CN111118164A - Marker, kit and detection method for early screening and diagnosis of tumor - Google Patents
Marker, kit and detection method for early screening and diagnosis of tumor Download PDFInfo
- Publication number
- CN111118164A CN111118164A CN202010134189.9A CN202010134189A CN111118164A CN 111118164 A CN111118164 A CN 111118164A CN 202010134189 A CN202010134189 A CN 202010134189A CN 111118164 A CN111118164 A CN 111118164A
- Authority
- CN
- China
- Prior art keywords
- pleural effusion
- diagnosis
- gene
- mrna
- ywhaz
- 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.)
- Pending
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 40
- 238000003745 diagnosis Methods 0.000 title claims abstract description 32
- 238000012216 screening Methods 0.000 title claims abstract description 31
- 238000001514 detection method Methods 0.000 title claims abstract description 19
- 239000003550 marker Substances 0.000 title claims abstract description 19
- 208000002151 Pleural effusion Diseases 0.000 claims abstract description 41
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims abstract description 32
- 201000005202 lung cancer Diseases 0.000 claims abstract description 32
- 208000020816 lung neoplasm Diseases 0.000 claims abstract description 32
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 27
- 101150076297 ywhaz gene Proteins 0.000 claims abstract description 25
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- 201000011510 cancer Diseases 0.000 claims abstract description 13
- 206010026673 Malignant Pleural Effusion Diseases 0.000 claims description 34
- 239000000523 sample Substances 0.000 claims description 25
- 238000002474 experimental method Methods 0.000 claims description 23
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 238000012408 PCR amplification Methods 0.000 claims description 12
- 238000011144 upstream manufacturing Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 9
- 238000010839 reverse transcription Methods 0.000 claims description 9
- 108010085238 Actins Proteins 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 230000001575 pathological effect Effects 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 206010019280 Heart failures Diseases 0.000 claims description 4
- 208000034767 Hypoproteinaemia Diseases 0.000 claims description 4
- 102000004271 Tryptophan 5-monooxygenases Human genes 0.000 claims description 4
- 108090000885 Tryptophan 5-monooxygenases Proteins 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 4
- 239000002299 complementary DNA Substances 0.000 claims description 4
- 230000007717 exclusion Effects 0.000 claims description 4
- 201000008827 tuberculosis Diseases 0.000 claims description 4
- 206010048612 Hydrothorax Diseases 0.000 claims description 3
- 239000013614 RNA sample Substances 0.000 claims description 3
- 238000002835 absorbance Methods 0.000 claims description 3
- 238000007664 blowing Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 108020004707 nucleic acids Proteins 0.000 claims description 3
- 102000039446 nucleic acids Human genes 0.000 claims description 3
- 150000007523 nucleic acids Chemical class 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 238000011895 specific detection Methods 0.000 claims description 3
- 230000001351 cycling effect Effects 0.000 claims description 2
- 239000000047 product Substances 0.000 claims description 2
- 239000003147 molecular marker Substances 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 208000005623 Carcinogenesis Diseases 0.000 abstract description 2
- 230000036952 cancer formation Effects 0.000 abstract description 2
- 231100000504 carcinogenesis Toxicity 0.000 abstract description 2
- 238000011161 development Methods 0.000 abstract description 2
- 238000013399 early diagnosis Methods 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 12
- 102100040685 14-3-3 protein zeta/delta Human genes 0.000 description 9
- 101000964898 Homo sapiens 14-3-3 protein zeta/delta Proteins 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 230000003211 malignant effect Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 201000003437 pleural cancer Diseases 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 206010073310 Occupational exposures Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 231100000675 occupational exposure Toxicity 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 210000003281 pleural cavity Anatomy 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
Images
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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Pathology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Hospice & Palliative Care (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Oncology (AREA)
- General Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a marker, a kit and a detection method for early screening and diagnosis of tumors, belonging to the technical field of biomedicine. Based on the effect of a cancer driver gene in tumorigenesis and development, the invention can achieve the purposes of early screening and diagnosis by detecting the expression of YWHAZ gene mRNA in pleural effusion of a subject by utilizing an auxiliary diagnosis molecular marker related to lung cancer, has the characteristics of high sensitivity, strong specificity, low cost, stable result and the like, is simple and quick to operate, and can provide a basis for early diagnosis of lung cancer, thereby guiding a clinician to provide an effective prevention means or treatment scheme for the subject; has good clinical application value for screening early lung cancer, wide clinical application prospect and great commercial value.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a marker, a kit and a detection method for early screening and diagnosis of tumors.
Background
Lung cancer is the first most common and lethal cancer worldwide, with about 208.8 million lung cancer cases worldwide each year and about 176.1 million people dying from lung cancer each year, as counted by the global cancer database (GLOBOCAN) 2018. Lung cancer patients often have no obvious clinical manifestations in the early stage, most of them are in the late stage when diagnosed, and the prognosis is very poor. The 5-year survival rate of lung cancer patients is closely related to the stage of the patients receiving the treatment, the 5-year survival rate of non-small cell lung cancer patients in stage I is 60-80%, and the 5-year survival rate of patients in stage IV is only 2%. Pleural effusion is divided into two major categories, benign and malignant, according to the cause of disease. Malignant Pleural Effusion (MPE) accounts for 38% -53% of the total pleural effusion, is a common complication of lung cancer, about 15% of patients with lung cancer have a first diagnosis, i.e., the malignant pleural effusion is combined, and the occurrence of the malignant pleural effusion usually indicates that the life cycle of the patients is shorter and the quality of life of the patients is worse. In recent years, with the factors of environmental pollution aggravation, smoking, occupational exposure and the like, the incidence rate and the fatality rate of lung cancer tend to increase, and the human life health is threatened more and more seriously.
Pathological tissue biopsy is the main basis for the definitive diagnosis of malignant tumor in lung, but at present, pathological tissue biopsy faces two main problems: on one hand, most of the patients are invasive operations and have high technical requirements for operating doctors, and on the other hand, some patients cannot tolerate the invasive operations and have relatively high cost. Thoracentesis is a commonly used diagnosis and treatment technique for examining the properties of pleural effusion, evacuating air, drawing fluid to relieve compression symptoms, or administering drug into pleural cavity by puncture. The pleural effusion is convenient to detect due to the abundant mRNA, has sensitive indexes and has obvious relevance with specific diseases. Therefore, by means of thoracentesis, a rapid, sensitive and specific quantitative detection method is adopted to screen mRNA which is specifically expressed or abnormally expressed in pleural effusion of a tumor disease patient as a molecular marker. Therefore, the thoracocentesis is used for searching for indexes directly or indirectly reflecting the benign and malignant indexes of the lung cancer pleural effusion, and auxiliary diagnostic kits for early and different stages of lung cancer are developed and used for evaluating the risk of the lung cancer and taking effective intervention measures, so that the thoracocentesis has significant social value for restraining the incidence trend of the lung cancer diseases.
Disclosure of Invention
The invention provides a marker for early screening and diagnosis of tumors to assist clinical diagnosis of lung cancer, and also provides a kit containing the marker. In addition, the invention also provides a detection method which has high sensitivity, strong specificity and low cost and can assist the clinical diagnosis of the lung cancer, has good clinical application value for the early screening of the lung cancer, and particularly provides a marker, a kit and a detection method for the early screening and diagnosis of tumors.
In order to solve the technical problems, the invention adopts the technical scheme that: a marker for early screening and diagnosis of tumors, wherein the marker is tryptophan 5-monooxygenase activating protein, and the tryptophan 5-monooxygenase activating protein is an expression product of mRNA of YWHAZ gene.
Furthermore, the marker for early screening and diagnosis of tumors, provided by the invention, comprises an upstream primer and a downstream primer of the YWHAZ gene, wherein the sequence of the upstream primer is SEQ ID NO.3, and the sequence of the downstream primer is SEQ ID NO. 4.
Furthermore, the invention also discloses a kit for early screening and diagnosis of tumors, which comprises the marker for early screening and diagnosis of tumors and a primer containing specific detection, wherein the primer is used for detecting the expression level of mRNA of the YWHAZ gene in a sample.
Furthermore, the kit for early screening and diagnosis of tumors, provided by the invention, uses β -actin gene as internal reference to detect YWHAZ gene mRNA expression level, namely delta CT value of YWHAZ gene mRNA expression, wherein the β -actin gene has an upstream primer and a downstream primer, the sequence of the upstream primer is SEQ ID NO. 1, and the sequence of the downstream primer is SEQ ID NO. 2.
Further, the kit for screening and diagnosing the early stage of the tumor is provided, wherein the detection sample is pleural effusion, and the tumor refers to lung cancer and comprises benign tumor and malignant tumor.
Further, the invention also discloses a detection method comprising the marker, and the specific detection method comprises the following steps:
(1) patients were selected according to exclusion criteria and signed informed consent, malignant pleural effusion selection criteria: finding cancer cells in the pleural effusion or finding cancer cells through pathological tissues; benign pleural effusion selection criteria: pleural effusion caused by tuberculosis or heart failure or hypoproteinemia;
(2) under the aseptic requirement, 10ml of pleural effusion per person of a patient is collected and put into a 15ml enzyme-removing centrifuge tube. Putting 15ml of enzyme-removed centrifuge tube containing the specimen into a low-temperature ultracentrifuge for centrifugation for 10min at the centrifugation condition of 4 ℃ and 1000 rpm;
(3) centrifuging, collecting precipitate, adding Trizol solution into 1.5ml centrifuge tube at a ratio of 1ml per 0.1ml hydrothorax precipitate, blowing, mixing, and standing at room temperature for 5 min;
(4) adding 0.2ml of chloroform into 1ml of Trizol solution, violently shaking for 15s, and standing for 2 min;
(5) placing the upper water phase in a new centrifuge tube, adding 0.5ml of isopropanol into each 1ml of Trizol solution, standing at room temperature for 10min, and centrifuging at 12000rpm for 10min at 4 deg.C;
(6) discarding supernatant, adding 75% ethanol at a ratio of 1ml to 1ml Trizol solution, mixing by vortex, centrifuging at 7500rpm at 4 deg.C for 5 min;
(7) carefully discarding the supernatant, drying at room temperature or under vacuum for 5-10min, and adding DEPC water to dissolve the precipitate;
(8) and (3) pure concentration detection: taking 2ul RNA samples, and determining absorbance value, namely OD value, on a NANODROP LITE nucleic acid protein detector, wherein the OD260/280 ratio is all in the range of 1.8-2.0;
(9) reverse transcription experiments: and respectively carrying out sample adding according to a reverse transcription test, and after sample adding, according to the following steps: carrying out reverse transcription experiments at 37 ℃, 15min → 85 ℃, 5s → 4 ℃ and infinity to obtain cDNA required by the subsequent PCR amplification experiment;
(10) PCR amplification experiments: and respectively carrying out sample adding according to a PCR amplification test, and after sample adding, according to the following steps: 94 ℃, 1min → 94 ℃, 30s → 56 ℃, 30s → 72 ℃ and cycling 40 times under the process conditions, then following: carrying out PCR amplification experiment at 72 ℃, 10min → 4 ℃ and infinity;
(11) and analyzing the receiver operating characteristic curve, the diagnosis index and the credible interval of the YWHAZ gene in the pleural effusion, determining the relative expression quantity of mRNA of the YWHAZ gene in the pleural effusion, and screening and diagnosing the type of the tumor through the relative expression quantity.
Further, in the detection method of the present invention, in the step (11) of screening and diagnosing tumors by using the relative expression level, the specific determination criterion is that the Δ CT value of mRNA expression of the ywaz gene in pleural effusion is within a range of 4 ± 0.47, which is considered as malignant pleural effusion, the expression of the relative expression level of mRNA of the ywaz gene in malignant pleural effusion is 1.641 ± 0.635 times higher than that of benign pleural effusion, and P <0.05 has statistical significance; the expression of mRNA of YWHAZ gene in the malignant pleural effusion of the stage III/IV of the lung cancer is higher than that of mRNA of YWHAZ gene in the malignant pleural effusion of the stage I/II of the lung cancer, the expression is 14.450 +/-0.859 times, and P <0.05 has statistical significance.
Compared with the prior art, the marker, the kit and the detection method for early screening and diagnosis of the tumor have the beneficial effects that: based on the role of a cancer driver gene in tumorigenesis and development, early screening and diagnosis can be achieved by detecting the expression of YWHAZ gene mRNA in pleural effusion of a subject by using an auxiliary diagnosis molecular marker related to lung cancer, and the detection method has the characteristics of high sensitivity, strong specificity, low cost, stable result and the like, is simple and quick to operate, can provide a basis for early diagnosis of lung cancer, and thus guides a clinician to provide an effective prevention means or treatment scheme for the subject; has good clinical application value for screening early lung cancer, wide clinical application prospect and great commercial value.
Drawings
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings, in which:
FIG. 1 is a schematic representation of the expression of YWHAZ in benign and malignant pleural effusion in an experimental example of the present invention;
FIG. 2 is a schematic representation of the expression of YWHAZ in malignant pleural effusion of stage I/II and III/IV lung cancer in an experimental example of the present invention;
FIG. 3 is a schematic diagram showing the expression of YWHAZ protein in benign and malignant pleural effusion according to an example of the present invention;
FIG. 4 is a schematic diagram showing the ROC curve and correct diagnosis index of YWHAZ for diagnosing malignant pleural effusion in the experimental example of the present invention.
Detailed Description
In order to more fully explain the practice of the invention, the invention will now be further described with reference to the accompanying drawings in which specific examples are shown, which are provided for illustration only and are not to be construed as limiting the invention. The following examples are examples of experimental methods not given specific conditions, and unless otherwise specified, the tests are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers, and the equipment and reagents involved are commercially available.
Example 1
First, prepare the sample
74 cases of malignant pleural effusion caused by the lung cancer which is not used after 4-month thoracocentesis in 2018 to 2019 in the first people hospital in Zunyi city and benign pleural effusion caused by other reasons are collected, 5 cases of malignant pleural effusion and 5 cases of benign pleural effusion specimens are randomly selected, and the general data of the patients are shown in Table 1.
TABLE 1 general patient data
Second, sample analysis
Centrifuging samples of malignant pleural effusion and benign pleural effusion, performing an RT-PCR experiment on collected supernate, taking β -actin gene as an internal reference, wherein the sequence of an upstream primer is SEQ ID NO. 1, the sequence of a downstream primer is SEQ ID NO. 2, the sequence of the upstream primer is 5'-GTTGCGTTACACCCTTTCTT-3', the sequence of the downstream primer is 5'-ACCACCCTGTTGCTGTAGCC-3', detecting the expression level of mRNA of YWHAZ gene, the sequence of the upstream primer is SEQ ID NO.3, the sequence of the downstream primer is SEQ ID NO. 4, the sequence of the upstream primer is 5'-GCAACCTCAGCCAAGTAA-3', and the sequence of the downstream primer is 5'-TGTTGTAGGAGCCCGTAG-3', wherein the conditions of a specific sequence list are shown in Table 2:
TABLE 2 sequence listing
The method comprises the following specific steps:
(1) patients were selected according to exclusion criteria and signed informed consent. Malignant pleural effusion selection criteria: cancer cells are found in pleural effusion or cancer cells are found in pathological tissues (lung). Benign pleural effusion selection criteria: pleural effusion caused by tuberculosis, heart failure or hypoproteinemia, etc.
(2) Strictly complying with the sterility requirements, 10ml of pleural effusion (benign or malignant) of a patient is collected in a 15ml enzyme-removing centrifuge tube. The 15ml enzyme-removed centrifuge tube with the specimen was placed in a low temperature ultracentrifuge and centrifuged (4 ℃, 1000rpm, 10 min).
(3) Centrifuging, collecting precipitate, adding Trizol solution into 1.5ml centrifuge tube at a ratio of 1ml per 0.1ml hydrothorax precipitate, blowing, mixing, and standing at room temperature for 5 min.
(4) Chloroform was added in an amount of 0.2ml per 1ml of Trizol solution, and the mixture was vigorously shaken for 15 seconds and allowed to stand for 2 min.
(5) Taking the upper water phase in a new centrifuge tube, adding 0.5ml of isopropanol into 1ml of Trizol solution, standing at room temperature for 10min, and centrifuging at 12000rpm for 10min at 4 ℃.
(6) Discarding supernatant, adding 75% ethanol at a ratio of 1ml to 1ml Trizol solution, mixing by vortex, centrifuging at 7500rpm at 4 deg.C for 5 min.
(7) The supernatant was carefully discarded, followed by drying at room temperature or under vacuum for 5-10min, and finally dissolving the precipitate with additional DEPC water.
(8) And (3) pure concentration detection: 2ul RNA samples were taken and absorbance values (OD values) were determined on a NANODROP LITE nucleic acid protein detector. The OD260/280 ratios were all in the range of 1.8-2.0.
(9) Reverse transcription experiments: samples were each loaded as per Table 3. After sample loading, the following steps are carried out: and carrying out reverse transcription experiments at 37 ℃, 15min → 85 ℃, 5s → 4 ℃ and infinity to obtain cDNA required by the subsequent PCR amplification experiment.
TABLE 3 reverse transcription reaction sample application System
Reagent | Amount of the composition used |
5×PrimeScript Buffer | 4μl |
PrimeScript RT Enzyme Mix | 1μl |
Olgo dT Prime(50μM) | 1μl |
Random 6 mers(100μM) | 1μl |
Total RAN(500ng/μl) | |
RNase Free dH20 | Up to 20μl |
(10) PCR amplification experiments: samples were each loaded as per Table 4. After sample loading, the following steps are carried out: PCR amplification experiments were carried out at 94 ℃, 1min → 94 ℃, 30s → 56 ℃, 30s → 72 ℃, 1min (94 ℃, 30s → 56 ℃, 30s → 72 ℃, 1min this step was co-cycled 40 times) → 72 ℃, 10min → 4 ℃ ∞.
TABLE 4 sample application System for PCR amplification test
Reagent | Amount of the composition used |
SYBR Premix Ex Taq Ⅱ(2×) | 12.5μl |
PCB Forward Primer(10μM) | 1μl |
PCB Reverse Primer(10μM) | 1μl |
cDNA solution | 2μl |
RNase Free dH20 | 8.5μl |
Total | 25μl |
Thirdly, experimental verification:
to increase the reliability of the assay, we performed immunoblot experiments on the collected supernatants to detect the YWHAZ protein expression level.
1. The specific operation steps are as follows:
(1) patients were selected according to exclusion criteria and signed informed consent. Malignant pleural effusion selection criteria: cancer cells are found in pleural effusion or cancer cells are found in pathological tissues (lung). Benign pleural effusion selection criteria: pleural effusion caused by tuberculosis, heart failure or hypoproteinemia, etc.
(2) Strictly complying with the sterility requirements, 10ml of pleural effusion (benign or malignant) of a patient is collected in a 15ml enzyme-removing centrifuge tube. A15 ml enzyme-removed centrifuge tube containing the specimen was placed in a low temperature ultracentrifuge and centrifuged (4 ℃ C., 1000rpm, 10 min).
(3) After centrifugation, the supernatant was discarded, and a protein lysate (RIPA: PMSF =100: 1) was added thereto, and vortexed 1 time at intervals of 5min for 30 seconds each time, and repeated 6 times.
(4) The samples were centrifuged at 12000-14000rpm for 15min at 4 ℃ and the supernatant was transferred to a fresh centrifuge tube.
(5) The collected supernatants were loaded using BCA kit. And (3) after sample addition, placing the sample in an oven at 37 ℃ for 30min, measuring the OD value at 562nm of a microplate reader, and calculating the protein concentration of the sample.
(6) Appropriate PBS was added for dilution, protein concentrations of different samples were pooled, and SDS-PAGE protein Loading Buffer (SDS-PAGE Sample Loading Buffer, 5X) was added to one-fourth of the Sample volume.
(7) Boiling the sample protein in boiling water for 10-15min to denature the sample protein.
(8) SDS-PAGE separation gel and concentrated gel are prepared, the loading amount is 10 ul/hole, and then electrophoresis and electrotransformation experiments are sequentially carried out.
(9) And respectively carrying out sealing, incubation primary antibody and incubation secondary antibody experiments on the protein band after the electric conversion, finally carrying out exposure, and finally carrying out statistics.
2. Statistical analysis was performed using GraphPad Prism 6.01 software, using one-way anova to determine whether statistical significance was present.
3. Analysis of the receiver operating characteristic curve (ROC curve) and correct diagnostic index and confidence interval was performed on ywaz in pleural effusion.
4. And (4) verification result:
according to the RT-PCR experimental result, as shown in figure 1, β -actin is used as an internal reference, the Delta CT value of the mRNA expression of YWHAZ in malignant pleural effusion is 4 +/-0.47, the relative expression quantity of the mRNA expression of YWHAZ in malignant pleural effusion is higher than that of benign pleural effusion and is about 1.641 +/-0.635 times, and P <0.05 has statistical significance.
According to the RT-PCR experimental results, as shown in FIG. 2, the expression of YWHAZ mRNA in the malignant pleural effusion of stage III/IV of lung cancer is higher than that in the malignant pleural effusion of stage I/II of lung cancer, which is about 14.450 +/-0.859 times, and P <0.05 has statistical significance.
According to the results of the immunoblot experiments, as shown in fig. 3, the expression of ywaz protein in malignant pleural effusion is higher than that of benign pleural effusion, and P <0.05 has statistical significance.
Finally, the PCR results of benign and malignant pleural effusion were analyzed, and as shown in fig. 4, the area under the ROC curve for ywaz diagnosing malignant pleural effusion was 0.920, the difference was statistically significant compared to 0.5 (P =0.028< 0.05), and the accuracy of using ywaz to diagnose malignant pleural effusion was higher (the area under the curve was greater than 0.9). Correct diagnostic index for ywaz diagnosis of malignant pleural effusion: the difference between the corresponding sensitivity and 1-specificity in this experimental example was 0.800 as the maximum correct diagnosis index corresponding to the cut-off point 11.800, and thus, it was considered as the optimum cut-off point. The 95% CI was 0.920. + -. 1.96X 0.093, i.e. [0.738, 1.102 ].
In conclusion, the delta CT value of the mRNA expression of YWHAZ in the pleural effusion (taking β -actin as an internal reference) is in the range of 4 +/-0.47, so that the YWHAZ gene can be considered as malignant pleural effusion, and the results show that the YWHAZ gene is a good auxiliary diagnosis molecular marker for identifying the benign and malignant pleural effusion and possibly has good clinical application value for screening early lung cancer.
Finally, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in the embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the claims and their equivalents. Although the present invention has been described with reference to the preferred embodiments thereof, it should be understood by those skilled in the art that any minor modifications, equivalents and improvements made in accordance with the technical solution of the present invention should be included in the protective scope of the technical solution of the present invention.
<110> Zunyi city first people hospital
<120> marker, kit and detection method for early screening and diagnosis of tumor
<160>4
<210>1
<211>20
<212>RNA
<213> Artificial sequence
<400>1
gttgcgttac accctttctt 20
<210>2
<211>20
<212>RNA
<213> Artificial sequence
<400>2
accaccctg tgctgtagcc 20
<210>3
<211>18
<212>RNA
<213> Artificial sequence
<400>3
gcaacctcag ccaagtaa 18
<210>4
<211>18
<212>RNA
<213> Artificial sequence
<400>4
tgttgtagga gcccgtag18
Claims (7)
1. A marker for early screening and diagnosis of tumors, characterized by: the marker is tryptophan 5-monooxygenase activating protein, and the tryptophan 5-monooxygenase activating protein is an expression product of mRNA of YWHAZ gene.
2. A marker for early screening and diagnosis of tumors according to claim 1, wherein: the primer of the YWHAZ gene comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is SEQ ID NO.3, and the sequence of the downstream primer is SEQ ID NO. 4.
3. A kit for early screening and diagnosis of tumors, characterized in that: comprising a marker for early screening and diagnosis of tumors as claimed in any one of claims 1 or 2, and a primer containing a specific detection for detecting the expression level of mRNA of the YWHAZ gene in a sample.
4. The kit for early screening and diagnosing of tumors as claimed in claim 3, wherein said primer uses β -actin gene as internal reference to detect the mRNA expression level of YWHAZ gene, i.e. Δ CT value of mRNA expression of YWHAZ gene, said β -actin gene has upstream primer and downstream primer, the sequence of its upstream primer is SEQ ID NO. 1, and the sequence of its downstream primer is SEQ ID NO. 2.
5. The kit for early screening and diagnosis of tumors according to claim 3, wherein: the detection sample is pleural effusion, and the tumor refers to lung cancer and comprises benign tumor and malignant tumor.
6. A detection method using the marker for early screening and diagnosis of tumor as claimed in any one of claims 1 or 2, characterized in that: the method comprises the following steps:
(1) patients were selected according to exclusion criteria and signed informed consent, malignant pleural effusion selection criteria: finding cancer cells in pleural effusion or finding cancer cells through pathological tissues, and selecting a standard for benign pleural effusion: pleural effusion caused by tuberculosis or heart failure or hypoproteinemia;
(2) under the aseptic requirement, 10ml of pleural effusion per person of a patient is collected and put into a 15ml enzyme-removing centrifuge tube. Putting 15ml of enzyme-removed centrifuge tube containing the specimen into a low-temperature ultracentrifuge for centrifugation for 10min at the centrifugation condition of 4 ℃ and 1000 rpm;
(3) centrifuging, collecting precipitate, adding Trizol solution into 1.5ml centrifuge tube at a ratio of 1ml per 0.1ml hydrothorax precipitate, blowing, mixing, and standing at room temperature for 5 min;
(4) adding 0.2ml of chloroform into 1ml of Trizol solution, violently shaking for 15s, and standing for 2 min;
(5) placing the upper water phase in a new centrifuge tube, adding 0.5ml of isopropanol into each 1ml of Trizol solution, standing at room temperature for 10min, and centrifuging at 12000rpm for 10min at 4 deg.C;
(6) discarding supernatant, adding 75% ethanol at a ratio of 1ml to 1ml Trizol solution, mixing by vortex, centrifuging at 7500rpm at 4 deg.C for 5 min;
(7) carefully discarding the supernatant, drying at room temperature or under vacuum for 5-10min, and adding DEPC water to dissolve the precipitate;
(8) and (3) pure concentration detection: taking 2ul RNA samples, and determining absorbance value, namely OD value, on a NANODROP LITE nucleic acid protein detector, wherein the OD260/280 ratio is all in the range of 1.8-2.0;
(9) reverse transcription experiments: and respectively carrying out sample adding according to a reverse transcription experiment, and after sample adding, according to the following steps: carrying out reverse transcription experiments at 37 ℃, 15min → 85 ℃, 5s → 4 ℃ and infinity to obtain cDNA required by the subsequent PCR amplification experiment;
(10) PCR amplification experiments: respectively carrying out sample adding according to a PCR amplification experiment, and after sample adding, according to the following steps: 94 ℃, 1min → 94 ℃, 30s → 56 ℃, 30s → 72 ℃ and cycling 40 times under the process conditions, then following: carrying out PCR amplification experiment at 72 ℃, 10min → 4 ℃ and infinity;
(11) and analyzing the receiver operating characteristic curve, the diagnosis index and the credible interval of the YWHAZ gene in the pleural effusion, determining the relative expression quantity of mRNA of the YWHAZ gene in the pleural effusion, and screening and diagnosing the type of the tumor through the relative expression quantity.
7. The detection method according to claim 6, characterized in that: in the process of screening and diagnosing the tumor through the relative expression level in the step (11), the specific judgment standard is that the delta CT value of the mRNA expression of the YWHAZ gene in the pleural effusion is considered as malignant pleural effusion when the delta CT value is in the range of 4 +/-0.47, the expression of the relative expression level of the mRNA of the YWHAZ gene in the malignant pleural effusion is 1.641 +/-0.635 times higher than that of benign pleural effusion, and P <0.05 has statistical significance; the expression of mRNA of YWHAZ gene in the malignant pleural effusion of the stage III/IV of the lung cancer is higher than that of mRNA of YWHAZ gene in the malignant pleural effusion of the stage I/II of the lung cancer, the expression is 14.450 +/-0.859 times, and P <0.05 has statistical significance.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010134189.9A CN111118164A (en) | 2020-03-02 | 2020-03-02 | Marker, kit and detection method for early screening and diagnosis of tumor |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010134189.9A CN111118164A (en) | 2020-03-02 | 2020-03-02 | Marker, kit and detection method for early screening and diagnosis of tumor |
Publications (1)
Publication Number | Publication Date |
---|---|
CN111118164A true CN111118164A (en) | 2020-05-08 |
Family
ID=70493598
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010134189.9A Pending CN111118164A (en) | 2020-03-02 | 2020-03-02 | Marker, kit and detection method for early screening and diagnosis of tumor |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111118164A (en) |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060194229A1 (en) * | 2005-01-25 | 2006-08-31 | Sky Genetics, Inc. | Cancer markers and detection methods |
US20070178503A1 (en) * | 2005-12-19 | 2007-08-02 | Feng Jiang | In-situ genomic DNA chip for detection of cancer |
US20110104062A1 (en) * | 2008-02-07 | 2011-05-05 | K. W. Michael Siu | Biomarkers for Head-And-Neck Cancers and Precancers |
CN102286370A (en) * | 2010-06-15 | 2011-12-21 | 希森美康株式会社 | Method of determining lymph node metastasis in lung cancer, apparatus for determining lymph node metastasis in lung cancer, and control system |
US20120100558A1 (en) * | 2008-09-08 | 2012-04-26 | Hanash Samir M | Lung cancer diagnosis |
US20180142303A1 (en) * | 2015-05-19 | 2018-05-24 | The Wistar Institute Of Anatomy And Biology | Methods and compositions for diagnosing or detecting lung cancers |
CN110114680A (en) * | 2016-05-05 | 2019-08-09 | 佰欧迪塞克斯公司 | For the composition of diagnosing, method and kit |
-
2020
- 2020-03-02 CN CN202010134189.9A patent/CN111118164A/en active Pending
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060194229A1 (en) * | 2005-01-25 | 2006-08-31 | Sky Genetics, Inc. | Cancer markers and detection methods |
US20070178503A1 (en) * | 2005-12-19 | 2007-08-02 | Feng Jiang | In-situ genomic DNA chip for detection of cancer |
US20110104062A1 (en) * | 2008-02-07 | 2011-05-05 | K. W. Michael Siu | Biomarkers for Head-And-Neck Cancers and Precancers |
US20120100558A1 (en) * | 2008-09-08 | 2012-04-26 | Hanash Samir M | Lung cancer diagnosis |
CN102286370A (en) * | 2010-06-15 | 2011-12-21 | 希森美康株式会社 | Method of determining lymph node metastasis in lung cancer, apparatus for determining lymph node metastasis in lung cancer, and control system |
US20110311981A1 (en) * | 2010-06-15 | 2011-12-22 | Sysmex Corporation | Method of determining lymph node metastasis in a lung cancer, apparatus for determining lymph node metastasis in a lung cancer, and computer program product |
US20180142303A1 (en) * | 2015-05-19 | 2018-05-24 | The Wistar Institute Of Anatomy And Biology | Methods and compositions for diagnosing or detecting lung cancers |
CN110114680A (en) * | 2016-05-05 | 2019-08-09 | 佰欧迪塞克斯公司 | For the composition of diagnosing, method and kit |
Non-Patent Citations (4)
Title |
---|
CHEN C.等: "A Novel Function of YWHAZ/β-catenin Axis in Promoting Epithelial-Mesenchymal Transition and Lung Cancer Metastasis" * |
DENG YONG等: "The clinical and prognostic significance of YWHAZ in non‐small–cell lung cancer patients: Immunohistochemical analysis" * |
FAN T.等: "\"Up-regulation of 14-3-3Z in Lung Cancer and Its Implication as Prognostic and Therapeutic Target\"" * |
刘潇莲;邢影;王校媛;孟庆威;蔡莉;: "14-3-3ζ和E-cadherin在Ⅲ期肺腺癌中的表达及其意义" * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN106434953B (en) | A kind of detection and application of gastric cancer New molecular marker object hsa_circ_0074362 | |
CN105671181B (en) | Gene marker, primer, probe and kit for detecting lung cancer | |
CN105256014B (en) | Breast cancer combined diagnosis marker and detection kit | |
CN105586399A (en) | Kit for serum/plasma lncRNA marker related to stomach cancer | |
CN107523647A (en) | Detect the LncRNA combinations of early stage cancer of the esophagus prognosis situation and the kit containing the combination | |
CN108660215B (en) | Application of reagent for detecting circMAN1A2 and circRNF13 and kit | |
CN109609634B (en) | Circulating miRNA marker related to endometrial cancer auxiliary diagnosis and application thereof | |
CN110628908B (en) | Biomarker and detection kit for prostate cancer diagnosis grading and benign and malignant prediction | |
Malik et al. | Comprehensive evaluation of microRNA as a biomarker for the diagnosis of hepatocellular carcinoma | |
CN105925703A (en) | Method for screening miRNA markers in PB (peripheral blood) of kidney cancer and kidney cancer diagnosis marker miR-210 | |
CN113528672A (en) | Biomarker combination for early screening of bladder cancer, kit and application | |
CN106119347B (en) | The primer and kit of colorectal cancer transfer detection based on serum exosomal microRNAs | |
CN111118164A (en) | Marker, kit and detection method for early screening and diagnosis of tumor | |
CN108103199B (en) | Circulating miRNA marker related to ovarian cancer auxiliary diagnosis and application thereof | |
CN108660213B (en) | Application of reagent for detecting three non-coding RNAs and kit | |
KR20230124671A (en) | Biomarker signature(s) for prevention and early detection of gastric cancer | |
CN111154880B (en) | Bladder cancer body fluid biopsy biomarker and application thereof | |
CN108034702A (en) | The expression of detection fusion gene M LL/CBP and the other oligonucleotides of pattern of fusion and application | |
CN109022586B (en) | Plasma miRNA marker related to cervical cancer auxiliary diagnosis and application thereof | |
CN113789379A (en) | Diagnostic kit for detecting prostate cancer, detection method and application thereof | |
CN108823308B (en) | Application of reagent for detecting circMAN1A2 and LOC284454 and kit | |
CN107858425A (en) | Applications and detection method of the miRNA 4741 as primary hepatic carcinoma diagnosis mark | |
CN111500733A (en) | Molecular marker for early diagnosis of non-small cell lung cancer in peripheral blood mononuclear cells | |
Sokoll et al. | A prospective, multicenter, NCI EDRN study of [-2] proPSA: improving prostate cancer detection and correlating with cancer aggressiveness | |
WO2019095541A1 (en) | Composition and method for diagnosing and predicting breast cancer bone metastases |
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 | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200508 |