CN111118164A

CN111118164A - A marker, kit and detection method for early screening and diagnosis of tumor

Info

Publication number: CN111118164A
Application number: CN202010134189.9A
Authority: CN
Inventors: 刘代顺; 浦盛蓝; 叶萌; 黄婷; 李才弘; 刘娅; 刘长路
Original assignee: Zunyi First Peoples Hospital
Current assignee: Zunyi First Peoples Hospital
Priority date: 2020-03-02
Filing date: 2020-03-02
Publication date: 2020-05-08

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

Marker, kit and detection method for early screening and diagnosis of tumor

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:

(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.

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<120> marker, kit and detection method for early screening and diagnosis of tumor

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Claims

1. a marker for early screening and diagnosis of tumors, characterized in that: the marker is a tryptophan 5-monooxygenase activating protein, and the tryptophan 5-monooxygenase activating protein It is the expression product of YWHAZ gene mRNA.

2. a kind of marker for early screening and diagnosis of tumor according to claim 1, is characterized in that: the primer of described YWHAZ gene has upstream primer and downstream primer, and upstream primer sequence is SEQ ID NO.3, The downstream primer sequence is SEQ ID NO.4.

3. A kit for early screening and diagnosis of tumors, characterized in that: comprising the markers for early screening and diagnosis of tumors as described in any one of claims 1 or 2, and containing specific A primer for detection, the primer is used to detect the expression level of YWHAZ gene mRNA in the sample.

4. a kind of test kit for tumor early screening and diagnosis according to claim 3, is characterized in that: described primer is internal reference with β-actin gene, detects YWHAZ gene mRNA expression level, namely YWHAZ gene mRNA expression The ΔCT value of the β-actin gene has an upstream primer and a downstream primer, the upstream primer sequence is SEQ ID NO.1, and the downstream primer sequence 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, including benign tumors and malignant tumors. 6 .

6. A detection method using the marker for early screening and diagnosis of tumors as claimed in any one of claims 1 or 2, characterized in that: comprising the following steps:

(1) Select patients according to the exclusion criteria and sign the informed consent form. Selection criteria for malignant pleural effusion: cancer cells are found in pleural effusion or cancer cells are found in pathological tissue. Selection criteria for benign pleural effusion: tuberculosis or cardiomyopathy Pleural effusion due to failure or hypoalbuminemia;

(2) In the case of sterility requirements, collect 10ml/person of pleural effusion from patients and put it in a 15ml de-enzyme centrifuge tube. Put the 15ml de-enzyme centrifuge tube containing the specimen, centrifuge in a low-temperature ultracentrifuge, and centrifuge at 4°C, 1000rpm, and centrifuge for 10min;

(3) After centrifugation, take the precipitate into a 1.5ml centrifuge tube, add Trizol solution at a ratio of 1ml per 0.1ml of pleural effusion, pipette, mix, and place at room temperature for 5min;

(4) Chloroform was added at a ratio of 0.2ml per 1ml of Trizol solution, and vigorously shaken for 15s, and left standstill for 2min;

(5) Take the upper water phase in a new centrifuge tube, add isopropanol at a rate of 0.5ml per 1ml of Trizol solution, place at room temperature for 10min, and then centrifuge at 12000rpm for 10min at 4°C;

(6) Discard the supernatant, add 75% ethanol at a ratio of 1 ml per 1 ml of Trizol solution, vortex to mix, and then centrifuge at 4°C, 7500 rpm for 5 min;

(7) Carefully discard the supernatant, then dry at room temperature or vacuum for 5-10 min, and then add DEPC water to dissolve the precipitate;

(8) Pure concentration detection: Take 2ul RNA samples and measure the absorbance value on the NANODROP LITE nucleic acid protein detector, that is, the OD value, and the OD260/280 ratio is all within the range of 1.8-2.0;

(9) Reverse transcription experiment: add samples according to the reverse transcription experiment, and after adding the samples, perform the reverse transcription experiment according to: 37°C, 15min→85°C, 5s→4°C, ∞, to obtain the cDNA required for the subsequent PCR amplification experiment;

(10) PCR amplification experiment: add samples according to the PCR amplification experiment, and after adding the samples, follow the following steps: 94°C, 1min→94°C, 30s→56°C, 30s→72°C, and cycle 40 times under this process condition , and then carry out the PCR amplification experiment according to: 72°C, 10min→4°C, ∞;

(11) To analyze the receiver operating characteristic curve, diagnostic index and confidence interval of YWHAZ gene in pleural effusion, to determine the relative expression of YWHAZ gene mRNA in pleural effusion, and to screen and diagnose the type of tumor by relative expression. .

7. detection method according to claim 6, is characterized in that: in described step (11) screening and diagnosing tumor process by relative expression amount, concrete criterion is, the mRNA expression of YWHAZ gene in pleural effusion When the ΔCT value is in the range of 4±0.47, it can be considered as malignant pleural effusion. The relative expression of YWHAZ gene mRNA in malignant pleural effusion is 1.641±0.635 times higher than that in benign pleural effusion, and P <0.05 is statistically significant; and the mRNA expression of YWHAZ gene in malignant pleural effusion of stage III/IV lung cancer is 14.450±0.859 times higher than that in malignant pleural effusion of stage I/II lung cancer, and P<0.05 was statistically significant.