酸；
    (ii)检测(i)的核酸中靶序列的CpG位点修饰情况，所述的靶序列是权利要求1或2所述的多核苷酸或由其转变而来的权利要求3或4所述的多核苷酸。
  15. 如权利要求14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、甲基化芯片法、qPCR法、数字PCR法、二代测序法、三代测序法、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、MassArray、甲基化特异PCR、或它们的组合以及SEQ ID NO:1或2所示序列中部分或全部甲基化位点的组合基因群组体外检测方法及体内示踪检测方法。
  16. 如权利要求14所述的方法，其特征在于，步骤(ii)包括：
    (1)对(i)的产物进行处理，使其中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)所述的核酸；
    (2)分析经(1)处理的核酸中所述的靶序列的修饰情况。
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CN109371138B (zh) * 2018-12-29 2022-10-25 上海奕谱生物科技有限公司 基于甲基化修饰的肿瘤标记物stamp-ep4
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012034170A1 (en) * 2010-09-13 2012-03-22 Clinical Genomics Pty. Ltd Epigenetic markers of colorectal cancers and diagnostic methods using the same
CN104411835A (zh) * 2012-05-11 2015-03-11 临床基因组学股份有限公司 用于结直肠癌的诊断性基因标记系列
CN109371138A (zh) * 2018-12-29 2019-02-22 上海奕谱生物科技有限公司 基于甲基化修饰的肿瘤标记物stamp-ep4

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008103763A2 (en) * 2007-02-20 2008-08-28 Sequenom, Inc. Methods and compositions for cancer diagnosis and treatment based on nucleic acid methylation
DK3608421T3 (da) * 2012-05-18 2022-09-05 Clinical Genomics Pty Ltd Fremgangsmåde til screening for colorektal cancer
CN108866192B (zh) * 2018-07-26 2021-06-22 上海奕谱生物科技有限公司 基于甲基化修饰的肿瘤标记物stamp-ep1
CN108866191B (zh) * 2018-07-26 2021-07-09 上海奕谱生物科技有限公司 基于甲基化修饰的肿瘤标记物stamp-ep2

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012034170A1 (en) * 2010-09-13 2012-03-22 Clinical Genomics Pty. Ltd Epigenetic markers of colorectal cancers and diagnostic methods using the same
CN104411835A (zh) * 2012-05-11 2015-03-11 临床基因组学股份有限公司 用于结直肠癌的诊断性基因标记系列
CN109371138A (zh) * 2018-12-29 2019-02-22 上海奕谱生物科技有限公司 基于甲基化修饰的肿瘤标记物stamp-ep4

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. SAMBROOK: "Molecular Cloning: A Laboratory Manual", 2002, SCIENCE PRESS
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