WO2024060775A1 - 新型的肿瘤检测标志物TAGMe及其应用 - Google Patents

新型的肿瘤检测标志物TAGMe及其应用 Download PDF

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WO2024060775A1
WO2024060775A1 PCT/CN2023/104682 CN2023104682W WO2024060775A1 WO 2024060775 A1 WO2024060775 A1 WO 2024060775A1 CN 2023104682 W CN2023104682 W CN 2023104682W WO 2024060775 A1 WO2024060775 A1 WO 2024060775A1
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nucleic acid
seq
samples
methylation
cancer
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French (fr)
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李振艳
董世华
毛湛睿
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上海奕谱生物科技有限公司
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q1/6869—Methods for sequencing
    • 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

Definitions

  • the present invention belongs to the field of gene epigenetic modification, and more specifically, the present invention relates to a new type of tumor detection marker and its application.
  • Endometrial cancer is one of the common malignant tumors in the female reproductive tract, and its incidence is second only to cervical cancer. With the improvement of living conditions, the incidence of EC is increasing year by year. It is estimated that by 2030, the global economic and medical burden caused by EC will increase by 60%. Although most EC occurs in postmenopausal women, the incidence of EC has recently increased significantly in women aged 40 years or younger, ranging from 2-14%. For young women of childbearing age, if EC is diagnosed early and there is no myometrial invasion and extrauterine spread, there is still a chance to preserve the uterus and/or ovaries. Therefore, early diagnosis of EC is crucial, which can reduce female mortality and gain treatment opportunities for young patients to maintain fertility or reproductive endocrine function.
  • Imaging examinations imaging examinations including magnetic resonance (pelvic MRI), CT, PET-CT, etc. can It can clarify information such as the size and specific location of lesions, but it is difficult to diagnose early EC or precancerous lesions and is generally used for typing and grading;
  • Cytology As a non-invasive detection method, it has high specificity and low sensitivity. The positive rate of exfoliated cytology examination is low, and the cancer cells exfoliated from the uterine cavity are prone to dissolution and degeneration, and are difficult to identify after staining.
  • Serum tumor markers There are no specific and sensitive diagnostic markers for endometrial cancer. Some patients may have CA125, CA19-9, CA153 or HE4 abnormalities, which are related to factors such as histological type, myometrial invasion depth and extrauterine invasion. There is correlation, but poor correlation with benign and malignant endometrium. In the absence of further minimally invasive triage techniques, all patients with abnormal vaginal bleeding must undergo invasive procedures—dilatation and curettage and/or hysteroscopic biopsy, where endometrial tissue is pathologically examined to confirm the diagnosis of endometrial cancer. Most endometrial cancers develop from endometrial hyperplasia to atypical hyperplasia (precancerous lesions), and the progression continues for many years. During the long course of disease progression, these patients with abnormal vaginal bleeding often undergo multiple invasive endometrial biopsies, resulting in triple physical, psychological, and financial pressures. Therefore, there is an urgent need to find new methods for early detection of cancer.
  • the purpose of the present invention is to provide a new set of DNA methylation tumor markers TAGMe-1 and TAGMe-2 and When used in combination, this group of markers are in a hypomethylated state in normal tissues and in a hypermethylated state in endometrial cancer samples, and can be used for the detection of endometrial cancer.
  • Test sample types include but are not limited to tissues, cervical exfoliated cells, uterine cavity scrapings, vaginal secretions, etc.
  • the use of isolated nucleic acids or nucleic acids converted therefrom in preparing reagents or kits for tumor detection is provided; wherein, the nucleic acid Be: (1) a nucleic acid or nucleic acid combination with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5; or (2) a nucleic acid or nucleic acid combination that is complementary in sequence to the nucleic acid of (1); wherein, The nucleic acid converted from the isolated nucleic acid is a nucleic acid corresponding to (1) or (2), and its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged.
  • the nucleic acid Be (1) a nucleic acid or nucleic acid combination with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5; or (2) a nucleic acid or nucleic acid combination that is complementary in sequence to the nucleic acid of (1); wherein, The nucleic acid converted from the isolated
  • the SEQ ID NO: 1 or SEQ ID NO: 5 also includes sequence variants thereof, or homologous sequences.
  • the sequence variant or homologous sequence is more than 80%, more than 85%, more than 90%, more than 92% compared with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5. , 95% or more, 96% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more sequence identity.
  • polynucleotides derived from the sequence variants or homologous sequence transformations unmodified cytosine is transformed into T or U, while the cytosine C at the modified CpG site remains unchanged are also included.
  • the nucleic acid complementary in sequence to the nucleic acid of (1) is the nucleic acid of the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6 respectively; the present invention also includes Sequence variants or homologous sequences of SEQ ID NO: 2 or SEQ ID NO: 6, which have more than 80%, more than 85%, Sequences with more than 90%, more than 92%, more than 95%, more than 96%, more than 98%, more than 99%, more than 99.5%, more than 99.8% sequence identity.
  • polynucleotides derived from the sequence variants or homologous sequence transformations unmodified cytosine is transformed into T or U, while the cytosine C at the modified CpG site remains unchanged are also included.
  • the tumors include: endometrial cancer, cervical cancer (cervical squamous cell carcinoma and cervical adenocarcinoma), pancreatic cancer, head and neck tumors, colon cancer, colorectal cancer, and esophageal cancer , lung cancer (lung squamous cell carcinoma, lung adenocarcinoma), cholangiocarcinoma.
  • the tumor detection is directed to tumors (eg, endometrial cancer) or precancerous lesions thereof (eg, atypical endometrial hyperplasia).
  • tumors eg, endometrial cancer
  • precancerous lesions thereof eg, atypical endometrial hyperplasia
  • samples for tumor detection include (but are not limited to): blood samples, tissue samples (such as paraffin-embedded samples), cervical samples, uterine cavity samples, pleural effusion samples, alveolar lavage Fluid sample, ascites sample, ascites lavage sample, bile sample, stool sample, urine sample, saliva sample, cerebrospinal fluid sample, cell smear sample, cell sample; preferably, the samples include (but are not limited to) : Cervical scraping or brushing sample, cervical swab, uterine cavity tissue (scratching), cervical exfoliated cells, uterine cavity scraping, uterine lavage fluid, vaginal secretions.
  • a method for detecting the methylation level of a sample to be tested includes: extracting nucleic acid of the sample to be tested; and detecting the CpG site modification of the target sequence or fragment thereof in the extracted nucleic acid.
  • the target sequence is a nucleic acid or nucleic acid combination converted from the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5, corresponding to the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5
  • the unmodified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged.
  • methods for detecting CpG site modifications of target sequences in extracted nucleic acids include: pyrosequencing, bisulfite conversion sequencing, methylation chip method, methylation Specific PCR, methylation-sensitive restriction enzyme digestion method, qPCR method, digital PCR method, second-generation sequencing method, third-generation sequencing method, whole-genome methylation Sequencing methods, DNA enrichment assays, simplified bisulfite sequencing technology, HPLC methods, MassArray, or combinations thereof.
  • the method for detecting CpG site modification of a target sequence in an extracted nucleic acid includes: (i) processing the extracted nucleic acid to convert unmodified cytosine is uracil.
  • the modification includes 5-methylation modification (5mC), 5-hydroxymethylation modification (5hmC), 5-aldehyde methylation modification (5-fC) or 5- Carboxymethylation modification (5-caC).
  • the nucleic acid of step (i) is treated with bisulfite; and (ii) the modification of the target sequence in the nucleic acid treated in (i) is analyzed.
  • the nucleic acid is a combination of "SEQ ID NO: 1 or its reverse complement” and "SEQ ID NO: 5 or its reverse complement”.
  • the abnormal methylation pattern refers to the hypermethylation of C in the nucleic acid CpG.
  • methylation detection methods and methylation detection methods newly developed in the future can also be applied in the present invention.
  • the methylation profile method is not a diagnostic method, that is, it is not intended to directly obtain diagnostic results of a disease.
  • the method for detecting the methylation profile of a sample is an in vitro method.
  • the methylation-sensitive restriction enzyme is a restriction enzyme that is sensitive to the presence of methylated bases in its recognition site; including but not limited to: HpaII, Acil, One or a combination of Bsu15I, Hin1I, Hin6I, HpyCH4IV, NarI, etc.
  • a method for preparing reagents for detection of tumors comprising: providing SEQ ID NO: 1 or SEQ For the nucleic acid with the nucleotide sequence shown in ID NO: 5, using the full length or fragment of the nucleic acid as the target sequence, a detection reagent is designed to specifically detect the modification of the CpG site of the target sequence.
  • the detection reagents include but are not limited to: primers, probes, chips or test strips.
  • one or more sets of reagents can be prepared for the full length or fragments of SEQ ID NO: 1.
  • one or more sets of reagents can be prepared for the full length or fragment of SEQ ID NO: 5.
  • the detection reagent is integrated on a chip.
  • a reagent or a combination of reagents that specifically detects the CpG site modification of a target sequence, and the target sequence is a nucleoside shown in SEQ ID NO: 1 or SEQ ID NO: 5 A nucleic acid or nucleic acid combination derived from a converted acid sequence.
  • the reagent or combination of reagents is directed to a gene sequence comprising the target sequence.
  • the gene sequence includes a gene panel or gene group.
  • the reagent or reagent combination is selected from one or more of the following groups: SEQ Primers for the sequences shown in ID NO: 9 and SEQ ID NO: 10; primers for the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12; primers for the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14; Or primers of the sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16.
  • the use of the reagent or reagent combination is provided for preparing a kit for detecting (including screening, diagnosis, detection or prognosis assessment) tumors; preferably, the tumor includes : Endometrial cancer, cervical cancer, colorectal cancer, lung cancer.
  • kits for tumor detection including screening, diagnosis, detection or prognosis evaluation
  • the kit comprises the reagent or reagent combination.
  • the kit may also include, but is not limited to: DNA purification reagents, DNA extraction reagents, bisulfite, and PCR amplification reagents.
  • the kit further includes instructions indicating the detection procedures and result determination criteria.
  • an isolated nucleic acid or a nucleic acid transformed therefrom is provided, the nucleic acid being: (1) a nucleic acid or a nucleic acid having a nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5 combination; or (2) a nucleic acid or nucleic acid combination that is complementary in sequence to the nucleic acid of (1); wherein the nucleic acid converted from the nucleic acid is a nucleic acid corresponding to (1) or (2), which is not modified Cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged.
  • the modified CpG sites include 5-aldehyde methylation modification, 5-hydroxymethylation modification, 5-methylation modification or 5-carboxymethylation modification. CpG sites.
  • Figure 1 Heat map of candidate markers for the diagnosis of endometrial cancer and precancerous lesions.
  • Figure 2A-D performance diagram of TAGMe-1 and TAGMe-2 as biomarkers in other cancer types in the TCGA database.
  • Figure 7 Sequence information of TAGMe-1 methylated tumor markers.
  • Figure 8 Sequence information of TAGMe-2 methylated tumor marker.
  • DNA methylation variation plays an important role in the occurrence and development of endometrial cancer and can be used as a marker for early screening, auxiliary diagnosis, efficacy evaluation, recurrence monitoring and other application scenarios of endometrial cancer.
  • new DNA methylation tumor markers were isolated and obtained.
  • the markers are in a hypomethylated state in normal tissues and are hypermethylated in tumor (especially endometrial cancer and other tumors) samples. Patients with tumors show significant differences in methylation status. , this difference is statistically significant. When the markers are used in combination for detection, the effect is even better.
  • the tumor markers of the present invention can be used as markers for the diagnosis, screening, typing, detection, and prognosis of clinical tumors. They can also be used as new molecules for clinical auxiliary diagnosis or prognosis of tumors, or can be used to design diagnostic reagents and kits. .
  • sample includes a substance suitable for detection of DNA methylation status obtained from any individual (preferably a human) or isolated tissue, cell or body fluid (such as plasma).
  • the samples may include but are not limited to: blood samples, tissue samples (such as paraffin-embedded samples), cervical samples, uterine cavity samples, pleural effusion samples, alveolar lavage fluid samples, ascites samples, ascites lavage fluid Samples, bile samples, stool samples, urine samples, saliva samples, cerebrospinal fluid samples, cell smear samples, cell samples; preferably, the samples include (but are not limited to): cervical scraping or brushing samples, cervical Swabs, uterine cavity tissue (scratches), cervical exfoliated cells, uterine cavity scrapes, uterine lavage fluid, vaginal secretions.
  • the term "high (degree) methylation” refers to the presence of high methylation, hydroxymethylation, aldehyde methylation or carboxymethylation modification of CpG in a gene sequence.
  • MSP methylation-specific PCR
  • a positive PCR result obtained by a PCR reaction performed with methylation-specific primers can indicate that the tested DNA (gene) region is in a high methylation state.
  • the determination of the high methylation state can be based on the relative value analysis of the methylation state of the control sample.
  • tumor refers to a type of tumor in which the SEQ ID NO: 1 or SEQ ID NO: 5 segment in the genome exhibits a hypermethylation state as described in the present invention.
  • detection includes screening, diagnosis, detection or prognostic assessment.
  • endometrial cancer broadly includes “endometrial cancer and its precancerous lesions, which are also called “endometrial atypical hyperplasia” or “early endometrium”. “cancer”; but it can be understood that in certain circumstances, such as in some embodiments, the "endometrial cancer” is classified with “endometrial cancer precancerous lesions", in which case “endometrial cancer” “Cancer” refers to cancer that has passed the precancerous stage, such as the advanced stage.
  • the present invention provides a set of methylation markers, TAGMe-1 and TAGMe-2, as molecular detection markers for endometrial cancer.
  • This marker is in a hypomethylated state in normal and paracancerous endometrial tissue, and in a hypermethylated state in endometrial cancer and precancerous lesions (atypical hyperplasia) tissue.
  • Non-invasive auxiliary diagnosis of early endometrial cancer and precancerous lesions can be achieved through exfoliated cells from the cervix and uterine cavity, filling the gap in existing endometrial cancer screening technology. And through further development, relatively simple, cheap, reliable and high-throughput clinical testing can be achieved.
  • This significant difference in the methylation status of the gene sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5 or its partial region is very significantly present in endometrial cancer (including early stages); at the same time, this Significant differences in methylation status also exist in several other tumors, including: cervical cancer (cervical squamous cell carcinoma and cervical adenocarcinoma), pancreatic cancer, head and neck cancer, colon cancer, colorectal cancer, esophageal cancer, lung cancer (lung cancer) Squamous cell carcinoma, lung adenocarcinoma), cholangiocarcinoma, etc.
  • the present invention also includes conservative or sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 (or its reverse complement) or SEQ ID NO: 5 (or its reverse complement).
  • the "higher sequence homology” is, for example, higher than 90%, higher than 92%, higher than 95%, higher than 98%, higher than 99%, etc. It should be understood that there may be differences in individual sequence positions between different biological individuals (for example, there may be some meaningless SNPs), but this does not affect the detection based on the overall scheme of the present invention.
  • the present invention provides a nucleic acid derived from a specific region of the human genome, which has the gene sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5 or a partial region thereof, and also includes its antisense strand.
  • 5-methylcytosine (5mC) is generated at the C position of the 5’-CpG-3’ base at multiple locations in the nucleic acid sequence.
  • the present invention also includes nucleic acid fragments of the nucleotide sequence, which include at least one methylated CpG site.
  • the at least one may include 2 to 53 of SEQ ID NO: 1 or its reverse complementary sequence, more specifically, 3, 5, 8, 10, 12, 15, 18, 20, 30, 40, 50.
  • the at least one may include 2 to 52 of SEQ ID NO: 5 or its reverse complementary sequence, more specifically such as 3, 5, 8, 10, 12, 15, 18, 20, 30, 40, 50.
  • the present invention also includes the gene Panel or gene group of the nucleotide sequence or sequence fragment shown in SEQ ID NO: 1 or SEQ ID NO: 5 or its complementary sequence (SEQ ID NO: 2 or SEQ ID NO: 6).
  • the characteristics of normal cells and tumor cells can also be obtained through DNA methylation status detection.
  • nucleic acids provided by the present invention can be used as key regions in the genome to analyze the methylation status. Their methylation status can be analyzed by various techniques known in the art, and then the occurrence or development of tumors can be analyzed.
  • nucleic acid described in SEQ ID NO: 1 or SEQ ID NO: 5 of the present invention or its fragment or its complementary sequence can be treated with bisulfite to convert unmethylated cytosine into uracil. , and the methylated cytosine constant. Therefore, the present invention also provides the nucleic acid obtained after bisulfite treatment of the above-mentioned nucleic acid (including its complementary strand (antisense strand)), including: the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 7 Nucleic acid or nucleic acid fragment, or nucleic acid or nucleic acid fragment of the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 8. These nucleic acids can serve as more direct targets for designing detection reagents or detection kits.
  • nucleic acid of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5 of the present invention and/or its complementary nucleic acid and/or one or more fragments thereof can be integrated into one or several entities, such as one Or a collection of several nucleic acids for those skilled in the art to use, such as selecting one or more nucleic acids or nucleic acid fragments from the nucleic acid collection to design targeted analysis reagents.
  • the designed targeted analysis reagents can also be integrated into one or several entities, such as one or several kits.
  • nucleic acid of the present invention and/or its complementary nucleic acid and/or one or more fragments of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5 is converted (for example, by bisulfite conversion)
  • Nucleic acids can also be integrated into one or several entities, such as one or several nucleic acid sets, for use by those skilled in the art, such as selecting one or more nucleic acids or nucleic acid fragments from the nucleic acid set to design targeted analysis Reagents.
  • the designed targeted analysis reagents can also be integrated into one or several entities, such as one or several kits, or one or several chips.
  • nucleic acid from the nucleotide sequence shown in SEQ ID NO: 1 and/or its complementary nucleic acid and/or one or more fragments thereof, and the nucleic acid from the nucleotide sequence shown in SEQ ID NO: 5 are combined for detection.
  • methylation-specific PCR MSP
  • QMSP quantitative methylation-specific PCR
  • qPCR Me-qPCR
  • second-generation sequencing method pyrosequencing method
  • Sanger sequencing method bisulfite conversion sequencing method
  • whole-genome methylation sequencing method DNA enrichment
  • Common methods in this field include detection methods, simplified bisulfite sequencing technology or HPLC methods, and combined gene group detection methods.
  • a method for detecting the methylation pattern of nucleic acid in a sample in vitro is also provided.
  • the principle on which the method is based is that bisulfite can convert unmethylated cytosine into uracil, which is then converted into thymine during the subsequent PCR amplification process, while the methylated cytosine remains unchanged; therefore, after the nucleic acid is treated with bisulfite, the methylated site produces a nucleic acid polymorphism (SNP) similar to a C/T.
  • SNP nucleic acid polymorphism
  • the method of the present invention includes: firstly providing a sample and extracting genomic DNA; secondly, using bisulfite to treat the genomic DNA described in step (a), thereby converting unmethylated cytosine in the genomic DNA into urine. Pyrimidine; again, analyze whether there is an abnormal methylation profile in the genomic DNA processed in step (b).
  • the method of the present invention can be used to: detect subject samples to evaluate whether the subject has tumors; or to differentiate between high-risk groups for tumors.
  • the method may be used for situations where the purpose is not to obtain direct disease diagnosis results, such as situations where the purpose is not to determine the final outcome of the disease, population regional analysis and research, scientific research, census, etc.
  • DNA methylation is detected through PCR amplification and pyrosequencing. Practical applications are not limited to this method. Other DNA methylation detection methods known in the art or being improved are also used. Can.
  • the primers used are not limited to those provided in the examples. Primers that are different in sequence from those provided in the examples of the present invention can also be obtained, but are still specific to the nucleic acid indicated in the present invention. Or primers corresponding to CpG sites.
  • a method for detecting the methylation status of nucleic acids in a sample in vitro is also provided.
  • the method is methylation sensitive restriction endonuclease (MSRE).
  • the methylation-sensitive restriction endonuclease cannot cut DNA when it contains a methylated base in its cutting site.
  • the MSRE method is based on the basic principle that methylation-sensitive type II restriction enzymes cannot cut sequences containing one or more methylation cut points. Fragments containing one or more methylated CpG sequences are cleaved with methyl-sensitive type II endonuclease and its isozymes (insensitive to methylation) and then analyzed by Southern blotting.
  • the advantages of this method include: it does not require detailed information about the primary structure of the target DNA, and it can provide direct evaluation of the methylation status of CpG islands, including obtaining some quantitative analysis information on the methylation of the tested genes.
  • the marker nucleic acid provided by the present invention, other detection methods and reagents known to those skilled in the art for determining the sequence of the genome, its variation and methylation status can be included in the present invention.
  • the invention provides a method for preparing a tumor detection reagent, which includes: providing the nucleic acid, using the full length or fragment of the nucleic acid as a target sequence, and designing a detection reagent for specifically detecting the target sequence; wherein, the The target sequence includes at least 1 methylated CpG site.
  • the detection reagents may include but are not limited to: chips, primers, probes, etc.; after obtaining the markers, the selection of detection reagents can be done by those skilled in the art.
  • the reagent is a primer, and the primer is preferably those listed in Table 2 and Table 6.
  • other diagnostic or detection reagents can also be prepared, including but not limited to probes, chips, etc.
  • the reagents may also be a combination of reagents, such as a primer combination.
  • the combination includes more than one set of primers, so that the above-mentioned plurality of nucleic acids can be amplified respectively.
  • the present invention also provides a kit for in vitro detection of the methylation profile of nucleic acids in samples.
  • 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, etc. and other reagents, such as sample processing reagents.
  • the kit may also include instructions for use, which indicate the detection operation steps and result judgment criteria for the convenience of use by those skilled in the art.
  • the methods and reagents of the present invention are very accurate when used to diagnose clinical tumors, which is reflected in the detection of clinical samples of various tumors in the embodiments of the present invention.
  • the present invention can be applied to the fields of pre-tumor screening, efficacy determination, auxiliary diagnosis, prognosis monitoring, etc., or situations where the purpose of obtaining direct disease diagnosis results is not as mentioned above.
  • the markers for tumors such as endometrial cancer provided by the present invention have very high specificity and sensitivity when used independently. However, if the two markers are used in combination, the sensitivity and specificity will be further improved. Therefore, the markers of the present invention have great application value in the fields of tumor auxiliary diagnosis, efficacy determination, prognosis monitoring, etc.
  • the present invention provides markers for tumors such as endometrial cancer, which can be applied to the detection of cervical exfoliated cells, etc.
  • the sample to be tested is easy and non-invasive to obtain. Compared with the clinical situation where surgery is required to obtain tissue samples, the subject's pain is greatly reduced, the compliance is good, and the clinician's operation is easier. Obviously, this represents a very significant improvement.
  • Degenerate expression reconstruction was used to The bisulfite sequencing (RRBS) method was used to analyze the methylation data at the whole genome level and screen specific DNA methylation markers for endometrial cancer and precancerous lesions.
  • RRBS bisulfite sequencing
  • a total of 43 frozen tissue samples were collected from five groups: normal endometrium - ordinary hyperplasia endometrium - atypical hyperplasia endometrium (precancerous lesions) - endometrial cancer - paired adjacent cancer. Each sample was taken as large as a soybean grain.
  • the extracted sample gDNA is digested with MspI endonuclease, and then magnetic beads are used to sort the fragments of appropriate size. The steps of end filling, adding dA, and adapters are then completed.
  • Each library sample is amplified using primers with different molecular tags, and the amplified products are sorted and purified using magnetic beads. After the concentration is measured, they are sequenced.
  • Illumina Hiseq 2000 was used to sequence 2 ⁇ 150bp genomic RRBS library of tissue samples. After the offline data were processed through quality control, comparison and other steps, basic information such as the effective number of sequences, the number of measured CpG sites, and the number of coverage layers in each sample was analyzed, as shown in Table 1.
  • the amount of offline data of the RRBS library reached 198.31G, after passing through the normal endometrium group (Group A, NE), ordinary hyperplasia group (Group B, EH), atypical hyperplasia group (Group C, AH), and endometrial cancer group ( An average of 16686194M, 16847536M, 14942502M, 14729170M and 14637777M sequencing sequences were obtained for group D, EC) and paracancerous tissue (group E, PC) respectively.
  • the numbers of all CpG sites with coverage layers greater than or equal to 5 are 4055958M, 4169395M, 4369673M, 3984048M and 4271290M respectively.
  • Statistical data and preliminary experimental analysis show that this RRBS sequencing total The body quality is good, the sequencing depth and CpG coverage are good, the data volume of each group is evenly distributed, and the results are highly reliable.
  • methylation sequencing data between different stages and combined with experimental analysis, specific methylation markers present in atypical hyperplasia endometrium (precancerous lesions) and/or cancer stages are screened.
  • Figure 1 a small part of the heat map of candidate markers for diagnosis of endometrial cancer and precancerous lesions is shown.
  • the inventors obtained two groups of endometrial cancer detection results: Precancer-DMRs for precancerous lesions and Cancer-DMRs for endometrial cancer.
  • Candidate methylation markers respectively correspond to DMRs that are significantly hypermethylated only in AH and EC or only in EC stages, but are hypomethylated in other stages including adjacent cancer tissues.
  • the obtained nucleotide sequence of the TAGMe-1 methylated tumor marker is as SEQ ID NO: 1, as shown in Figure 7.
  • the sites marked with background color are potential methylated CG sites (group 53).
  • SEQ ID NO: 1 The reverse complementary sequence of SEQ ID NO: 1 is such as SEQ ID NO: 2, as shown in Figure 7.
  • the sites marked with background color are CG sites that are potentially methylated.
  • SEQ ID NO:3 The sequence of SEQ ID NO:1 after sulfite conversion is as SEQ ID NO:3, as shown in Figure 7.
  • the sites marked with the background color are potential CG sites for methylation, and Y represents C or T.
  • SEQ ID NO: 4 The sequence of the reverse complementary sequence of SEQ ID NO: 3 after sulfite conversion is as SEQ ID NO: 4, see Figure 7.
  • the sites marked with the background color are potential CG sites for methylation, and Y represents C or T.
  • the nucleotide sequence of the obtained TAGMe-2 methylated tumor marker is shown in SEQ ID NO: 5, see Figure 8, and the sites marked with background color are CG sites that are potentially methylated (52 groups).
  • the reverse complementary sequence of SEQ ID NO: 5 is such as SEQ ID NO: 6.
  • the sites marked with background color are CG sites that are potentially methylated.
  • SEQ ID NO: 7 The sequence of SEQ ID NO: 1 after sulfite conversion is as SEQ ID NO: 7, as shown in Figure 8.
  • the sites marked with the background color are potential CG sites for methylation, and Y represents C or T.
  • the reverse complementary sequence of SEQ ID NO: 7 is the sulfite-converted sequence such as SEQ ID NO: 8, as shown in Figure 8.
  • the sites marked with background color are potential CG sites for methylation, and Y represents C or T. .
  • the markers of the present invention were compared with other Precancer-DMRs that were also analyzed to have methylation differences and significance (Figure 1 Heat Map Precancer-DMRs Ranked top), comparative experiments were conducted using pyrosequencing technology in additionally collected clinical tissue samples. 45 clinical tissue samples were collected from clinics, including 14 subjects with normal endometrium (NE), 11 subjects with hyperplasia (without atypical hyperplasia, EH), and 5 subjects with atypical hyperplasia (AH). and 15 endometrial cancer (EC) subjects.
  • NE normal endometrium
  • EH without atypical hyperplasia
  • AH atypical hyperplasia
  • EC endometrial cancer
  • the AUCs of the two DMRs sites TAGMe-1 and TAGMe-2 are 0.938 and 0.925 respectively, both higher than 0.9. It is surprising that patients with early-stage endometrial cancer (Precancer) can reach a very high AUC. Its performance as a methylation marker for early screening of endometrial cancer has been demonstrated to be excellent.
  • the TCGA database includes data on a variety of tumors; for a certain tumor, it records a series of expression data, miRNA expression data, methylation data, mutation data, and copy number data. Therefore, the inventor used this database to perform performance verification on TAGMe-1 and TAGMe-2.
  • Download the methylation data of multiple cancer types in the TCGA database Download the methylation 450K chip data of all tumor samples in the TCGA database, including 11,087 tumors and control normal samples. A total of 35 tumor types and sample sizes involved are shown in Table 4.
  • Calculation of methylation values of TAGMe-1 and TAGMe-2 First, calculate the average methylation values of TAGMe-1 and TAGMe-2 in different types of tumor tissue samples and control normal tissue samples, and then calculate TAGMe-1 and the difference in the average methylation value of TAGMe-2 in tumor samples of this type and control samples.
  • TAGMe-1 and TAGMe-2 have diagnostic value in many types of tumors.
  • Example 3 DNA methylation detection in endometrial cancer-adjacent tissue samples: target-NGS method
  • the target-NGS method was used to conduct DNA methylation analysis in endometrial cancer-paracancerous tissue samples. Proceed as follows:
  • the DNA of the experimental group and the control group were extracted respectively; in this experiment, Epiprobe Genomic DNA Extraction Kit (Epiprobe Biotech, K-21) was used for DNA extraction (but the present invention is not limited to this method).
  • the extracted DNA was modified with bisulphite.
  • PCR primers designed for the target region after bisulfite treatment are used for amplification and enrichment.
  • the primer sequences are shown in Table 6.
  • the detection was performed using Illumia's HiSeq X high-throughput sequencer, and the procedures were strictly followed in accordance with the instructions (but the present invention is not limited to this model of high-throughput sequencer).
  • Target NGS sequencing data contains the methylation status of all single CpG sites in the target region.
  • tissue Target NGS The results of tissue Target NGS are shown in Figure 3.
  • the results of TAGMe-1 are shown in Figure 3A.
  • the methylation values of TAGMe-1 in atypical hyperplasia and cancer tissue samples were significantly higher than those in the control group.
  • the ROC analysis results of TAGMe-1 are shown in Figure 3B.
  • the AUC of TAGMe-1 for distinguishing the endometrial cancer group and the control group is 0.995, and the AUC for distinguishing the atypical hyperplasia group and the control group is 0.983, both of which have good distinguishing performance.
  • the sensitivity and specificity of TAGMe-1 for distinguishing the endometrial cancer group from the control group are 95% and 100%, respectively; the sensitivity and specificity for distinguishing atypical hyperplasia from the control group are 83.33% and 90%, respectively.
  • the TAGMe-2 results are shown in Figure 3C.
  • the methylation values in atypical hyperplasia and cancer tissue samples were significantly higher than those in the control group.
  • the ROC analysis results of TAGMe-2 are shown in Figure 3D.
  • the AUC of TAGMe-2 in distinguishing the endometrial cancer group from the control group is 0.990, and the AUC in distinguishing the atypical hyperplasia group from the control group is 0.967, both of which have good discriminatory performance.
  • the sensitivity and specificity of TAGMe-2 in distinguishing the endometrial cancer group and the control group are 95% and 100% respectively, and the sensitivity and specificity in distinguishing atypical hyperplasia and the control group are respectively 95% and 100%. is 83.33% and 100%.
  • TAGMe-1 and TAGMe-2 were jointly tested, and the joint performance is shown in Table 7.
  • pyrosequencing is used to detect DNA methylation in endometrial cancer-paracancerous tissue samples and perform marker analysis. Proceed as follows:
  • DNA extraction extract DNA from clinical samples; in this experiment, Epiprobe Genomic DNA Extraction Kit (Epiprobe Biotech, K-21) was used for DNA extraction (but the present invention is not limited to this method).
  • the extracted DNA was modified with bisulfurous acid.
  • ZYMO Research Company's EZ DNA Methylation-Gold Kit, product number D5006 was used in strict accordance with the steps of the user manual (but the present invention is not limited to this method).
  • amplification primers and pyrosequencing primers were designed respectively to detect the methylation value of the corresponding CpG site on the target sequence as its Representatives of methylation values, PCR primer amplification sequences, pyrosequencing primer sequences, pyrosequencing on-machine detection sequences and detection sites are the same as in Example 1.
  • the bisulfite-treated samples were used as PCR templates for PCR amplification, and the amplified products were used to identify the specificity of PCR amplification by agarose gel electrophoresis.
  • Detection was carried out using QIAGEN's PyroMarkQ96ID pyrosequencer, and the operation was strictly carried out in accordance with the instructions.
  • Pyrosequencing can independently detect the methylation status of individual CpG sites in the target region, and calculate the average methylation value of all CpG sites as the methylation value of TAGMe-1 and TAGMe-2 in the sample.
  • TAGMe-1 analysis results are shown in Figure 4A.
  • methylation values of TAGMe-1 in atypical hyperplasia and cancer tissue samples were significantly higher than those in the control group.
  • ROC analysis results are shown in Figure 4B.
  • TAGMe-1 differentiates endometrial cancer.
  • the AUC between the atypical hyperplasia group and the control group was 0.955, and the AUC for distinguishing the atypical hyperplasia group from the control group was 0.952, both with good discriminatory performance.
  • the sensitivity and specificity of TAGMe-1 in distinguishing the endometrial cancer group and the control group are 100% and 84% respectively, and the sensitivity and specificity in distinguishing atypical hyperplasia and the control group are respectively 100% and 84%. is 100% and 84%.
  • the TAGMe-2 analysis results are shown in Figure 4C.
  • the methylation values of TAGMe-2 in atypical hyperplasia and cancer tissue samples were significantly higher than those in the control group.
  • the ROC analysis results of TAGMe-2 are shown in Figure 4D.
  • the AUC of TAGMe-2 in distinguishing the endometrial cancer group from the control group is 0.943, and the AUC in distinguishing the atypical hyperplasia group from the control group is 0.928, both of which have good discriminatory performance.
  • the sensitivity and specificity of TAGMe-2 in distinguishing the endometrial cancer group from the control group are 86.67% and 88% respectively, and the sensitivity and specificity in distinguishing atypical hyperplasia from the control group are respectively 86.67% and 88%. to 100% and 80%.
  • TAGMe-1 and TAGMe-2 were jointly detected to further analyze the sensitivity of methylation markers in detecting endometrial cancer and precancerous lesions.
  • the combined performance is shown in Table 8.
  • Para-cancerous samples cannot simply be regarded as normal samples. During clinical sampling, para-cancerous samples are easily mixed with some cancer tissues or undergo certain epigenetic mutations due to the influence of the cancer site, which can easily lead to a decrease in specificity. , so the specificity in some samples in the table will fluctuate compared with the previous embodiment. Nevertheless, the combined detection of two markers can significantly improve the specificity, overall coincidence rate and positive predictive value in detecting atypical endometrial hyperplasia.
  • the Me-qPCR method is used to detect DNA methylation in endometrial cancer-cervical exfoliated cell samples and perform marker analysis. Proceed as follows:
  • DNA extraction extract DNA from clinical samples; in this experiment, Epiprobe Genomic DNA Extraction Kit (Epiprobe Biotech, K-21) was used for DNA extraction (but the present invention is not limited to this method).
  • Enzyme digestion reaction Use methylation-sensitive restriction endonuclease to digest DNA. The unmethylated enzyme cutting sites will be cut off.
  • HpaII enzyme (NEB, R0171) was used for the enzyme digestion reaction. (But the present invention is not limited to this method).
  • Primer design According to the TAGMe-1 sequence and TAGMe-2 sequence, design corresponding amplification primers to amplify the products digested by methylation-sensitive restriction enzymes, and detect the abundance of DNA fragments after digestion, respectively. As a representative of TAGMe-1 and TAGMe-2 methylation values, the GAPDH gene was used as an internal reference.
  • qPCR amplification Use the digested sample as a qPCR template to perform qPCR amplification. In this experiment, Thermo The company's ABI 7500 qPCR instrument is used for detection (but the present invention is not limited to this method).
  • Cervical cell Me-qPCR results are shown in Figure 5.
  • the TAGMe-1 results are shown in Figure 5A.
  • the ROC analysis results of TAGMe-1 are shown in Figure 5B.
  • the AUC of TAGMe-1 in distinguishing the endometrial cancer group from the control group is 0.916, and the AUC in distinguishing the atypical hyperplasia group from the control group is 0.870, both of which have good discriminatory performance.
  • the sensitivity and specificity of TAGMe-1 in distinguishing the endometrial cancer group and the control group are 84.85% (68.1%-94.89%) and 89.47% (75.2%-97.06%) respectively.
  • the sensitivity and specificity for distinguishing atypical hyperplasia from controls were 87.5% (47.35%-99.68%) and 76.32% (59.76%-88.56%) respectively.
  • the TAGMe-2 results are shown in Figure 5C.
  • the methylation values of TAGMe-2 in atypical hyperplasia and cancer tissue samples were significantly higher than those in the control group.
  • the ROC analysis results of TAGMe-2 are shown in Figure 5D.
  • the AUC of TAGMe-2 in distinguishing the endometrial cancer group from the control group is 0.921, and the AUC in distinguishing the atypical hyperplasia group from the control group is 0.845, both of which have good discriminatory performance.
  • the sensitivity and specificity of TAGMe-2 in distinguishing the endometrial cancer group from the control group are 87.88% and 86.84% respectively, and the sensitivity and specificity in distinguishing atypical hyperplasia from the control group are respectively is 100% and 57.89%.
  • TAGMe-1 and TAGMe-2 joint analysis TAGMe-1 and TAGMe-2 were jointly detected to further analyze the sensitivity of methylation markers in detecting endometrial cancer and precancerous lesions.
  • the performance of the combined model is as shown in the table 9.
  • the combined detection of the two markers can significantly improve the overall coincidence rate in detecting atypical endometrial hyperplasia, as well as the sensitivity and coincidence rate in detecting endometrial cancer.
  • Me-qPCR results of uterine cavity cells are shown in Figure 6.
  • the TAGMe-1 results are shown in Figure 6A.
  • the ROC analysis results are shown in Figure 6B.
  • the AUC of TAGMe-1 in distinguishing the endometrial cancer group from the control group is 0.988, and the AUC in distinguishing the atypical hyperplasia group from the control group is 0.921, both of which have good discriminatory performance.
  • TAGMe-1 distinguishes the uterus
  • the sensitivity and specificity of endometrial cancer group and control group were 90.48% and 100% respectively, and the sensitivity and specificity of distinguishing atypical hyperplasia and control group were 83.33% and 94.74% respectively.
  • the results of TAGMe-2 are shown in Figure 6C.
  • the methylation values of TAGMe-2 in atypical hyperplasia and cancer tissue samples were significantly higher than those in the control group.
  • the results of ROC analysis are shown in Figure 6D.
  • the AUC of TAGMe-2 for distinguishing the endometrial cancer group and the control group was 0.985, and the AUC for distinguishing the atypical hyperplasia group and the control group was 0.973, both of which had good distinguishing performance.
  • the sensitivity and specificity of TAGMe-2 for distinguishing the endometrial cancer group and the control group were 95.24% and 94.74%, respectively, and the sensitivity and specificity for distinguishing the atypical hyperplasia group and the control group were 100% and 84.21%, respectively.
  • TAGMe-1 and TAGMe-2 were jointly detected to further analyze the sensitivity of methylation markers in detecting endometrial cancer and precancerous lesions.
  • the performance of the joint model is shown in Table 10.
  • the combined detection of the two markers can significantly improve the overall coincidence rate in detecting atypical endometrial hyperplasia.

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Abstract

一种肿瘤检测标志物TAGMe及其应用。基于对大量临床样品进行分析研究的基础上，分离获得了新型的DNA甲基化肿瘤标志物，所述标志物在正常组织中均处于低甲基化状态，在肿瘤样本中呈高甲基化状态患有肿瘤的患者中表现出显著性的甲基化状态的差异，这种差异统计学意义显著。所述标志物当联合应用于检测时，效果更为优异。该肿瘤标志物可作为临床肿瘤的诊断、筛查、分型、检测、预后的标志物，也可作为肿瘤临床辅助诊断或预后的新型分子，或可用于设计诊断试剂及试剂盒。

Description

新型的肿瘤检测标志物TAGMe及其应用
本申请要求2022年9月22日提交、发明名称为“新型的肿瘤检测标志物TAGMe及其应用”、申请号为CN 202211157754.9的优先权，其全部内容并入本文。
技术领域
本发明属于基因表观修饰领域，更具体地，本发明涉及一种新型的肿瘤检测标志物及其应用。
背景技术
子宫内膜癌(EC)是女性生殖道常见的恶性肿瘤之一，其发病率仅次于宫颈癌。随着生活条件的改善，EC的发病率逐年上升。据估计，到不久后的2030年，全球因EC发病所致的经济和医疗负担将增加60％。尽管大多数EC发生在绝经后的妇女，但最近EC的发生率在40岁或40岁以下的妇女中显著增加，范围为2-14％。对于育龄年轻女性，如果在EC早期诊断，没有子宫肌层侵犯和子宫外扩散，仍有机会保留子宫和/或卵巢。因此，早期诊断EC至关重要，这可以降低女性死亡率，争取年轻患者的治疗机会，以保持生育能力或生殖内分泌功能。
然而，目前临床缺乏可靠有效且无创的早期筛查和辅助诊断手段。在子宫内膜癌早期，多数患者没有明显的相关阳性体征，多项研究表明，90％子宫内膜癌的患者会出现各种阴道流血，但出现异常阴道流血的女性中，仅有5-10％会被诊断为子宫内膜癌及癌前病变。且目前临床缺乏可靠有效且无创的筛查和辅助诊断手段。现有的传统检测方法包括：(1)超声检查，经阴道超声(TVS)往往是绝经后出血患者的初步检查，也是最常用的无创辅助检查方法，然而由于其高假阳性率，它不能可靠地区分良性和恶性子宫内膜，同时EC也可发生在子宫内膜不增厚的女性；(2)影像学检查：包括磁共振(盆腔MRI)、CT、PET-CT等的影像学检查能够明确病变大小、具体位置等信息，但很难诊断早期EC或癌前病变，一般作分型分级用；(3)细胞学检查：作为一种无创检测手段、特异性高，灵敏性低，阴道脱落细胞学检查阳性率低，而宫腔脱落的癌细胞容易发生溶解、变性，染色后不易辨认。(4)血清肿瘤标志物：子宫内膜癌无特异敏感的诊断标志物，部分患者可出现CA125、CA19-9、CA153或HE4异常，与组织学类型、肌层浸润深度及子宫外侵袭等因素具有相关性，与子宫内膜良恶性相关性较差。由于缺乏进一步的微创分诊手法，所有异常阴道流血患者必须通过侵入性检查——刮宫和/或宫腔镜活检，对子宫内膜组织进行病理学检查以确诊子宫内膜癌。大多数子宫内膜癌是由子宫内膜增生发展为非典型增生(癌前病变)，进展持续多年。在漫长的疾病进展过程中，这些异常阴道流血患者往往经受了多次有侵袭性子宫内膜活检，导致生理、心理和经济的三重压力。因此，迫切需要寻找用于癌症早期检测的新方法。
发明内容
本发明的目的在于提供一组新型的DNA甲基化肿瘤标志物TAGMe-1和TAGMe-2及 其联合应用，该组标志物在正常组织中均处于低甲基化状态，在子宫内膜癌样本中呈高甲基化状态，可用于子宫内膜癌的检测。检测样本类型包括但不限于组织、宫颈脱落细胞、宫腔刮出物、阴道分泌物等。
在本发明的第一方面，提供分离的核酸或其转变而来的核酸在制备肿瘤检测(包括筛查、诊断、检测或预后评估)的试剂或试剂盒中的用途；其中，所述的核酸为：(1)SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列的核酸或核酸组合；或(2)与(1)的核酸在序列上互补的核酸或核酸组合；其中，所述由分离的核酸转变而来的核酸为对应于(1)或(2)的核酸，其非修饰的胞嘧啶转变为T或U，而其修饰的CpG位点的胞嘧啶C不变。
在一种或多种实施方式中，所述SEQ ID NO：1或SEQ ID NO：5还包括其序列变体，或同源序列。较佳地，所述序列变体或同源序列为与所述SEQ ID NO：1或SEQ ID NO：5所示序列相比，具有80％以上、85％以上、90％以上、92％以上、95％以上、96％以上、98％以上、99％以上、99.5％以上、99.8％以上序列同一性的序列。相应地，也包括由所述序列变体或同源序列转变(非修饰的胞嘧啶转变为T或U，而其修饰的CpG位点的胞嘧啶C不变)而来的多核苷酸。
在一种或多种实施方式中，所述与(1)的核酸在序列上互补的核酸分别是SEQ ID NO：2或SEQ ID NO：6所示核苷酸序列的核酸；本发明也包括SEQ ID NO：2或SEQ ID NO：6的序列变体或同源序列，其与所述SEQ ID NO：2或SEQ ID NO：6所示序列相比，具有80％以上、85％以上、90％以上、92％以上、95％以上、96％以上、98％以上、99％以上、99.5％以上、99.8％以上序列同一性的序列。相应地，也包括由所述序列变体或同源序列转变(非修饰的胞嘧啶转变为T或U，而其修饰的CpG位点的胞嘧啶C不变)而来的多核苷酸。
在一种或多种实施方式中，所述的肿瘤包括：子宫内膜癌，宫颈癌(宫颈鳞癌与宫颈腺癌)，胰腺癌，头颈部肿瘤，结肠癌，结直肠癌，食管癌、肺癌(肺鳞癌，肺腺癌)，胆管癌。
在一种或多种实施方式中，所述肿瘤检测针对肿瘤(如子宫内膜癌)或其癌前病变(如子宫内膜非典型增生)。
在一种或多种实施方式中，肿瘤检测针对的样本包括(但不限于)：血液样本、组织样本(如石蜡包埋样本)、宫颈样本、宫腔样本、胸腔积液样本、肺泡灌洗液样本、腹水样本、腹水灌洗液样本、胆汁样本、粪便样本、尿液样本、唾液样本、脑脊液样本、细胞涂片样本、细胞样本；较佳地，所述的样本包括(但不限于)：宫颈刮片或刷片样本、宫颈拭子、宫腔组织(刮出物)、宫颈脱落细胞、宫腔刮出物、宫腔灌洗液、阴道分泌物。
在本发明的另一方面，提供一种检测待测样品的甲基化水平的方法，包括：提取待测样品的核酸；以及检测所提取的核酸中靶序列或其片段的CpG位点修饰情况，所述的靶序列是SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列经转变而来的核酸或核酸组合，对应于SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列，其非修饰的胞嘧啶转变为T或U，而其修饰的CpG位点的胞嘧啶C不变。
在一种或多种实施方式中，检测所提取的核酸中靶序列的CpG位点修饰情况的方法包括：焦磷酸测序法、重亚硫酸盐转化测序法、甲基化芯片法、甲基化特异PCR、甲基化敏感限制性内切酶酶切法、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，或它们的组合。
在一种或多种实施方式中，所述检测所提取的核酸中靶序列的CpG位点修饰情况的方法包括：(i)对所提取的核酸进行处理，使其中未发生修饰的胞嘧啶转化为尿嘧啶。
在一种或多种实施方式中，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-羧甲基化修饰(5-caC)。
在一种或多种实施方式中，利用重亚硫酸盐处理步骤(i)所述的核酸；和(ii)分析经(i)处理的核酸中所述的靶序列的修饰情况。
在一种或多种实施方式中，所述的核酸为“SEQ ID NO：1或其反向互补序列”与“SEQ ID NO：5或其反向互补些”的组合。
在一种或多种实施方式中，甲基化谱式异常是指该核酸CpG中的C发生高度甲基化。
在一种或多种实施方式中，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。
在一种或多种实施方式中，所述的甲基化谱式的方法不是诊断性的方法，也即其不以直接获得疾病的诊断结果为目的。
在一种或多种实施方式中，所述检测样品的甲基化谱式的方法为体外方法。
在一种或多种实施方式中，所述甲基化敏感限制性内切酶为对其识别位点含有甲基化碱基敏感的限制性内切酶；包括但不限于：HpaII、AciI、Bsu15I、Hin1I、Hin6I、HpyCH4IV、NarI等其中1种或多种的组合。
在本发明的另一方面，提供一种制备试剂的方法，所述试剂用于肿瘤的检测(包括筛查、诊断、检测或预后评估)，所述方法包括：提供SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列的核酸，以所述核酸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。
在一种或多种实施方式中，所述的检测试剂包括但不限于：引物，探针，芯片或试纸。
在一种或多种实施方式中，对于SEQ ID NO：1的全长或片段，可以制备一组或多组试剂。
在一种或多种实施方式中，对于SEQ ID NO：5的全长或片段，可以制备一组或多组试剂。
在一种或多种实施方式中，所述的检测试剂被整合于芯片上。
在本发明的另一方面，提供一种试剂或试剂组合，其特异性检测靶序列的CpG位点修饰情况，所述的靶序列是SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列经转变而来的核酸或核酸组合。
在一种或多种实施方式中，所述的试剂或试剂组合针对包含所述靶序列的基因序列。
在一种或多种实施方式中，所述的基因序列包括基因Panel或基因群组。
在一种或多种实施方式中，较佳地，所述的试剂或试剂组合选自以下一组或多组：SEQ  ID NO：9和SEQ ID NO：10所示序列的引物；SEQ ID NO：11和SEQ ID NO：12所示序列的引物；SEQ ID NO：13和SEQ ID NO：14所示序列的引物；或SEQ ID NO：15和SEQ ID NO：16所示序列的引物。
在本发明的另一方面，提供所述的试剂或试剂组合的用途，用于制备检测(包括筛查、诊断、检测或预后评估)中瘤的试剂盒；较佳地，所述的肿瘤包括：子宫内膜癌，宫颈癌，结直肠癌，肺癌。
在本发明的另一方面，提供一种用于进行肿瘤检测(包括筛查、诊断、检测或预后评估)的试剂盒，所述试剂盒中包括所述的试剂或试剂组合。
在一种或多种实施方式中，所述试剂盒中还可包括但不限于：DNA纯化试剂，DNA提取试剂，重亚硫酸盐，PCR扩增试剂。
在一种或多种实施方式中，所述的试剂盒中还包括：用于标明检测操作步骤和结果判定标准的说明书。
在本发明的另一方面，提供分离的核酸或其转变而来的核酸，所述的核酸为：(1)SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列的核酸或核酸组合；或(2)与(1)的核酸在序列上互补的核酸或核酸组合；其中，所述由核酸转变而来的核酸为对应于(1)或(2)的核酸，其非修饰的胞嘧啶转变为T或U，而其修饰的CpG位点的胞嘧啶C不变。
在一种或多种实施方式中，所述修饰的CpG位点包括发生5-醛甲基化修饰、5-羟甲基化修饰、5-甲基化修饰或5-羧甲基化修饰的CpG位点。
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、子宫内膜癌及癌前病变诊断候选标志物热图。
图2A-D、TAGMe-1和TAGMe-2在TCGA数据库其他癌种中作为生物标志物的性能图。
图3A-D、实施例3中组织Target NGS结果。
图4A-D、实施例4中TAGMe-1分析结果。
图5A-D、实施例5中宫颈细胞Me-qPCR结果。
图6A-D、实施例6中宫腔细胞Me-qPCR结果。
图7、TAGMe-1甲基化肿瘤标志物的序列信息。
图8、TAGMe-2甲基化肿瘤标志物的序列信息。
具体实施方式
本发明披露，DNA甲基化变异在子宫内膜癌的发生发展过程中发挥重要作用，可以作为子宫内膜癌早期筛查、辅助诊断、疗效评估、复发监测等应用场景的标志物。本发明中，基于对大量临床样品进行分析研究的基础上，分离获得了新型的DNA甲基化肿瘤标志物， 所述标志物在正常组织中均处于低甲基化状态，在肿瘤(尤其子宫内膜癌等肿瘤)样本中呈高甲基化状态患有肿瘤的患者中表现出显著性的甲基化状态的差异，这种差异统计学意义显著。所述标志物当联合应用于检测时，效果更为优异。因此，本发明的肿瘤标志物可作为临床肿瘤的诊断、筛查、分型、检测、预后的标志物，也可作为肿瘤临床辅助诊断或预后的新型分子，或可用于设计诊断试剂及试剂盒。
本发明中，术语“样品”或“样本”包括从任何个体(较佳地为人)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。例如，所述的样本可以包括但不限于：血液样本、组织样本(如石蜡包埋样本)、宫颈样本、宫腔样本、胸腔积液样本、肺泡灌洗液样本、腹水样本、腹水灌洗液样本、胆汁样本、粪便样本、尿液样本、唾液样本、脑脊液样本、细胞涂片样本、细胞样本；较佳地，所述的样本包括(但不限于)：宫颈刮片或刷片样本、宫颈拭子、宫腔组织(刮出物)、宫颈脱落细胞、宫腔刮出物、宫腔灌洗液、阴道分泌物。
本发明中，术语“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。
本发明中，术语“肿瘤”是指一类肿瘤，其基因组中SEQ ID NO：1或SEQ ID NO：5区段呈现如本发明所述的高甲基化状态。
如本发明中所用，所述的“检测”包括筛查、诊断、检测或预后评估。
如本发明中所用，所述的“子宫内膜癌”在广义上包括了“子宫内膜癌及其癌前病变，后者也称为“子宫内膜非典型增生”或“早期子宫内膜癌”；但可以理解的是，在特定的情况下，例如一些实施例中，所述的“子宫内膜癌”被与“子宫内膜癌癌前病变”分列，此时“子宫内膜癌”指已经过了癌前病变阶段的癌症，例如已处于进展期。
本发明中，提出一些有意义的表观遗传学变化是子宫内膜癌发生的重要因素。与DNA突变相比，基因组特定区域呈现高甲基化状态，该高甲基化状态在肿瘤的早期诊断中发生得更早且更稳定，异常的DNA甲基化模式可用于预测非典型增生向子宫内膜癌转变的风险。所述的DNA高甲基化状态贯穿于正常细胞变为肿瘤细胞的全过程，且发生于肿瘤超早期，超前于基因突变。因此，基于DNA高甲基化状态的肿瘤检测，可应用于肿瘤超早期筛查、辅助诊断、疗效评估、复发监测，覆盖肿瘤诊疗的全流程。
在此基础上，本发明提供了一组甲基化标志物，TAGMe-1和TAGMe-2，作为子宫内膜癌的分子检测标志物。该标志物在正常及癌旁子宫内膜组织中处于低甲基化状态，在子宫内膜癌及癌前病变(非典型增生)组织中呈高甲基化状态。可以通过宫颈和宫腔处的脱落细胞，实现对早期子宫内膜癌和癌前病变的无创辅助诊断，填补了现有子宫内膜癌筛查技术的空白。并且可通过进一步发展，实现相对简单、廉价、可靠和高通量的临床测试。
本发明中，SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列或其部分区域(片段)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，当检测到所述基因序列区域存在异常高甲基化状态，则可认为该受检者罹患肿瘤或属于肿瘤高危人群。SEQ ID NO：1或SEQ ID NO：5所示基因序列或其部分区域呈现的这种甲基化状态的显著差异非常显著地存在于子宫内膜癌中(包括早期阶段)；同时，这种甲基化状态的显著差异也存在于其它一些肿瘤中，包括：宫颈癌(宫颈鳞癌与宫颈腺癌)，胰腺癌，头颈部肿瘤，结肠癌，结直肠癌，食管癌、肺癌(肺鳞癌，肺腺癌)，胆管癌等。
本发明中，也包括具有与SEQ ID NO：1(或括其反向互补序列)或SEQ ID NO：5(或其反向互补序列)所示核苷酸序列具有保守性的或序列同一性较高的、SEQ ID NO：1(或其反向互补序列)或SEQ ID NO：5(或其反向互补序列)序列的“保守性变异序列”。所述“序列一源性较高”例如高于90％、高于92％、高于95％、高于98％高于99％等。应理解，不同的生物个体之间，可能在个别序列位点上存在不同(例如可能存在一些无意义的SNP)，但这不影响基于本发明的总体方案的检测。
根据上述，本发明提供了来源于人基因组特定区域的核酸，其具有SEQ ID NO：1或SEQ ID NO：5所示基因序列或其部分区域，同时还包括其反义链。在肿瘤细胞内，该核酸序列中多处5’-CpG-3’的碱基C位置上生成5-甲基胞嘧啶(5mC)。
针对本发明提供的一个或多个CpG进行检测均是可以的，因此本发明也包含所述核苷酸序列的核酸的片段，且其中包括至少1个甲基化CpG位点。所述的至少一个可以包括SEQ ID NO：1或其反向互补序列中2～53个，更具体如3，5，8，10，12，15，18，20，30，40，50个。所述的至少一个可以包括SEQ ID NO：5或其反向互补序列中2～52个，更具体如3，5，8，10，12，15，18，20，30，40，50个。本领域人员可以理解的是，在本发明提供了基于一条DNA链的CpG编号后，与之互补的DNA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。
根据本发明所提供的人基因组中所述特定片段的信息后，本领域技术人员可以方便地获得所述CpG位点，加以应用。本发明的实施例中提供了一系列的含有CpG位点的序列片段，这些片段可以作为较佳实施方式的一些例子，但是应理解，根据本发明提供的信息，人们可以作出变化，例如选择更长的、但含有本发明的序列的序列，或选择与本发明相对序列在区域上有交叉的序列。
本发明也包括SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列或序列片段或其互补序列(SEQ ID NO：2或SEQ ID NO：6)的基因Panel或基因群组。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。
应理解，多种多样的可用于分析甲基化状态的技术均可被应用于本发明中，本发明对此类检测技术没有特别的限制。本发明提供的核酸可以作为基因组中分析甲基化状态的关键区域，可通过各种本领域已知的技术来分析它们的甲基化状态，进而分析肿瘤的发生或发展情况。
本发明的SEQ ID NO：1或SEQ ID NO：5所述的核酸或其片段或与其互补的序列，可在经过重亚硫酸盐处理后，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核酸(包括其互补链(反义链))经过重亚硫酸盐处理后获得的核酸，包括：SEQ ID NO：3或SEQ ID NO：7所示核苷酸序列的核酸或核酸片段，或如SEQ ID NO：4或SEQ ID NO：8所示核苷酸序列的核酸或核酸片段。这些核酸可作为设计检测试剂或检测试剂盒的更为直接的靶点。
本发明的SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列的核酸和/或其互补核酸和/或其一或多条片段可被整合于一个或若干个整体中，如一个或若干个核酸集中，供本领域技术人员运用，如从该核酸集中选择一条或多条核酸或核酸的片段，设计靶向分析试剂。所设计的靶向分析试剂，也可被整合于一个或若干个整体中，如一个或若干个试剂盒中。
本发明的由SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列的核酸和/或其互补核酸和/或其一或多条片段转变(例如经重亚硫酸盐转化)而来的核酸，也可被整合于一个或若干个整体中，如一个或若干个核酸集中，供本领域技术人员运用，如从该核酸集中选择一条或多条核酸或核酸的片段，设计靶向分析试剂。所设计的靶向分析试剂，也可被整合于一个或若干个整体中，如一个或若干个试剂盒中，或一片或若干片芯片中。
作为本发明的优选方式，来自SEQ ID NO：1所示核苷酸序列的核酸和/或其互补核酸和/或其一或多条片段，与来自SEQ ID NO：5所示核苷酸序列的核酸和/或其互补核酸和/或其一或多条片段被联合用于检测。
在本发明的提供了靶基因及其表观特点的基础上，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，来实施甲基化水平的检测。测定核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。例如，检测甲基化水平时可使用定量甲基化特异性PCR(QMSP)的方法，其基于一种荧光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。此外，其他可用的技术还有：qPCR(Me-qPCR)法、二代测序法、焦磷酸测序法、Sanger测序法、重亚硫酸盐转化测序法、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。尽管本发明的实施例中提供了一些优选的方式，但本发明的总体方案并不限于此。
作为本发明的优选方式，还提供了一种体外检测样品中核酸的甲基化谱式的方法。所述的方法基于的原理是：重亚硫酸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，经过重亚硫酸盐处理核酸后，甲基化的位点产生类似于一个C/T的核酸多态性(SNP)。基于上述原理来鉴定检测样品中核酸的甲基化谱式，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：首先提供样品、提取基因组DNA；其次，利用重亚硫酸盐处理步骤(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；再次，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：对受试者样品进行检测，评估受试者是否患有肿瘤；或用于区分肿瘤高危人群。所述的方法可以是不以获得直接的疾病诊断结果为目的的情形，例如不以判断疾病最终结果为目的的情形、人群地域性分析研究、科学研究、人口普查等。
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，实际应用中并不限于该方法，本领域已知的或正在改进的其它DNA甲基化检测方法亦可。在进行PCR扩增中，所应用的引物并不限于是实施例中所提供的，还可以获得与本发明实施例所提供的引物在序列上存在不同，但仍然针对于本发明所指示的核酸或相应CpG位点的引物。
作为本发明的优选方式，还提供了一种体外检测样品中核酸的甲基化情况的方法，所述方法为甲基化敏感限制性内切酶酶切法(methylation sensitive restriction endonuclease，MSRE)。当在其切割位点中含有一个甲基化碱基时，所述的甲基化敏感限制性内切酶不能切割DNA。MSRE法是基于甲基化敏感II型限制性内切酶不能切割含有一个或多个甲基化切点序列的基本原理。用甲基敏感II型内切酶及其同工酶(对甲基化不敏感)切割含有一个或多个甲基化CpG序列的片段，然后用DNA印迹法分析。这一方法的优点包括：无须知道靶DNA一级结构的详细信息，并可提供CpG岛甲基化状态的直接评价，包括取得一些对被检基因甲基化的定量分析信息。
围绕本发明所提供的标记物核酸，其它本领域技术人员已知的确定基因组的序列、其变异及甲基化状态的检测方法和试剂均可包括在本发明中。
本发明提供了一种制备肿瘤检测试剂的方法，包括：提供所述的核酸，以所述核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，所述的靶序列中包括至少1个甲基化CpG位点。所述的检测试剂可以包括但不限于：芯片，引物，探针，等等；在获得了所述的标记物后，检测试剂的选择是本领域技术人员可以做到的。
在得知了核酸的序列后，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)。在本发明优选的实施例中，所述的试剂是引物，所述的引物优选地为被列于表2和表6中的那些。除了引物，其它的诊断或检测试剂也是可以制备的，包括但不限于探针、芯片等。
所述的试剂也可以是试剂组合，例如引物组合。例如，所述的组合包括多于一组的引物，从而可分别扩增上述的多条核酸。
本发明还提供了体外检测样品中核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。
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以及其它试剂，例如样本处理试剂。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。
本发明的方法及试剂用于诊断临床肿瘤时，准确性非常高，这在本发明实施例中针对多种肿瘤临床样本的检测中得以体现。本发明可应用于肿瘤前期筛查、疗效判定、辅助诊断、预后监测等等领域，或如前所述的不以获得直接的疾病诊断结果为目的的情形。
本发明提供的子宫内膜癌等肿瘤的标志物，独立应用时特异性和敏感性非常高，而若是将两种标志物进行联合应用，其敏感性和特异性还会呈现进一步的提高。因此，本发明的标志物在肿瘤辅助诊断、疗效判定、预后监测等等领域极具应用价值。
本发明提供了子宫内膜癌等肿瘤的标志物，可以适用于针对宫颈脱落细胞等的检测， 待测样本的获得是容易的，且是无创的。这与临床上需要手术获取组织样本的情形相比，受试者的痛苦大大减少、依从性佳，且临床医师的操作也更为容易。显然，这呈现了非常显著的进步。
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，科学出版社中所述的条件，或按照制造厂商所建议的条件。
实施例1、子宫内膜癌检测标志物的筛选
从临床收集了正常子宫内膜-普通增生子宫内膜-非典型增生子宫内膜(癌前病变)-子宫内膜癌-配对癌旁的五组共43例冰冻组织样本，利用简并表达重亚硫酸盐测序(RRBS)方法，对其全基因组水平上的甲基化数据进行分析，筛选子宫内膜癌及癌前病变特异性DNA甲基化标志物。
1、获取临床样本
收集正常子宫内膜-普通增生子宫内膜-非典型增生子宫内膜(癌前病变)-子宫内膜癌-配对癌旁的五组共43例冰冻组织样本，每例样本取黄豆粒大小。
2、DNA提取
用奕谱基因组DNA提取试剂盒(Epiprobe Biotech，K-21)进行样本基因组DNA提取(但本发明核酸提取技术方案不限于该方法)。
3、RRBS测序文库构建
提取好的样本gDNA利用MspI内切酶进行酶切，随后用磁珠分选大小合适的片段，后续完成末端补平、加dA、加接头等步骤。
4、重亚硫酸盐处理
上述文库样品使用重亚硫酸进行修饰，本实验中用ZYMO Research公司的EZ DNA Methylation-Gold Kit，货号D5006，严格按照使用手册步骤操作(但本发明不限于该试剂盒)。
5、PCR扩增与产物纯化
每个文库样本选用带有不同分子标签的引物进行扩增，利用磁珠分选并纯化扩增产物，测定浓度后上机测序。
6、RRBS测序
利用Illumina Hiseq 2000对组织样本基因组RRBS文库进行2×150bp测序。下机数据通过质控、比对等步骤，分析出每个样本中的有效测序数、所测CpG位点数量、覆盖层数等基本信息如表1所示。RRBS文库的下机数据量达到198.31G，经过正常内膜组(A组，NE)、普通增生组(B组，EH)、非典型增生组(C组，AH)、子宫内膜癌组(D组，EC)、癌旁组织(E组，PC)分别平均获得16686194M、16847536M、14942502M、14729170M和14637777M的测序序列。覆盖层数大于等于5层的所有CpG位点数分别为4055958M、4169395M、4369673M、3984048M和4271290M。统计数据及初步实验分析表明，本次RRBS测序总 体质量较好，测序深度和CpG覆盖层数良好，各组数据量分布均匀，结果可信度高。
表1

7、标志物筛选
通过对不同阶段间甲基化测序数据的分析、结合实验分析，筛选在非典型增生子宫内膜(癌前病变)和/或癌症阶段存在的特异性甲基化标志物。如图1展示了其中一小部分子宫内膜癌及癌前病变诊断候选标志物热图，本发明人得到癌前病变Precancer-DMRs和子宫内膜癌Cancer-DMRs两组子宫内膜癌检测的候选甲基化标志物，分别对应仅在AH和EC或仅在EC阶段显著性高甲基化，而在其它阶段包括癌旁组织中均低甲基化的DMRs。
(1)TAGMe-1
所获得的TAGMe-1甲基化肿瘤标志物的核苷酸序列如SEQ ID NO：1，见图7，底色标示的位点为潜在发生甲基化的CG位点(53组)。
SEQ ID NO：1的反向互补序列如SEQ ID NO：2，见图7，底色标示的位点为潜在发生甲基化的CG位点。
SEQ ID NO：1序列经亚硫酸盐转化后的序列如SEQ ID NO：3，见图7，底色标示的位点为潜在发生甲基化的CG位点，Y代表C或T。
SEQ ID NO：3的反向互补序列经亚硫酸盐转化后的序列如SEQ ID NO：4，见图7，底色标示的位点为潜在发生甲基化的CG位点，Y代表C或T。
(2)TAGMe-2
所获得的TAGMe-2甲基化肿瘤标志物的核苷酸序列如SEQ ID NO：5，见图8，底色标示的位点为潜在发生甲基化的CG位点(52组)。
SEQ ID NO：5的反向互补序列如SEQ ID NO：6，底色标示的位点为潜在发生甲基化的CG位点。
SEQ ID NO：1序列经亚硫酸盐转化后的序列如SEQ ID NO：7，见图8，底色标示的位点为潜在发生甲基化的CG位点，Y代表C或T。
SEQ ID NO：7的反向互补序列亚硫酸盐转化后的序列如SEQ ID NO：8，见图8，底色标示的位点为潜在发生甲基化的CG位点，Y代表C或T。
8、焦磷酸测序法验证候选DNA甲基化生物标志物临床性能
为了进一步验证上述筛选到的候选标志物是否具有临床应用的价值，将本发明的标志物与其它也被分析认为具有甲基化差异和显著性的Precancer-DMRs(图1热图Precancer-DMRs里排名靠前)，利用焦磷酸测序技术在另外收集的临床组织样品中进行比较实验。从临床收集了45例临床组织样品，其中包括14例普通内膜(NE)受试者、11例增生(不伴非典型增生，EH)受试者、5例伴非典型增生(AH)受试者和15例子宫内膜癌(EC)受试者的组织。
焦磷酸测序扩增引物如表2。
表2
临床组织样本中候选甲基化标志物诊断子宫内膜癌前病变及以上的性能指标如表3。
表3
根据上述，两种DMRs位点TAGMe-1和TAGMe-2的AUC分别为0.938和0.925，均高于0.9，对于子宫内膜癌早期(Precancer)患者就能达到很高的AUC，令人意外，证明其作为子宫内膜癌早期筛查甲基化标志物的性能非常优异。
实施例2、TAGMe-1和TAGMe-2的性能验证
TCGA数据库作为公认的肿瘤数据库，包括了多种肿瘤的数据；针对某种肿瘤，其记录了一系列表达数据、miRNA表达数据、甲基化数据、突变数据和拷贝数数据。因此，本发明人以该数据库对TAGMe-1和TAGMe-2进行性能验证。
TCGA数据库中多癌种甲基化数据下载：下载了TCGA数据中全部肿瘤样品的甲基化450K芯片数据，其中包括11087例肿瘤及对照正常样本。共涉及到的35种肿瘤类型及样本量见表4。
表4、35种肿瘤甲基化数据集信息

TAGMe-1和TAGMe-2的甲基化值计算：首先分别计算出TAGMe-1和TAGMe-2在不同类型肿瘤组织样本与对照正常组织样本中的平均甲基化值，然后计算出TAGMe-1和TAGMe-2在该类型肿瘤样本与对照样本中平均甲基化值的差值。
TAGMe-1和TAGMe-2在其它癌症中的诊断性能分析：TAGMe-1和TAGMe-2的甲基化值在不同癌种肿瘤样本与对照样本间的差异如图2A、2C所示，通过双侧Mann-Whitney t-test分析，TAGMe-1在包括子宫内膜癌、宫颈癌、胆管癌、结直肠癌(结肠癌、直肠癌)、食管癌、肺癌(肺鳞癌、肺腺癌)、头颈部肿瘤、胰腺癌等多种肿瘤类型中存在肿瘤组与对照组间的统计学显著的甲基化水平差异。而TAGMe-2则分别在结直肠癌、子宫内膜癌和宫颈癌中存在肿瘤组与对照组间的统计学显著的甲基化水平差异。其对应诊断的AUC及性能见图2B、2D及表5。
表5、其他癌种中作为生物标志物的性能表
因此，TAGMe-1和TAGMe-2在多种类的肿瘤中具有诊断价值。
实施例3、子宫内膜癌-癌旁组织样本中DNA甲基化检测：target-NGS法
本实施例中，利用target-NGS法，进行子宫内膜癌-癌旁组织样本中DNA甲基化分析。步骤如下：
1、获取临床样本
从临床获取20例正常子宫内膜组织/癌旁组织，6例子宫内膜非典型增生样本和20例子宫内膜癌组织样本，正常/癌旁样本作为对照组，非典型增生及癌组织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；本实验用奕谱基因组DNA提取试剂盒(Epiprobe Biotech，K-21)进行DNA提取(但本发明不限于该方法)。
3、重亚硫酸盐处理
提取后的DNA使用重亚硫酸进行修饰，本实验中用ZYMO Research公司的EZ DNA Methylation-Gold Kit，货号D5006，严格按照使用手册步骤操作(但本发明不限于该试剂盒)。
4、PCR扩增与建库
完成修饰后先用针对重亚硫酸盐处理后的目标区域设计的PCR引物进行扩增富集，引物序列如表6。
表6
随后进行产物纯化、测序接头连接来构建NGS测序文库。
5、NGS测序
通过Illumia公司的HiSeq X高通量测序仪进行检测，严格按照说明书步骤进行操作(但本发明不限于该型号高通量测序仪)。
6、TAGMe-1和TAGMe-2甲基化值计算
Target NGS测序下机数据中包含目标区域内所有单个CpG位点的甲基化情况，TAGMe-1和TAGMe-2甲基化水平的计算方式为：单个位点甲基化水平＝该位点甲基化reads数/总reads数，目标区域的甲基化水平＝所有位点甲基化水平的平均值。
7、单一标志物结果分析
组织Target NGS结果如图3。比较癌旁/正常对照组与肿瘤实验组中的TAGMe-1和TAGMe-2甲基化值水平差异，并进行ROC分析。TAGMe-1结果如图图3A，在子宫内膜癌临床样本中，TAGMe-1在非典型增生及癌组织样本中的甲基化值均显著高于对照组。TAGMe-1的ROC分析结果如图3B，TAGMe-1区分子宫内膜癌组和对照组的AUC为0.995，区分非典型增生组和对照组的AUC为0.983，均具有良好的区分性能。取约登系数最大时为阈值，则TAGMe-1区分子宫内膜癌组与对照组的灵敏性和特异性分别为95％和100％；区分非典型增生与对照组的灵敏性和特异性分别为83.33％和90％。
TAGMe-2结果如图3C，在子宫内膜癌临床样本中，在非典型增生及癌组织样本中的甲基化值均显著高于对照组。TAGMe-2的ROC分析结果如图3D，TAGMe-2区分子宫内膜癌组和对照组的AUC为0.990，区分非典型增生组和对照组的AUC为0.967，均具有良好的区分性能。取约登系数最大时为阈值，则TAGMe-2区分子宫内膜癌组和对照组的灵敏性和特异性分别为95％和100％，区分非典型增生和对照组的灵敏性和特异性分别为83.33％和100％。
8、标志物的联合应用分析
将TAGMe-1和TAGMe-2进行联合检测，联合性能如表7。
表7、TAGMe-1和TAGMe-2联合分析性能表
结果显示，两者联用具有协同作用，进一步增加甲基化标志物检测子宫内膜癌及癌前病变的敏感性。
实施例4、子宫内膜癌-癌旁组织样本中DNA甲基化检测：焦磷酸测序法
本实施例中，利用焦磷酸测序法进行子宫内膜癌-癌旁组织样本中DNA甲基化检测，进行标志物分析。步骤如下：
1、获取临床样本：从临床获取25例正常宫颈脱落细胞样品，5例子宫内膜非典型增生患者宫颈脱落细胞样本和15例子宫内膜癌患者宫颈脱落细胞样本，正常宫颈脱落细胞样品作为对照组，非典型增生及子宫内膜癌患者宫颈脱落细胞样本作为肿瘤检测实验组。
2、DNA提取：提取临床样品DNA；本实验用奕谱基因组DNA提取试剂盒(Epiprobe Biotech，K-21)进行DNA提取(但本发明不限于该方法)。
3、重亚硫酸盐处理
提取后的DNA使用重亚硫酸进行修饰，本实验中用ZYMO Research公司的EZ DNA Methylation-Gold Kit，货号D5006，严格按照使用手册步骤操作(但本发明不限于该方法)。
4、引物设计
根据TAGMe-1和TAGMe-2序列(SEQ ID NO：1和SEQ ID NO：5)，分别设计扩增引物与焦磷酸测序引物，检测靶序列上对应CpG位点的甲基化值，作为其甲基化值的代表，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同实施例1中。
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的模板，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PyroMarkQ96ID焦磷酸测序仪进行检测，严格按照说明书步骤进行操作。
7、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TAGMe-1和TAGMe-2在该样本中的甲基化值。
8、结果分析
比较癌旁/正常对照组与肿瘤实验组中的TAGMe-1和TAGMe-2甲基化值水平差异，并进行ROC分析。
TAGMe-1分析结果如图4A，在子宫内膜癌临床样本中，TAGMe-1在非典型增生及癌组织样本中的甲基化值均显著高于对照组。ROC分析结果如图4B，TAGMe-1区分子宫内膜癌 组和对照组的AUC为0.955，区分非典型增生组和对照组的AUC为0.952，均具有良好的区分性能。取约登系数最大时为阈值，则TAGMe-1区分子宫内膜癌组和对照组的灵敏性和特异性分别为100％和84％，区分非典型增生和对照组的灵敏性和特异性分别为100％和84％。
TAGMe-2分析结果如图4C，在子宫内膜癌临床样本中，TAGMe-2在非典型增生及癌组织样本中的甲基化值均显著高于对照组。TAGMe-2的ROC分析结果如图4D，TAGMe-2区分子宫内膜癌组和对照组的AUC为0.943，区分非典型增生组和对照组的AUC为0.928，均具有良好的区分性能。取约登系数最大时为阈值，则TAGMe-2区分子宫内膜癌组和对照组的灵敏性和特异性分别为86.67％和88％，区分非典型增生和对照组的灵敏性和特异性分别为100％和80％。
9、TAGMe-1及TAGMe-2联用分析
将TAGMe-1和TAGMe-2进行联合检测，进一步分析甲基化标志物检测子宫内膜癌及癌前病变的敏感性，联合性能如表8。
表8、TAGMe-1和TAGMe-2联合分析性能表
癌旁样本并不能简单认为正常样本，癌旁样本在临床取样时由于容易混杂部分癌组织、或由于癌灶部位的影响而发生一定的表观遗传学变异，由此易于使得特异性有所下降，故表中部分样本中的特异性相比前述实施例会有浮动。尽管如此，两种标志物的联合检测能明显提高检测子宫内膜非典型增生时的特异性、总体符合率和阳性预测值。
实施例5、子宫内膜癌-宫颈脱落细胞样本中DNA甲基化检测
本实施例中，利用Me-qPCR法进行子宫内膜癌-宫颈脱落细胞样本中DNA甲基化检测，进行标志物分析。步骤如下：
1、获取临床样本：从临床获取38例正常宫颈脱落细胞样品，8例子宫内膜非典型增生患者宫颈脱落细胞样本和33例子宫内膜癌患者宫颈脱落细胞样本，正常宫颈脱落细胞样品作为对照组，非典型增生及子宫内膜癌患者宫颈脱落细胞样本作为肿瘤检测实验组。
2、DNA提取：提取临床样品DNA；本实验用奕谱基因组DNA提取试剂盒(Epiprobe Biotech，K-21)进行DNA提取(但本发明不限于该方法)。
3、酶切反应：利用甲基化敏感限制性内切酶对DNA进行酶切，未被甲基化的酶切位点会被切断，本实验用HpaII酶(NEB，R0171)进行酶切反应(但本发明不限于该方法)。
4、引物设计：根据TAGMe-1序列和TAGMe-2序列，设计对应扩增引物，对甲基化敏感限制性内切酶酶切产物进行扩增，检测酶切后的DNA片段丰度，分别作为TAGMe-1和TAGMe-2甲基化值的代表，以GAPDH基因作为内参。
5、qPCR扩增：以酶切后的样本作为qPCR的模板，进行qPCR扩增，本实验用Thermo 公司的ABI 7500qPCR仪进行检测(但本发明不限于该方法)。
6、甲基化值计算：通过如下公式评估每个样本的DNA甲基化水平：ΔCt_本发明标记物＝Ct_本发明标记物-Ct_GAPDH，ΔCt越小代表甲基化水平越高。
7、结果分析：比较对照组与肿瘤实验组中的TAGMe-1和TAGMe-2甲基化值水平差异，并进行ROC分析。
宫颈细胞Me-qPCR结果如图5。TAGMe-1结果如图5A，在子宫内膜癌临床样本中，TAGMe-1在非典型增生及癌组织样本中的甲基化值均显著高于对照组。TAGMe-1ROC分析结果如图5B，TAGMe-1区分子宫内膜癌组和对照组的AUC为0.916，区分非典型增生组和对照组的AUC为0.870，均具有良好的区分性能。取约登系数最大时为阈值，则TAGMe-1区分子宫内膜癌组和对照组的灵敏性和特异性分别为84.85％(68.1％-94.89％)和89.47％(75.2％-97.06％)，区分非典型增生和对照组的灵敏性和特异性分别为87.5％(47.35％-99.68％)和76.32％(59.76％-88.56％)。TAGMe-2结果如图5C，在子宫内膜癌临床样本中，TAGMe-2在非典型增生及癌组织样本中的甲基化值均显著高于对照组。TAGMe-2ROC分析结果如图5D，TAGMe-2区分子宫内膜癌组和对照组的AUC为0.921，区分非典型增生组和对照组的AUC为0.845，均具有良好的区分性能。取约登系数最大时为阈值，则TAGMe-2区分子宫内膜癌组和对照组的灵敏性和特异性分别为87.88％和86.84％，区分非典型增生和对照组的灵敏性和特异性分别为100％和57.89％。
8、TAGMe-1和TAGMe-2联用分析：将TAGMe-1和TAGMe-2进行联合检测，进一步分析甲基化标志物检测子宫内膜癌及癌前病变的敏感性，联合模型性能如表9。
表9、TAGMe-1和TAGMe-2联合分析性能表
根据上述，两种标志物的联合检测能明显提高检测子宫内膜非典型增生时的总体符合率，以及检测子宫内膜癌时的灵敏性和符合率。
实施例6、子宫内膜癌-宫腔脱落细胞样本中DNA甲基化检测(Me-qPCR法)
从临床获取19例正常宫颈脱落细胞样品，6例子宫内膜非典型增生患者宫颈脱落细胞样本和21例子宫内膜癌患者宫颈脱落细胞样本，正常宫颈脱落细胞样品作为对照组，非典型增生及子宫内膜癌患者宫颈脱落细胞样本作为肿瘤检测实验组。按照实施例5的Me-qPCR检测步骤，比较对照组与肿瘤实验组中TAGMe-1和TAGMe-2的甲基化值水平差异，并进行ROC分析。
宫腔细胞Me-qPCR结果如图6。TAGMe-1结果如图6A，在子宫内膜癌临床样本中，TAGMe-1在非典型增生及癌组织样本中的甲基化值均显著高于对照组。ROC分析结果如图6B，TAGMe-1区分子宫内膜癌组和对照组的AUC为0.988，区分非典型增生组和对照组的AUC为0.921，均具有良好的区分性能。取约登系数最大时为阈值，则TAGMe-1区分子宫 内膜癌组和对照组的灵敏性和特异性分别为90.48％和100％，区分非典型增生和对照组的灵敏性和特异性分别为83.33％和94.74％。
TAGMe-2结果如图6C，在子宫内膜癌临床样本中，TAGMe-2在非典型增生及癌组织样本中的甲基化值均显著高于对照组。ROC分析结果如图6D，TAGMe-2区分子宫内膜癌组和对照组的AUC为0.985，区分非典型增生组和对照组的AUC为0.973，均具有良好的区分性能。取约登系数最大时为阈值，则TAGMe-2区分子宫内膜癌组和对照组的灵敏性和特异性分别为95.24％和94.74％，区分非典型增生和对照组的灵敏性和特异性分别为100％和84.21％。
将TAGMe-1和TAGMe-2进行联合检测，以进一步分析甲基化标志物检测子宫内膜癌及癌前病变的敏感性。联合模型性能如表10。
表10、TAGMe-1和TAGMe-2联合分析性能表
根据上述，两种标志物的联合检测能明显提高检测子宫内膜非典型增生时的总体符合率。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。同时，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。

Claims (15)

  1. 分离的核酸或其转变而来的核酸在制备肿瘤检测的试剂或试剂盒中的用途；
    其中，所述的核酸为：(1)SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列的核酸或核酸组合；或(2)与(1)的核酸在序列上互补的核酸或核酸组合；
    其中，所述由分离的核酸转变而来的核酸为对应于(1)或(2)的核酸，其非修饰的胞嘧啶转变为T或U，而其修饰的CpG位点的胞嘧啶C不变。
  2. 如权利要求1所述的用途，其特征在于，所述与(1)的核酸在序列上互补的核酸分别是SEQ ID NO：2或SEQ ID NO：6所示核苷酸序列的核酸。
  3. 如权利要求1所述的用途，其特征在于，所述的肿瘤包括：子宫内膜癌，宫颈癌，胰腺癌，头颈部肿瘤，结肠癌，结直肠癌，食管癌、肺癌，胆管癌。
  4. 如权利要求1所述的用途，其特征在于，所述肿瘤检测针对肿瘤或其癌前病变。
  5. 如权利要求1所述的用途，其特征在于，肿瘤检测针对的样本包括：血液样本、组织样本、宫颈样本、宫腔样本、胸腔积液样本、肺泡灌洗液样本、腹水样本、腹水灌洗液样本、胆汁样本、粪便样本、尿液样本、唾液样本、脑脊液样本、细胞涂片样本、细胞样本；较佳地，所述的样本包括：宫颈刮片或刷片样本、宫颈拭子、宫腔组织、宫颈脱落细胞、宫腔刮出物、宫腔灌洗液、阴道分泌物。
  6. 一种检测待测样品的甲基化水平的方法，其特征在于，所述方法包括：
    提取待测样品的核酸；以及
    检测所提取的核酸中靶序列或其片段的CpG位点修饰情况，所述的靶序列是SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列经转变而来的核酸或核酸组合，对应于SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列，其非修饰的胞嘧啶转变为T或U，而其修饰的CpG位点的胞嘧啶C不变。
  7. 如权利要求6所述的方法，其特征在于，检测所提取的核酸中靶序列的CpG位点修饰情况的方法包括：焦磷酸测序法、重亚硫酸盐转化测序法、甲基化芯片法、甲基化特异PCR、甲基化敏感限制性内切酶酶切法、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，或它们的组合。
  8. 如权利要求6所述的方法，其特征在于，所述检测所提取的核酸中靶序列的CpG位点修饰情况的方法包括：(i)对所提取的核酸进行处理，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。
  9. 如权利要求8所述的方法，其特征在于，利用重亚硫酸盐处理步骤(i)所述的核酸；和(ii)分析经(i)处理的核酸中所述的靶序列的修饰情况。
  10. 一种制备试剂的方法，所述试剂用于肿瘤的检测，其特征在于，所述方法包括：
    提供SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列的核酸，以所述核酸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。
  11. 如权利要求10所述的方法，其特征在于，所述的检测试剂包括但不限于：引物，探针，芯片或试纸。
  12. 一种试剂或试剂组合，其特异性检测靶序列的CpG位点修饰情况，所述的靶序列是SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列经转变而来的核酸或核酸组合；较佳地，所述的试剂或试剂组合针对包含所述靶序列的基因序列，较佳地所述的基因序列包括基因Panel或基因群组；较佳地，所述的试剂或试剂组合选自：
    SEQ ID NO：9和SEQ ID NO：10所示序列的引物；
    SEQ ID NO：11和SEQ ID NO：12所示序列的引物；
    SEQ ID NO：13和SEQ ID NO：14所示序列的引物；或
    SEQ ID NO：15和SEQ ID NO：16所示序列的引物。
  13. 权利要求12所述的试剂或试剂组合的用途，用于制备检测肿瘤的试剂盒；较佳地，所述的肿瘤包括：子宫内膜癌，宫颈癌，胰腺癌，头颈部肿瘤，结肠癌，结直肠癌，食管癌、肺癌，胆管癌。
  14. 一种用于进行肿瘤检测的试剂盒，其特征在于，所述试剂盒中包括权利要求12所述的试剂或试剂组合。
  15. 分离的核酸或其转变而来的核酸，其特征在于，所述的核酸为：
    (1)SEQ ID NO：1或SEQ ID NO：5所示核苷酸序列的核酸或核酸组合；或
    (2)与(1)的核酸在序列上互补的核酸或核酸组合；
    其中，所述由核酸转变而来的核酸为对应于(1)或(2)的核酸，其非修饰的胞嘧啶转变为T或U，而其修饰的CpG位点的胞嘧啶C不变。
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