CN116790761B - Biomarker for benign and malignant lesions of endometrium and application of biomarker - Google Patents
Biomarker for benign and malignant lesions of endometrium and application of biomarker Download PDFInfo
- Publication number
- CN116790761B CN116790761B CN202311054535.2A CN202311054535A CN116790761B CN 116790761 B CN116790761 B CN 116790761B CN 202311054535 A CN202311054535 A CN 202311054535A CN 116790761 B CN116790761 B CN 116790761B
- Authority
- CN
- China
- Prior art keywords
- gene
- methylation
- endometrial cancer
- endometrial
- gypc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000003902 lesion Effects 0.000 title claims abstract description 54
- 239000000090 biomarker Substances 0.000 title claims abstract description 23
- 230000003211 malignant effect Effects 0.000 title claims abstract description 15
- 210000004696 endometrium Anatomy 0.000 title description 7
- 206010014759 Endometrial neoplasm Diseases 0.000 claims abstract description 161
- 206010014733 Endometrial cancer Diseases 0.000 claims abstract description 157
- 230000011987 methylation Effects 0.000 claims abstract description 150
- 238000007069 methylation reaction Methods 0.000 claims abstract description 150
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 74
- 206010020718 hyperplasia Diseases 0.000 claims abstract description 64
- 230000002357 endometrial effect Effects 0.000 claims abstract description 51
- 238000001514 detection method Methods 0.000 claims abstract description 23
- 238000003745 diagnosis Methods 0.000 claims abstract description 23
- 101150054984 GYPC gene Proteins 0.000 claims abstract description 21
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 4
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 4
- 238000002360 preparation method Methods 0.000 claims abstract description 3
- 101000905336 Homo sapiens Glycophorin-C Proteins 0.000 claims description 55
- 102100023849 Glycophorin-C Human genes 0.000 claims description 54
- 239000000523 sample Substances 0.000 claims description 47
- 238000012360 testing method Methods 0.000 claims description 40
- 238000000034 method Methods 0.000 claims description 15
- 102100029136 Collagen alpha-1(II) chain Human genes 0.000 claims description 10
- 101000771163 Homo sapiens Collagen alpha-1(II) chain Proteins 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 5
- 239000012502 diagnostic product Substances 0.000 claims description 4
- 238000009472 formulation Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 101000916547 Homo sapiens Zinc finger and BTB domain-containing protein 38 Proteins 0.000 description 48
- 102100028125 Zinc finger and BTB domain-containing protein 38 Human genes 0.000 description 47
- 108020004414 DNA Proteins 0.000 description 36
- 101000784535 Homo sapiens Zinc finger and SCAN domain-containing protein 12 Proteins 0.000 description 30
- 102100020922 Zinc finger and SCAN domain-containing protein 12 Human genes 0.000 description 30
- 239000003153 chemical reaction reagent Substances 0.000 description 30
- 238000006243 chemical reaction Methods 0.000 description 23
- 102100026358 Lipoma-preferred partner Human genes 0.000 description 19
- 208000028183 atypical endometrial hyperplasia Diseases 0.000 description 18
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 15
- 201000006828 endometrial hyperplasia Diseases 0.000 description 14
- 238000003753 real-time PCR Methods 0.000 description 14
- 102000053602 DNA Human genes 0.000 description 13
- 230000000295 complement effect Effects 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 9
- 230000036961 partial effect Effects 0.000 description 8
- 101100433128 Homo sapiens ZBTB38 gene Proteins 0.000 description 7
- 101150074251 lpp gene Proteins 0.000 description 7
- 238000007400 DNA extraction Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000012188 paraffin wax Substances 0.000 description 5
- 230000001575 pathological effect Effects 0.000 description 5
- 101100240528 Caenorhabditis elegans nhr-23 gene Proteins 0.000 description 4
- 206010049678 Endometrial dysplasia Diseases 0.000 description 4
- 101100495925 Schizosaccharomyces pombe (strain 972 / ATCC 24843) chr3 gene Proteins 0.000 description 4
- 101150111181 ZSCAN12 gene Proteins 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical class NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 101150066838 12 gene Proteins 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical class O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 108700019961 Neoplasm Genes Proteins 0.000 description 2
- 102000048850 Neoplasm Genes Human genes 0.000 description 2
- 101150106031 TRI gene Proteins 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 108010008629 CA-125 Antigen Proteins 0.000 description 1
- 102000007269 CA-125 Antigen Human genes 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 230000007067 DNA methylation Effects 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 208000006994 Precancerous Conditions Diseases 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical compound OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- -1 ZCAN 12 Proteins 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 201000003914 endometrial carcinoma Diseases 0.000 description 1
- 208000011099 endometrial disease Diseases 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- 230000004049 epigenetic modification Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 201000010255 female reproductive organ cancer Diseases 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000000762 glandular Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000003234 polygenic effect Effects 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
Classifications
-
- 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
Abstract
The application provides a biomarker for benign and malignant endometrial lesions and application thereof, wherein the biomarker at least comprises a nucleic acid sequence methylated in at least one target region of GYPC gene. The biomarkers of benign and malignant endometrial lesions are applied to the preparation of endometrial cancer diagnosis products. The GYPC gene is screened out as a detection target gene (target gene), and the optimal methylation region in each gene is determined, so that the GYPC gene can be combined with each other to be used for early detection of endometrial cancer. Due to various endometrial cancer types, the multiple gene methylation regions are selected for joint detection, so that functional complementation is formed, and the diagnosis performance of endometrial cancer is remarkably improved. The detection has clinical value for detecting endometrial cancer and atypical hyperplasia.
Description
Technical Field
The application relates to the technical field of medical detection, in particular to a biomarker for benign and malignant lesions of endometrium and application thereof.
Background
Endometrial cancer (EC, endometrial carcinoma) is a common gynaecological cancer worldwide, with the third incidence of female reproductive cancers, being arranged behind cervical and ovarian cancers. At present, the number of endometrial cancer patients increases year by year, the onset age tends to be younger, and the death rate is high. About 14.2 tens of thousands of women suffer from endometrial cancer and 4.2 tens of thousands die from the disease every year worldwide. Endometrial cancer is classified into type i and type ii according to histopathological characteristics and pathogenesis. Type I accounts for more than 80% of the total number of endometrial cancers, is mostly developed from endometrial tissues in the proliferation stage, belongs to hormone-dependent type, and has better prognosis. Type II is mostly developed from intimal tissue in secretory phase, and belongs to non-hormone-dependent type with poor prognosis. Atypical hyperplasia of the endometrium refers to the glandular cells of the endometrium, which are currently in an intermediate stage of progression from simple hyperplasia to endometrial cancer. Once a woman performs uterine curettage and shows atypical hyperplasia of endometrium, attention must be paid to the condition that is currently in precancerous lesions. If the treatment is not timely, the endometrial cancer is easy to develop, and the life and health of women are endangered.
The gold standard for endometrial cancer diagnosis is pathological examination of an extracellular metastasis or surgical excision tissue specimen, which are all invasive, and cause a certain damage to patients. The lack of markers for early detection of endometrial cancer in the clinic today relies mainly on imaging examinations, detection of mutated genes and related proteins. The literature reports that the sensitivity and specificity of ultrasonic diagnosis to early endometrial cancer is not high, false negatives account for 40%, and are interfered by other factors. The gene mutation related to the carcinogenesis of the two types of endometrial cancers comprises dozens of types, the most effective detection method is high-throughput sequencing, the operation is complex, the cost is high, and the method is not suitable for early detection of endometrial cancers. The protein detection items are mainly sugar chain antigen CA125, human epididymal protein HE4 and the like. Many gynecological benign diseases can cause increase of CA125 in serum and cause excessive diagnosis and treatment; and only 11% -33% of endometrial cancer patients have elevated serum CA125, which is easy to miss diagnosis. HE4 is expressed in epithelium of the reproductive system and also marks in epithelium of normal glands of other tissues such as respiratory system, so HE4 has poor specificity. In addition, early endometrial cancer CA125 and HE4 expression did not change, and expression levels were increased only in middle and advanced cancers. These methods therefore make it difficult to detect endometrial cancer early. In addition, atypical hyperplasia of the endometrial precancerous condition is even less an effective biomarker. If the early treatment is not found early, the best prevention time is missed, so that endometrial cancer is easy to develop, and the life and health of women are even endangered. The incidence rate of domestic endometrial cancer is high, and a means for efficient early detection is needed.
Recent studies have shown that the most predominant form of epigenetic modification of DNA methylation can be involved in regulating vital activities associated with the development of cancer such as differentiation, proliferation, invasion, etc. of cells. Early endometrial cancer is often accompanied by methylation changes in related genes. The methylation level of a gene promoter region is low, and the gene is expressed normally; high methylation level, inhibition of gene expression, and even silencing. The methylation level of the tumor suppressor gene is reduced in early cancer, and detection of early endometrial cancer can be realized.
The following deficiencies exist in the prior art for early detection of endometrial cancer:
(1) neglecting endometrial atypical hyperplasia: atypical hyperplasia of endometrium is a state of precancerous lesions of endometrium, with about 14% -30% of the probability of transformation into endometrial cancer.
(2) Neglecting different types of endometrial cancer: endometrial cancer is classified into type i and type ii according to histopathological characteristics and causes of the disease.
(3) There was no predictive assessment of normal, atypical hyperplasia of the endometrium and endometrial cancer.
Disclosure of Invention
The application provides a biomarker for benign and malignant endometrial lesions and application thereof, wherein the biomarker comprises a methylated nucleic acid sequence in at least one target region of a GYPC gene, and the target region is selected from any one of the following methylated sequences and regions in the GYPC gene:
the chr2:127413772-127413893 region of the GYPC gene;
the Chr2:127413870-127413996 region of the GYPC gene.
Use of a biomarker of benign and malignant endometrial lesions as described above in the preparation of a diagnostic endometrial cancer product.
Optionally, the diagnostic product includes any one of a kit, a formulation, and a chip.
Optionally, the kit at least comprises a second primer pair and a second probe, wherein the second primer pair is used for detecting GYPC genes, the nucleotide sequence of the second primer pair is shown as SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of the second probe is shown as SEQ ID NO. 9;
alternatively, the nucleotide sequence of the second primer pair is shown as SEQ ID NO.10 and SEQ ID NO.11, and the nucleotide sequence of the second probe is shown as SEQ ID NO. 12.
Optionally, in the process of detecting biomarkers of benign and malignant endometrial lesions by using the diagnostic product, the results of diagnosing endometrial cancer and atypical hyperplasia by gene methylation are interpreted as follows:
the methylation level of a specific gene target is reflected by a Δcp value of:
ΔCp(GYPC[1])= Cp(GYPC[1])- Cp(COL2A1)，ΔCp(GYPC[2])= Cp(GYPC[2])- Cp(COL2A1)；
the methylation of delta Cp is less than or equal to k, and the result is positive methylation, namely that the tested sample is endometrial cancer, atypical hyperplasia or endometrial cancer; the gene methylated ΔCp > k is interpreted as methylation negative, i.e., indicating that the test sample is not endometrial cancer and atypical hyperplasia or endometrial cancer; where k is a particular value.
Optionally, in the process of detecting the biomarkers of benign and malignant endometrial lesions by using the diagnostic product, the method for predicting and judging endometrial lesions is as follows:
compared with the prior art, the application has the following beneficial effects:
the GYPC gene is screened out as a detection target gene (target gene), and the optimal methylation region in each gene is determined, so that the GYPC gene can be combined with each other to be used for early detection of endometrial cancer. Due to various endometrial cancer types, the multiple gene methylation regions are selected for joint detection, so that functional complementation is formed, and the diagnosis performance of endometrial cancer is remarkably improved. The detection has clinical value for detecting endometrial cancer and atypical hyperplasia.
In addition to the objects, features and advantages described above, the present application has other objects, features and advantages. The present application will be described in further detail with reference to the drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 (a) is a box line diagram showing the methylation value ΔCp of ZBTB38[1] (Chr 3: 141130440-141130560) in experimental example 1 of the present application in benign lesions, atypical hyperplasia of endometrium, endometrial cancer;
FIG. 1 (b) is a box line graph showing the methylation value ΔCp of ZBTB38[2] (Chr 3: 141122572-141122691) in experimental example 1 of the present application in benign lesions, atypical hyperplasia of endometrium, endometrial cancer;
FIG. 1 (c) is a box line diagram showing the methylation value ΔCp of GYPC [1] (Chr 2: 127413772-127413893) in experimental example 1 of the present application in benign lesions, atypical hyperplasia of endometrium, endometrial cancer;
FIG. 1 (d) is a box line diagram showing the methylation value ΔCp of GYPC 2 (Chr 2: 127413870-127413996) in experimental example 1 of the present application in benign lesions, atypical hyperplasia of endometrium, endometrial cancer;
FIG. 1 (e) is a box plot of ZSCAN12[1] (Chr 6: 28367484-28367600) methylation values ΔCp in experimental example 1 of the present application for benign lesions, endometrial dysplasia, endometrial cancer;
FIG. 1 (f) is a box plot of ZSCAN12[2] (Chr 6: 28367510-28367631) methylation values ΔCp in experimental example 1 of the present application for benign lesions, endometrial dysplasia, endometrial cancer;
FIG. 1 (g) is a box line diagram showing the methylation value ΔCp of LPP 2 (Chr 3: 188157018-188157138) in experimental example 1 of the present application in benign lesions, atypical hyperplasia of endometrium, endometrial cancer;
FIG. 2 (a) is a graph showing the methylation of ZBTB38[1] (Chr 3: 141130440-141130560) gene of experimental example 1 of the present application for diagnosing endometrial cancer;
FIG. 2 (b) is a graph showing the methylation of ZBTB38[2] (Chr 3: 141122572-141122691) gene in experimental example 1 of the present application for diagnosing endometrial cancer;
FIG. 2 (c) is a graph showing the methylation of GYPC 1 (Chr 2: 127413772-127413893) gene of experimental example 1 of the present application for diagnosing endometrial cancer;
FIG. 2 (d) is a graph showing the methylation of GYPC 2 (Chr 2: 127413870-127413996) gene of experimental example 1 of the present application for diagnosing endometrial cancer;
FIG. 2 (e) is a graph of a subject diagnosed with endometrial cancer by ZSCAN12[1] (Chr 6: 28367484-28367600) gene methylation in experimental example 1 of the present application;
FIG. 2 (f) is a graph of a subject diagnosed with endometrial cancer by ZSCAN12[2] (Chr 6: 28367510-28367631) gene methylation in experimental example 1 of the present application;
FIG. 2 (g) is a graph showing the methylation of LPP 2 (Chr 3: 188157018-188157138) gene in experimental example 1 of the present application for diagnosing endometrial cancer;
FIG. 3 (a) is a graph showing the methylation of ZBTB38[1] (Chr 3: 141130440-141130560) gene in experimental example 1 of the present application for diagnosing endometrial cancer and atypical hyperplasia;
FIG. 3 (b) is a graph showing the methylation of ZBTB38[2] (Chr 3: 141122572-141122691) gene in experimental example 1 of the present application for diagnosing endometrial cancer and atypical hyperplasia;
FIG. 3 (c) is a graph showing the methylation of GYPC 1 (Chr 2: 127413772-127413893) gene in experimental example 1 of the present application for diagnosing endometrial cancer and atypical hyperplasia;
FIG. 3 (d) is a graph showing the methylation of GYPC 2 (Chr 2: 127413870-127413996) gene in experimental example 1 of the present application for diagnosing endometrial cancer and atypical hyperplasia;
FIG. 3 (e) is a graph of a subject diagnosed with endometrial cancer and atypical hyperplasia by ZSCAN12[1] (Chr 6: 28367484-28367600) gene methylation in experimental example 1 of the present application;
FIG. 3 (f) is a graph of a subject diagnosed with endometrial cancer and atypical hyperplasia by ZSCAN12[2] (Chr 6: 28367510-28367631) gene methylation in experimental example 1 of the present application;
FIG. 3 (g) is a graph showing the methylation of LPP 2 (Chr 3: 188157018-188157138) gene in experimental example 1 of the present application for diagnosing endometrial cancer and atypical hyperplasia;
FIG. 4 (a) is a box line graph showing the methylation value ΔCp of ZBTB38[1] (Chr 3: 141130440-141130560) in experimental example 2 of the present application in benign lesions, atypical hyperplasia of endometrium, endometrial cancer;
FIG. 4 (b) is a box line graph showing the methylation value ΔCp of ZBTB38[2] (Chr 3: 141122572-141122691) in experimental example 2 of the present application in benign lesions, atypical hyperplasia of endometrium, endometrial cancer;
FIG. 4 (c) is a box line graph showing the methylation value ΔCp of GYPC [1] (Chr 2: 127413772-127413893) in experimental example 2 of the present application in benign lesions, atypical hyperplasia of endometrium, endometrial cancer;
FIG. 4 (d) is a box plot of GYPC 2 (Chr 2: 127413870-127413996) methylation values ΔCp in experimental example 2 of the present application for benign lesions, atypical hyperplasia of the endometrium, endometrial cancer;
FIG. 4 (e) is a box plot of ZSCAN12[1] (Chr 6: 28367484-28367600) methylation values ΔCp in benign lesions, endometrial dysplasia, endometrial cancer in Experimental example 2 of the application;
FIG. 4 (f) is a box plot of ZSCAN12[2] (Chr 6: 28367510-28367631) methylation values ΔCp in benign lesions, endometrial dysplasia, endometrial cancer in Experimental example 2 of the application;
FIG. 4 (g) is a box plot of the methylation value ΔCp of LPP 2 (Chr 3: 188157018-188157138) in Experimental example 2 of the present application in benign lesions, atypical hyperplasia of endometrium, endometrial cancer;
FIG. 5 (a) is a graph showing the methylation of ZBTB38[1] (Chr 3: 141130440-141130560) in experimental example 2 of the present application for diagnosing endometrial cancer;
FIG. 5 (b) is a graph showing the methylation of ZBTB38[2] (Chr 3: 141122572-141122691) in experimental example 2 of the present application for diagnosing endometrial cancer;
FIG. 5 (c) is a graph showing the methylation of GYPC 1 (Chr 2: 127413772-127413893) in experimental example 2 of the present application for diagnosing endometrial cancer;
FIG. 5 (d) is a graph showing the methylation of GYPC 2 (Chr 2: 127413870-127413996) in experimental example 2 of the present application for diagnosing endometrial cancer;
FIG. 5 (e) is a graph of a subject with ZSCAN12[1] (Chr 6: 28367484-28367600) methylation diagnosis of endometrial cancer in experimental example 2 of the application;
FIG. 5 (f) is a graph of a subject with ZSCAN12[2] (Chr 6: 28367510-28367631) methylation diagnosis of endometrial cancer in experimental example 2 of the application;
FIG. 5 (g) is a graph of the subject diagnosed with endometrial cancer by LPP 2 (Chr 3: 188157018-188157138) methylation in experimental example 2 of the application;
FIG. 6 (a) is a graph of a subject diagnosed with endometrial cancer and atypical hyperplasia by methylation of ZBTB38[1] (Chr 3: 141130440-141130560) in Experimental example 2 of the present application;
FIG. 6 (b) is a graph of a subject diagnosed with endometrial cancer and atypical hyperplasia by methylation of ZBTB38[2] (Chr 3: 141122572-141122691) in Experimental example 2 of the present application;
FIG. 6 (c) is a graph of a subject diagnosed with endometrial cancer and atypical hyperplasia by GYPC [1] (Chr 2: 127413772-127413893) methylation in Experimental example 2 of the present application;
FIG. 6 (d) is a graph of a subject diagnosed with endometrial cancer and atypical hyperplasia by GYPC 2 (Chr 2: 127413870-127413996) methylation in Experimental example 2 of the present application;
FIG. 6 (e) is a graph of a subject diagnosed with endometrial cancer and atypical hyperplasia by ZCAN 12[1] (Chr 6: 28367484-28367600) methylation in experimental example 2 of the application;
FIG. 6 (f) is a graph of a subject diagnosed with endometrial cancer and atypical hyperplasia by ZCAN 12[2] (Chr 6: 28367510-28367631) methylation in experimental example 2 of the application;
FIG. 6 (g) is a graph of a subject diagnosed with endometrial cancer and atypical hyperplasia by LPP 2 (Chr 3: 188157018-188157138) methylation in Experimental example 2 of the present application;
FIG. 7 (a) is a stacked view showing the methylation of ZBTB38[1] (Chr 3: 141130440-141130560) in experimental example 2 of the present application to predict the degree of endometrial lesion;
FIG. 7 (b) is a stacked view showing the methylation of ZBTB38[2] (Chr 3: 141122572-141122691) in experimental example 2 of the present application to predict the degree of endometrial lesion;
FIG. 7 (c) is a stacked view showing the methylation of GYPC [1] (Chr 2: 127413772-127413893) in Experimental example 2 of the present application for predicting the degree of endometrial lesion;
FIG. 7 (d) is a stacked view showing the methylation of GYPC 2 (Chr 2: 127413870-127413996) in Experimental example 2 of the present application for predicting the degree of endometrial lesion;
FIG. 7 (e) is a stacked graph of ZCAN 12[1] (Chr 6: 28367484-28367600) methylation predicted endometrial lesion level in experimental example 2 of the application;
FIG. 7 (f) is a stacked graph of ZCAN 12[2] (Chr 6: 28367510-28367631) methylation predicted endometrial lesion level in experimental example 2 of the application;
FIG. 7 (g) is a stacked view showing the methylation of LPP 2 (Chr 3: 188157018-188157138) in Experimental example 2 of the present application for predicting the degree of endometrial lesion;
FIG. 8 is a stacked graph showing the prediction of the degree of endometrial lesion by the methylation of the three genes ZBTB38[1] (Chr 3: 141130440-141130560), GYPC [1] (Chr 2: 127413772-127413893), ZCAN 12[2] (Chr 6: 28367510-28367631) in a cervical exfoliated cell sample according to Experimental example 2 of the present application.
Detailed Description
The following are specific embodiments of the present application and the technical solutions of the present application will be further described with reference to the accompanying drawings, but the present application is not limited to these embodiments.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
Example 1:
the biomarker for early screening diagnosis of endometrial cancer (particularly comprises screening diagnosis of atypical hyperplasia of endometrium and atypical hyperplasia or endometrial cancer), which at least comprises a nucleic acid sequence methylated in at least one target region of ZBTB38 gene, GYPC gene, ZSCAN12 gene and LPP gene, wherein the target region is selected from any one of the following methylation sequences and regions in each of the ZBTB38 gene, GYPC gene, ZSCAN12 gene and LPP gene, and the methylation sequences and the regions are shown in table 1.
TABLE 1 methylation sequences and regions in ZBTB38 gene, GYPC gene, ZCAN 12 gene, LPP gene
Note that: the methylation sequences and regions followed by [1] and [2] each represent a different target region of the same gene.
It should be noted that, whatever the method used to detect the methylation of the entire length of any one of the ZBTB38, GYPC, ZCAN 12, LPP genes or a partial region thereof is used for diagnosis of endometrial cancer, it is within the scope of the present application.
Designing a primer probe for PCR amplification aiming at the target gene and the region:
the reagents for detecting the above target genes are classified into a first reagent for detecting ZBTB38 gene, a second reagent for detecting GYPC gene, a third reagent for detecting ZSCAN12 gene, and a fourth reagent for detecting LPP gene; wherein:
the first reagent is a reagent suitable for detecting the methylation of the ZBTB38 gene by adopting a PCR method, the first target area detected by the first reagent is a partial area selected from the group consisting of Chr3 and 141130440-141130560, and the second target area detected by the first reagent is a partial area selected from the group consisting of Chr3 and 141122572-141122691;
the second reagent is a reagent suitable for detecting GYPC gene methylation by adopting a PCR method, the first target area detected by the second reagent is a partial area selected from the group consisting of Chr2 and 127413772-127413893, and the second target area detected by the second reagent is a partial area selected from the group consisting of Chr2 and 127413870-127413996;
the third reagent is a reagent suitable for detecting ZCAN 12 gene methylation by adopting a PCR method, the first target area detected by the third reagent is a partial area selected from the group consisting of Chr6 and 28367484-28367600, and the second target area detected by the third reagent is a partial area selected from the group consisting of Chr6 and 28367510-28367631;
the fourth reagent is a reagent suitable for detecting LPP gene methylation by a PCR method, and the second target region detected by the fourth reagent is a partial region selected from the group consisting of Chr3: 188157018-188157138.
The first reagent comprises a first primer pair and a first probe, and the sequences of the first primer pair and the first probe for detecting the various first target areas are shown in table 2; the second reagent comprises a second primer pair and a second probe, and the sequences of the second primer pair and the second probe for detecting the various second target areas are shown in table 3; the third reagent comprises a third primer pair and a third probe, and the sequences of the third primer pair and the third probe for detecting the various third target regions are shown in table 4; the fourth reagent includes a fourth primer pair and a fourth probe, and the sequences of the fourth primer pair and the fourth probe for detecting the above-described various fourth target regions are shown in Table 5.
TABLE 2 first primer pair and base sequence of first probe for detecting methylation of ZBTB38 gene
Specifically, ZBTB38[1] (Chr 3: 141130440-141130560) is selected from UCSC Genome Browser on Human (GRCh 37/hg 19), > hg19_dnarange=chr3: 141130440-141130560 5' pad=0 strand= +repeat mask=none:
AGTCTGCAAAAGCCATGACGTGGCACTGAAATGAAGCCCTGTGTAGGGTTTTACTCAGACGTTAGAGAACTGGCTGCCCCTGAGTGCTGTGCTAACTAGGGAAAGAAAAAAAACCCTGACC
the DNA strand was subjected to bisulfite conversion (unmethylated C to T, methylated C remained unchanged), yielding a converted DNA strand:
AGTTTGTAAAAGTTATGACGTGGTATTGAAATGAAGTTTTGTGTAGGGTTTTATTTAGACGTTAGAGAA TTGGTTGTTTTTGAGTGTTGTGTTAATTAGGGAAAGAAAAAAAATTTTGATT
the underlined sequence is the sequence of the primer probe of ZBTB38[1] (Chr 3: 141130440-141130560) finally obtained.
Specifically, ZBTB38[2] (chr3: 141122572-141122691) is selected from the DNA strand in UCSC Genome Browser on Human (GRCh 37/hg 19), > hg19_dnarange=chr3: 141122572-141122691, 5 'pad=0, 3' pad=0 strand= +repeat mask=none:
TTCTATAGCTGTAAAATGGGGGAATGTTCCCTACATCTGAAGGTTGTTATGAAGACAACACGAAACCACATATGTCAACCACTTTGTGGGTGTTTGACTTATGTGTTCAATATGTGCTAA
the DNA strand was subjected to bisulfite conversion (unmethylated C to T, methylated C remained unchanged), yielding a converted DNA strand:
TTTTATAGTTGTAAAATGGGGGAATGTTTTTTATATTTGAAGGTTGTTATGAAGATAATACGAAATTATATATGTTAATTATTTTGTGGGTGTTTGATTTATGTGTTTAATATGTGTTAA
the underlined sequence is the sequence of the primer probe of ZBTB38[2] (Chr 3: 141122572-141122691) finally obtained.
TABLE 3 first primer pair and first probe base sequence for detecting GYPC Gene methylation
Specifically, GYPC [1] (Chr2: 127413772-127413893) is selected from UCSC Genome Browser on Human (GRCh 37/hg 19), > hg19_dnarange=chr2: 127413772-127413893 5' pad=0 strand= +repeat mask=none:
CCTCCCCCGGCCCGGCCTGGCCCGGCCTGGCCAGTCCCCGCGGTCTCTGCCCGGGCTGACGCCCAGGAATGTGGTCGACGAGAAGCCCCAACAGCACGGCGTGGCCTCTCAGCCTCGGTGAG
the DNA strand was subjected to bisulfite conversion (unmethylated C to T, methylated C remained unchanged), yielding a converted DNA strand:
TTTTTTTCGGTTCGGTTTGGTTCGGTTTGGTTAGTTTTCGCGGTTTTTGTTCGGGTTGACGTTTAGGAA TGTGGTCGACGAGAAGTTTTAATAGTACGGCGTGGTTTTTTAGTTTCGGTGAG
the underlined sequence is the sequence of the primer probe of GYPC [1] (Chr 2: 127413772-127413893) finally obtained.
Specifically, GYPC [2] (Chr2: 127413870-127413996) is selected from UCSC Genome Browser on Human (GRCh 37/hg 19), > hg19_dnarange=chr2: 127413870-127413996 5' pad=0 strand= +repeat mask=none:
GCGTGGCCTCTCAGCCTCGGTGAGTACCCGCCGTGGGGAAGGGTCCTGGGGACCCACTGGAGGCCGCGGCCCGCAGCAGCCAGGGGCCGAGCCACGGCCACGGACGCCCTGGTGTCCCGGTCCGTGC
finding the complementary strand of the DNA according to the selected DNA strand:
GCACGGACCGGGACACCAGGGCGTCCGTGGCCGTGGCTCGGCCCCTGGCTGCTGCGGGCCGCGGCCTCCAGTGGGTCCCCAGGACCCTTCCCCACGGCGGGTACTCACCGAGGCTGAGAGGCCACGC
the complementary strand of the DNA was subjected to bisulfite conversion (unmethylated C to T, methylated C remained unchanged), to give a converted complementary strand of DNA:
GTACGGATCGGGATATTAGGGCGTTCGTGGTCGTGGTTCGGTTTTTGGTTGTTGCGGGTCGCGGTTTTTAGTGGGTTTTTAGGATTTTTTTTTACGGCGGGTATTTATCGAGGTTGAGAGGTTACGT
the underlined sequence is the sequence of the primer probe of GYPC 2 (Chr 2: 127413870-127413996) finally obtained.
TABLE 4 base sequences of first primer pair and first probe for detecting ZCAN 12 Gene methylation
Specifically, ZSCAN12[1] (chr6: 28367484-28367600) is selected from UCSC Genome Browser on Human (GRCh 37/hg 19), > hg19_dnarange=chr6: 28367484-2836750 5' pad=0 strand= +repeat mask=dna strand in none:
GAGGCCTCGAACCCTTTTGGGACCCGGAACCCATCAAAAGTGACCCACAAAGGCCGGAAGCGGCCACGGGGGGTCTAAGAACCAGCCCGCGCGGGGCGCACTTCCGCGGCCGCTCTA
finding the complementary strand of the DNA according to the selected DNA strand:
TAGAGCGGCCGCGGAAGTGCGCCCCGCGCGGGCTGGTTCTTAGACCCCCCGTGGCCGCTTCCGGCCTTTGTGGGTCACTTTTGATGGGTTCCGGGTCCCAAAAGGGTTCGAGGCCTC
the complementary strand of the DNA was subjected to bisulfite conversion (unmethylated C to T, methylated C remained unchanged), to give a converted complementary strand of DNA:
TAGAGCGGTCGCGGAAGTGCGTTTCGCGCGGGTTGGTTTTTAGATTTTTCGTGGTCGTTTTCGGTTTTT GTGGGTTATTTTTGATGGGTTTCGGGTTTTAAAAGGGTTCGAGGTTTT
the underlined sequence is the sequence of the primer probe of ZCAN 12[1] (Chr 6: 28367484-28367600) finally obtained.
Specifically, ZSCAN12[2] (chr6: 28367510-28367631) is selected from UCSC Genome Browser on Human (GRCh 37/hg 19), > hg19_dnarange=chr6: 28367510-28367531 5' pad=0 strand= +repeat mask=dna strand in none:
GAACCCATCAAAAGTGACCCACAAAGGCCGGAAGCGGCCACGGGGGGTCTAAGAACCAGCCCGCGCGGGGCGCACTTCCGCGGCCGCTCTAGGAAGGGAGCGAAAGGGGCTTTCAACTCGGT
finding the complementary strand of the DNA according to the selected DNA strand: ACCGAGTTGAAAGCCCCTTTCGCTCCCTTCCTAGAGCGGCCGCGGAAGTGCGCCCCGCGCGGGCTGGTTCTTAGACCCCCCGTGGCCGCTTCCGGCCTTTGTGGGTCACTTTTGATGGGTTC
The complementary strand of the DNA was subjected to bisulfite conversion (unmethylated C to T, methylated C remained unchanged), to give a converted complementary strand of DNA:
ATCGAGTTGAAAGTTTTTTTCGTTTTTTTTTTAGAGCGGTCGCGGAAGTGCGTTTCGCGCGGGTTGGTT TTTAGATTTTTCGTGGTCGTTTTCGGTTTTTGTGGGTTATTTTTGATGGGTTT
the underlined sequence is the sequence of the primer probe of ZCAN 12[2] (Chr 6: 28367510-28367631) obtained finally.
TABLE 5 base sequences of first primer pair and first probe for detecting methylation of LPP Gene
Specifically, LPP [2] (Chr3: 188157018-188157138) is selected from UCSC Genome Browser on Human (GRCh 37/hg 19), > hg19_dnarange=chr3: 188157018-188157138 5' pad=0 strand= +repeat mask=none:
CTATTAATAATGTTAAAGGGGGTTACAGCCGTGACTCACTCCAGGAAGTCAACCATCATTCGCTTCCTCAATCTCACCAACCAGTCCCCATGTACAAAATCAGCGACTTCTGCAAATGAGT
finding the complementary strand of the DNA according to the selected DNA strand:
ACTCATTTGCAGAAGTCGCTGATTTTGTACATGGGGACTGGTTGGTGAGATTGAGGAAGCGAATGATGGTTGACTTCCTGGAGTGAGTCACGGCTGTAACCCCCTTTAACATTATTAATAG
the complementary strand of the DNA was subjected to bisulfite conversion (unmethylated C to T, methylated C remained unchanged), to give a converted complementary strand of DNA:
ATTTATTTGTAGAAGTCGTTGATTTTGTATATGGGGATTGGTTGGTGAGATTGAGGAAGCGAATGATGGTTGATTTTTTGGAGTGAGTTACGGTTGTAATTTTTTTTAATATTATTAATAG
the underlined sequence is the sequence of the primer probe of ZCAN 12[2] (Chr 6: 28367510-28367631) obtained finally.
Note that: the first target region and the second target region of the same gene are shown in tables 2 to 5 after the names of the target genes [1] and [2], respectively, and the probe sequences shown in tables 2 to 5 are not labeled with a fluorescent group and a quenching group.
Example 2:
the kit provided by the application is used for a detection test method for detecting endometrial cancer inhibiting ZBTB38 gene, GYPC gene, ZSCAN12 gene and LPP gene, and comprises the following steps:
1. sample DNA extraction
The method adopts paraffin tissue slice sample DNA extraction reagent or shed cell sample DNA extraction reagent to extract genome DNA, and simultaneously carries out DNA quality monitoring (yield, purity and integrity), wherein the total amount of DNA is between 1ug and 10ug, OD260/280 is between 1.9 and 2.0, the yield and purity of the DNA electrophoresis gel are indicated to be better at more than 200bp, and the next test can be carried out.
2. DNA bisulfite conversion
The DNA of the extracted number is subjected to bisulfite conversion by adopting a bisulfite conversion kit (namely a methylation detection sample pretreatment kit (20200131. Hunan Hongya Gene technology Co., ltd.) which is autonomously developed by the applicant, wherein unmethylated cytosine (C, cytosine) in the DNA is converted into unmethylated uracil (U, uracil) and methylated cytosine (C) is unchanged, so that DNA (B-DNA) after bisulfite conversion is obtained. The conversion efficiency of the bisulfite in the example is 99.7-100.0%, which is higher than that of most medium sulfite conversion kits on the market.
3. Methylation specific fluorescent quantitative PCR amplification
After a fluorescent quantitative PCR reaction system is configured (the fluorescent quantitative PCR reaction system is shown in table 6), fluorescent quantitative PCR reaction is carried out according to fluorescent quantitative PCR upper program parameters (the fluorescent quantitative PCR upper program parameters are shown in table 7).
TABLE 6 fluorescent quantitative PCR reaction system (20 ul/person)
Note that: sample DNA after bisulfite conversion, or negative quality control product, or positive quality control product.
TABLE 7 fluorescent quantitative PCR on-machine program parameters
Note that: the adaptive model is a Roche real-time fluorescent quantitative PCR instrument LC480 or a Siemens Feibi 7500 real-time fluorescent quantitative PCR instrument.
4. Gene methylation result interpretation
4.1, qualified quality control: the internal control gene is amplified in an S type, and Cp is less than or equal to q (more than or equal to 20 and less than or equal to 40) tested by LC480 is in control;
4.2, the methylation level of a target region of a specific gene is reflected with ΔCp values of the 4 genes:
ΔCp(ZBTB38[1])= Cp(ZBTB38[1])- Cp(COL2A1)，ΔCp(ZBTB38[2])= Cp(ZBTB38[2])- Cp(COL2A1)；
ΔCp(GYPC[1])= Cp(GYPC[1])- Cp(COL2A1)，ΔCp(GYPC[2])= Cp(GYPC[2])- Cp(COL2A1)；
ΔCp(ZSCAN12[1])= Cp(ZSCAN12[1])- Cp(COL2A1)，ΔCp(ZSCAN12[2])= Cp(ZSCAN12[2])- Cp(COL2A1)；
ΔCp(LPP[2])= Cp(LPP[2])- Cp(COL2A1)。
note that: if the PCR reaction performed by the real-time fluorescent quantitative PCR instrument of the Siemens Fei ABI7500 is adopted, the Ct value in the real-time fluorescent quantitative PCR instrument of the Siemens Fei ABI7500 is replaced by the Cp value.
Wherein: cp is proposed by Roche, C represents Cycle, p represents point, and Cp is the number of cycles of the amplification curve in each reaction tube passing through the threshold (i.e., the point at which the fluorescence detection value increases significantly);
the Δcp value is the difference between the Cp values of the target gene and the corresponding reference gene.
Ct is proposed by the Siemens femto company, C represents Cycle, t represents threshold, and Ct is the number of cycles undergone by the fluorescent signal in each reaction tube when reaching a set threshold.
4.3 results of diagnosing endometrial cancer and atypical hyperplasia by gene methylation are shown in Table 8.
TABLE 8 diagnosis of endometrial cancer and atypical hyperplasia by methylation of genes
Note that: gene methylation（/>A particular number) is interpreted as methylation positive, i.e., indicating that the test sample is endometrial cancer and atypical hyperplasia or endometrial cancer; gene methylation>The interpretation is methylation negative, i.e., it indicates that the test sample is not endometrial cancer and atypical hyperplasia or endometrial cancer.
4.4, prediction and interpretation of endometrial lesions
TABLE 9 value ranges for the methylation levels of genes divided into low, medium and high methylation
4.5 The polygenic combination predicted endometrial lesions interpretation is shown in table 10.
Table 10 polygene joint prediction endometrial lesion interpretation
Note that: (1) ZBTB38[1], GYPC [1], ZCAN 12[2] respectively represent methylation level, low methylation value 0, medium methylation value 1, high methylation value 2, and substitution formula operation;
(2) MV is the predicted value calculation of ZBTB38[1], GYPC [1], ZCAN 12[2] methylation combined prediction endometrium disease.
Experimental example 1:
the specific process of endometrial cancer gene methylation detection based on paraffin tissue section specimens is as follows:
96 endometrial cancer paraffin tissue section samples were taken for which clear pathological information results are known: 20 cases are benign endometrial samples; 30 cases are atypical endometrial hyperplasia samples; samples of 46 endometrial cancer, 38 of which were type i endometrial cancer and 8 of which were type ii endometrial cancer. According to sample DNA extraction, DNA bisulfite conversion and methylation specific fluorescence quantitative PCR amplification, the distribution of gene methylation in a sample is shown in FIG. 1 (a) -FIG. 1 (g), NS: p is more than 0.05, and no obvious difference exists;：0.01＜P＜0.05；/>：0.001＜P＜0.01；/>：P＜0.001。
the ability of the primer probe reagents of the application to identify endometrial cancer is shown in FIGS. 2 (a) -2 (g) (where AUC is expressed as the area under the subject's working curve) and is analyzed as follows:
the ZBTB38[1] (Chr 3: 141130440-141130560) methylation test identified an Area (AUC) of the subject operating curve ROC of endometrial cancer of 0.910.
The ZBTB38[2] (Chr 3: 141122572-141122691) methylation test identifies a subject with endometrial cancer with an Area (AUC) of the working curve ROC of 0.883.
GYPC [1] (Chr 2: 127413772-127413893) methylation test the Area (AUC) of the working curve ROC of subjects identified endometrial cancer was 0.928.
GYPC [2] (Chr 2: 127413870-127413996) methylation test the Area (AUC) of the working curve ROC of subjects identified endometrial cancer was 0.895.
ZSCAN12[1] (Chr 6: 28367484-28367600) methylation test identifies a subject with endometrial cancer with an area of working curve ROC (AUC) of 0.906.
ZSCAN12[2] (Chr 6: 28367510-28367631) methylation test identifies a subject with endometrial cancer with an area of working curve ROC (AUC) of 0.897.
LPP 2 (Chr 3: 188157018-188157138) methylation test identifies a subject with endometrial cancer with an Area (AUC) of the working curve ROC of 0.881.
In paraffin tissue section specimens, the ability of the primer probe reagent of the present application to identify endometrial cancer and atypical hyperplasia is shown in fig. 3 (a) -3 (g), and analyzed as follows:
the ZBTB38[1] (Chr 3: 141130440-141130560) methylation test identified an area of the subject working curve ROC for endometrial cancer and atypical hyperplasia (AUC) of 0.995; the critical value of negative and positive of the methylation test result of the target region of the gene is delta Cp=8.0, delta Cp is less than or equal to 8.0, and the result is judged to be positive; the positive rate in endometrial cancer specimens was 100.0% (46/46), in endometrial atypical hyperplasia specimens was 83.3% (25/30), and in endometrial benign specimens was 0.0% (0/20).
The ZBTB38[2] (Chr 3: 141122572-141122691) methylation test identified an area of the subject working curve ROC for endometrial cancer and atypical hyperplasia (AUC) of 0.886; the critical value of negative and positive of the methylation test result of the target region of the gene is delta Cp=15.0, delta Cp is less than or equal to 15.0, and the result is judged to be positive; the positive rate in endometrial cancer specimens was 100.0% (46/46), in endometrial atypical hyperplasia specimens was 60.0% (18/30), and in endometrial benign specimens was 5.0% (1/20).
GYPC [1] (Chr 2: 127413772-127413893) methylation test identified an Area (AUC) of the subject working curve ROC for endometrial cancer and atypical hyperplasia of 1.000; the critical value of negative and positive of the methylation test result of the target region of the gene is DeltaCp=11.5, deltaCp is less than or equal to 11.5, and the result is judged to be positive; the positive rate in the endometrial cancer specimen was 100.0% (46/46), the positive rate in the endometrial atypical hyperplasia specimen was 100.0% (30/30), and the positive rate in the endometrial benign specimen was 0.0% (0/20).
GYPC [2] (Chr 2: 127413870-127413996) methylation test identifies a subject with endometrial cancer and atypical hyperplasia with an area of the working curve ROC (AUC) of 0.983; the critical value of negative and positive of the methylation test result of the target region of the gene is DeltaCp=11.0, deltaCp is less than or equal to 11.0, and the result is judged to be positive; the positive rate in endometrial cancer specimens was 97.8% (45/46), in endometrial atypical hyperplasia specimens was 90.0% (27/30), and in endometrial benign specimens was 5.0% (1/20).
ZSCAN12[1] (Chr 6: 28367484-28367600) methylation test identifies a subject with endometrial cancer and atypical hyperplasia with an area of the working curve ROC (AUC) of 0.896; the critical value of negative and positive of the methylation test result of the target region of the gene is delta Cp=15.2, delta Cp is less than or equal to 15.2, and the result is judged to be positive; the positive rate in endometrial cancer specimens was 100.0% (46/46), in endometrial atypical hyperplasia specimens was 60.0% (18/30), and in endometrial benign specimens was 0.0% (0/20).
ZSCAN12[2] (Chr 6: 28367510-28367631) methylation test identifies a subject working curve ROC Area (AUC) of 0.999 for endometrial cancer and atypical hyperplasia; the critical value of the methylation test result of the target region of the gene is DeltaCp=9.2, deltaCp is less than or equal to 9.2, and the result is judged to be positive; the positive rate in endometrial cancer specimens was 97.8% (45/46), in endometrial atypical hyperplasia specimens was 100.0% (30/30), and in endometrial benign specimens was 0.0% (0/20).
LPP 2 (Chr 3: 188157018-188157138) methylation test identifies a subject working curve ROC for endometrial cancer and atypical hyperplasia with an Area (AUC) of 0.942; the critical value of negative and positive of the methylation test result of the target region of the gene is delta Cp=15.2, delta Cp is less than or equal to 15.2, and the result is judged to be positive; the positive rate in endometrial cancer specimens was 100.0% (46/46), in endometrial atypical hyperplasia specimens was 70% (21/30), and in endometrial benign specimens was 5.0% (1/20).
Experimental example 2:
the specific process of endometrial cancer gene methylation detection based on cervical exfoliated cell samples is as follows:
150 cervical exfoliated cell samples were taken for which clear pathological information results are known: 57 cases are normalA sample; 19 cases are atypical endometrial hyperplasia samples; samples of 74 endometrial cancer, 58 of which were type i endometrial cancer and 16 of which were type ii endometrial cancer. According to sample DNA extraction, DNA bisulfite conversion and methylation specific fluorescence quantitative PCR amplification, the distribution of gene methylation in cervical exfoliated cells with different pathological degrees of endometrium is shown in the figures 4 (a) -4 (g), wherein: NS: p is more than 0.05, and no obvious difference exists;：0.01＜P＜0.05；/>：0.001＜P＜0.01；/>：P＜0.001。
the ability of the primer probe reagent of the present application to identify endometrial cancer and atypical hyperplasia is shown in fig. 5 (a) -5 (g) and fig. 6 (a) -6 (g), and is analyzed as follows:
the ZBTB38[1] (Chr 3: 141130440-141130560) methylation test identified an Area (AUC) of the subject working curve ROC of endometrial cancer of 0.955.
The ZBTB38[2] (Chr 3: 141122572-141122691) methylation test identifies a subject with endometrial cancer with an Area (AUC) of the working curve ROC of 0.940.
GYPC [1] (Chr 2: 127413772-127413893) methylation test the Area (AUC) of the working curve ROC of subjects identified endometrial cancer was 0.950.
GYPC [2] (Chr 2: 127413870-127413996) methylation test identifies a subject with endometrial cancer with an Area (AUC) of the working curve ROC of 0.938.
ZSCAN12[1] (Chr 6: 28367484-28367600) methylation test identifies a subject with endometrial cancer with an area of working curve ROC (AUC) of 0.942.
ZSCAN12[2] (Chr 6: 28367510-28367631) methylation test identifies a subject with endometrial cancer with an area of working curve ROC (AUC) of 0.950.
LPP 2 (Chr 3: 188157018-188157138) methylation test identifies a subject with endometrial cancer with an Area (AUC) of the working curve ROC of 0.942.
The ZBTB38[1] (Chr 3: 141130440-141130560) methylation test identified an area of the subject working curve ROC for endometrial cancer and atypical hyperplasia (AUC) of 0.976.
The ZBTB38[2] (Chr 3: 141122572-141122691) methylation test identifies a subject with endometrial cancer with an area of the working curve ROC (AUC) of 0.958.
GYPC [1] (Chr 2: 127413772-127413893) methylation test identifies the Area (AUC) of the working curve ROC of a subject with endometrial cancer and atypical hyperplasia as 0.975.
GYPC 2 (Chr 2: 127413870-127413996) methylation test identifies a subject with endometrial cancer and atypical hyperplasia with an area of the working curve ROC (AUC) of 0.952.
ZSCAN12[1] (Chr 6: 28367484-28367600) methylation test identifies a subject with endometrial cancer and atypical hyperplasia with an area of the working curve ROC (AUC) of 0.943.
ZSCAN12[2] (Chr 6: 28367510-28367631) methylation test identifies the area of the subject working curve ROC for endometrial cancer and atypical hyperplasia (AUC) as 0.990.
LPP 2 (Chr 3: 188157018-188157138) methylation test identifies a subject with endometrial cancer and atypical hyperplasia with an area of the working curve ROC (AUC) of 0.938.
Alternatively, the positive rate of gene methylation in cervical exfoliated cells with different lesions of the endometrium according to the threshold value taken in experimental example 1 is shown in table 11.
TABLE 11 Positive Rate of Gene methylation in cervical exfoliated cells in pathological types
Alternatively, the clinical diagnostic performance of the 7-pair primer probe reagent of the present application on endometrial cancer and atypical hyperplasia according to the threshold value taken in experimental example 1 is shown in table 12.
Table 12 clinical Properties of Gene methylation in cervical exfoliated cells for diagnosis of endometrial cancer and atypical hyperplasia
Alternatively, two gene combinations were performed based on the optimal thresholds ZBTB38[1] (Chr 3: 141130440-141130560), ZBTB38[2] (Chr 3: 141122572-141122691), GYPC [1] (Chr 2: 127413772-127413893), GYPC [2] (Chr 2: 127413870-127413996), ZSCAN12[1] (Chr 6: 28367484-28367600), ZSCAN12[2] (Chr 6: 28367510-28367631), LPP 2] (Chr 3: 188157018-188157138), and the clinical performance analyses of endometrial cancer and atypical hyperplasia were performed by selecting the four target region combinations of ZBTB38[1], GYPC [2], ZSCAN12[2] based on the threshold value of experimental example 1, and the results of the clinical performance analyses of endometrial cancer and atypical hyperplasia are shown in tables 13 and 14.
Table 13 clinical performance of double Gene methylation in cervical exfoliated cells in combination with diagnosis of endometrial cancer and atypical hyperplasia
Table 14 clinical performance of dual gene methylation in cervical exfoliated cells in combination with diagnosis of endometrial cancer
Wherein: the OR in tables 13 and 14 is: trust positive, any positive result is judged to be positive, and double negative results are judged to be negative; the "sum" in tables 13 and 14 is: trust is negative, any negative result is judged to be negative, and the double positive result is judged to be positive.
Alternatively, three gene combinations were performed based on the optimal thresholds ZBTB38[1] (Chr 3: 141130440-141130560), ZBTB38[2] (Chr 3: 141122572-141122691), GYPC [1] (Chr 2: 127413772-127413893), GYPC [2] (Chr 2: 127413870-127413996), ZSCAN12[1] (Chr 6: 28367484-28367600), ZSCAN12[2] (Chr 6: 28367510-28367631), LPP 2] (Chr 3: 188157018-188157138), and the clinical performance analyses of endometrial cancer and atypical hyperplasia were performed by selecting the four target region combinations of ZBTB38[1], GYPC [2], ZSCAN12[2] based on the threshold value of experimental example 1, and the results of the clinical performance analyses of endometrial cancer and atypical hyperplasia are shown in tables 15 and 16.
Table 15 clinical Properties of Tri-Gene methylation in cervical exfoliated cells in combination with diagnosis of endometrial cancer and atypical hyperplasia
Table 16 clinical performance of Tri-Gene methylation in cervical exfoliated cells in combination with diagnosis of endometrial cancer
Alternatively, four gene combinations were performed based on the optimal thresholds of ZBTB38[1] (Chr 3: 141130440-141130560), ZBTB38[2] (Chr 3: 141122572-141122691), GYPC [1] (Chr 2: 127413772-127413893), GYPC [2] (Chr 2: 127413870-127413996), ZCAN 12[1] (Chr 6: 28367484-28367600), ZCAN 12[2] (Chr 6: 28367510-28367631), LPP 2] (Chr 3: 188157018-188157138), and the clinical performance analyses of ZBTB38[1], GYPC [2], ZCAN 12[2] were selected based on the experimental threshold values and were performed as shown in Table 17.
Table 17 clinical diagnosis of endometrial cancer and atypical hyperplasia by combination of tetragenic methylation in cervical exfoliated cells
Alternatively, the methylation values ΔCp of ZBTB38[1] (Chr 3: 141130440-141130560), ZBTB38[2] (Chr 3: 141122572-141122691), GYPC [1] (Chr 2: 127413772-127413893), GYPC [2] (Chr 2: 127413870-127413996), ZCAN 12[1] (Chr 6: 28367484-28367600), ZCAN 12[2] (Chr 6: 28367510-28367631), LPP [2] (Chr 3: 188157018-188157138) have significant differences in the distribution of different endometrial lesions (normal or benign lesions, atypical hyperplasia, endometrial cancer) (see FIGS. 1 (a) -1 (g) and FIGS. 4 (a) -4 (g)), and after two suitable threshold values are taken for each gene target region, the predicted endometrial lesion degree of each gene is shown in FIGS. 7 (a) -7 (g).
Optionally, according to ZBTB38[1] (Chr 3: 141130440-141130560), ZBTB38[2] (Chr 3: 141122572-141122691), GYPC [1] (Chr 2: 127413772-127413893), GYPC [2] (Chr 2: 127413870-127413996), ZCAN 12[1] (Chr 6: 28367484-28367600), ZCAN 12[2] (Chr 6: 28367510-28367631), LPP 2] (Chr 3: 188157018-188157138) methylation value ΔCp has significant difference in distribution of different endometrial lesions (normal or benign lesions, atypical hyperplasia, endometrial cancer) (see FIG. 1 (a) -FIG. 1 (g) and FIG. 4 (a) -FIG. 4 (g), after two suitable critical values are taken from each gene target area, the degree of endometrial lesions can be predicted by two, three and four genes in combination, experiment example 2 selects ZBTB38[1], GYPC [1], ZCAN 12[2] three genes in combination, the degree of endometrial lesions can be predicted by taking the critical values of FIG. 7 (a) -FIG. 7 (g), the combined prediction rate of the critical values MV is 8.98.93%, the prediction rate of endometrial lesions is the typical prediction rate of endometrial lesions is shown as being 3.93.93.3% of the prediction accuracy of the combined prediction rate of the normal endometrial lesions; wherein: mv=w1×zbtb38[1] +w2×gypc [1] +w3×zscan12[2], where w1=3.27, w2=2.74, w3=4.14.
The advantages of the present application over the prior art include:
(1) aiming at differential methylation genes of endometrial cancer, a more efficient primer probe is designed and developed;
(2) the single gene has high AUC in diagnosis of endometrial cancer in endometrial paraffin tissue specimens and cervical exfoliated cell specimens, and has good detection rate for atypical hyperplasia of the endometrium;
(3) the gene combination has complementarity to the detection of different endometrial cancers, not only detects EC in early stage, but also has predictive value to atypical hyperplasia, and improves the clinical application value.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (4)
1. A biomarker for benign and malignant endometrial lesions, said biomarker comprising at least a nucleic acid sequence methylated in at least one target region of a GYPC gene, wherein said target region is selected from the group consisting of the following methylated sequences and regions of the GYPC gene:
the chr2:127413870-127413996 region of the GYPC gene;
the biomarker is applied to preparation of endometrial cancer diagnosis products, and in the detection process of the biomarker of benign and malignant endometrial lesions by using the diagnosis products, the method for judging and reading the results of endometrial cancer and atypical hyperplasia diagnosis by gene methylation is as follows:
the methylation level of a specific gene target is reflected by a Δcp value of:
ΔCp(GYPC[2])＝Cp(GYPC[2])-Cp(COL2A1)；
the methylation of delta Cp is less than or equal to k, and the result is positive methylation, namely that the tested sample is endometrial cancer, atypical hyperplasia or endometrial cancer; the gene methylated ΔCp > k is interpreted as methylation negative, i.e., indicating that the test sample is not endometrial cancer and atypical hyperplasia or endometrial cancer; where k is a particular value.
2. The biomarker for benign and malignant endometrial lesions according to claim 1, wherein the diagnostic product comprises any one of a kit, a formulation and a chip.
3. The biomarker for benign and malignant endometrial lesions according to claim 2, wherein the kit comprises at least a second primer pair for detecting the GYPC gene and a second probe, the nucleotide sequence of the second primer pair is shown in SEQ ID No.10 and SEQ ID No.11, and the nucleotide sequence of the second probe is shown in SEQ ID No. 12.
4. The biomarker for benign and malignant endometrial lesions according to claim 1, wherein the diagnosis product is applied to detect the biomarker for benign and malignant endometrial lesions in the following manner:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311054535.2A CN116790761B (en) | 2023-08-22 | 2023-08-22 | Biomarker for benign and malignant lesions of endometrium and application of biomarker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311054535.2A CN116790761B (en) | 2023-08-22 | 2023-08-22 | Biomarker for benign and malignant lesions of endometrium and application of biomarker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116790761A CN116790761A (en) | 2023-09-22 |
| CN116790761B true CN116790761B (en) | 2023-11-17 |
Family
ID=88044021
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311054535.2A Active CN116790761B (en) | 2023-08-22 | 2023-08-22 | Biomarker for benign and malignant lesions of endometrium and application of biomarker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116790761B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001077373A2 (en) * | 2000-04-06 | 2001-10-18 | Epigenomics Ag | Detection of variations in the dna methylation profile |
| CA3127329A1 (en) * | 2019-01-24 | 2020-07-30 | Mayo Foundation For Medical Education And Research | Detecting endometrial cancer |
| CN114480661A (en) * | 2022-04-18 | 2022-05-13 | 北京起源聚禾生物科技有限公司 | Endometrium benign and malignant lesion combined marker, detection primer probe set and kit |
| CN114729399A (en) * | 2019-10-31 | 2022-07-08 | 梅约医学教育与研究基金会 | Detection of ovarian cancer |
| CN116783309A (en) * | 2020-06-17 | 2023-09-19 | Ucl商业有限责任公司 | Methods for detecting and predicting cancer and/or CIN3 |
-
2023
- 2023-08-22 CN CN202311054535.2A patent/CN116790761B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001077373A2 (en) * | 2000-04-06 | 2001-10-18 | Epigenomics Ag | Detection of variations in the dna methylation profile |
| CA3127329A1 (en) * | 2019-01-24 | 2020-07-30 | Mayo Foundation For Medical Education And Research | Detecting endometrial cancer |
| CN113557308A (en) * | 2019-01-24 | 2021-10-26 | 梅约医学教育与研究基金会 | Detection of endometrial cancer |
| CN114729399A (en) * | 2019-10-31 | 2022-07-08 | 梅约医学教育与研究基金会 | Detection of ovarian cancer |
| CN116783309A (en) * | 2020-06-17 | 2023-09-19 | Ucl商业有限责任公司 | Methods for detecting and predicting cancer and/or CIN3 |
| CN114480661A (en) * | 2022-04-18 | 2022-05-13 | 北京起源聚禾生物科技有限公司 | Endometrium benign and malignant lesion combined marker, detection primer probe set and kit |
Non-Patent Citations (3)
| Title |
|---|
| A Simple Cervicovaginal Epigenetic Test for Screening and Rapid Triage of Women With Suspected Endometrial Cancer: Validation in Several Cohort and Case/Control Sets;Chiara Herzog;《J Clin Oncol》;第3828-3838页 * |
| Obesity and menopause modify the epigenomic profile of breast cancer;Ana B Crujeiras;《Endocrine-Related Cancer》;第331–343页 * |
| 影响儿童急性淋巴细胞白血病化疗疗效及预后的分子生物学因素;宁芳;何晓莲;才绍江;;中国小儿血液与肿瘤杂志(第05期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116790761A (en) | 2023-09-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220073994A1 (en) | Dna methylation biomarkers for early detection of cervical cancer | |
| EP2210942B1 (en) | Gene associated with liver cancer, and method for determination of the risk of acquiring liver cancer | |
| EP2831270B1 (en) | Biomarker for bladder cancer | |
| JP2005509445A (en) | Prostate cancer detection | |
| WO2016115354A1 (en) | Methods for cancer diagnosis and prognosis | |
| CN112567049A (en) | Method and kit for identifying gastric cancer status | |
| CN108977544A (en) | For identifying kit and its application of gastric cancer and/or polyp of stomach | |
| CN111630187A (en) | Method and kit for identifying colorectal cancer status | |
| US20210404018A1 (en) | Unbiased dna methylation markers define an extensive field defect in histologically normal prostate tissues associated with prostate cancer: new biomarkers for men with prostate cancer | |
| CN109082467A (en) | For identifying kit and its application of breast cancer status or Precancerous Lesions of Breast | |
| JP2011160711A (en) | Method for using frequency of methylation of specific gene as biomarker for gynecologic cancer | |
| CN111363811A (en) | Lung cancer diagnostic agent and kit based on FOXD3 gene | |
| Kipp et al. | Assessing the value of reflex fluorescence in situ hybridization testing in the diagnosis of bladder cancer when routine urine cytological examination is equivocal | |
| EP3368684B1 (en) | Biomarker for breast cancer | |
| CN116790761B (en) | Biomarker for benign and malignant lesions of endometrium and application of biomarker | |
| CN116121387A (en) | Combined marker for colorectal cancer detection and application thereof | |
| JP2011530287A (en) | Prostate cancer methylation analysis | |
| CN117070629A (en) | Biomarker for benign and malignant lesions of endometrium and application of biomarker | |
| CN116875700A (en) | Biomarker for benign and malignant lesions of endometrium and application of biomarker | |
| Miura et al. | A diagnostic evaluation of serum human telomerase reverse transcriptase mRNA as a novel tumor marker for gynecologic malignancies | |
| CN113234821A (en) | Method and kit for identifying esophageal cancer status | |
| WO2022013880A1 (en) | Blood based biomarkers for early diagnosis and follow up of breast cancer | |
| SG185254A1 (en) | 3.4 kb mitochondrial dna deletion for use in the detection of cancer | |
| WO2014160829A2 (en) | Unbiased dna methylation markers define an extensive field defect in histologically normal porstate tissues associated with prostate cancer: new biomarkers for men with prostate cancer | |
| CN113186282B (en) | Methods and kits for identifying pancreatic cancer status |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 410205 the first floor of all complex buildings and workshops, No. 19, Lugu Lutian Road, Changsha high tech Development Zone, Changsha City, Hunan Province Patentee after: HUNAN HONGYA GENE TECHNOLOGY CO.,LTD. Address before: 410205 Changsha High tech Development Zone, Changsha City, Hunan Province, China, No. 19 Lugu Lutian Road, Comprehensive Building, First Floor of Workshop Patentee before: HUNAN HONGYA GENE TECHNOLOGY CO.,LTD. |