CN110042162B - Marker for early diagnosis and metastasis early warning of lung cancer and kit prepared from marker - Google Patents
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Abstract
The invention relates to a marker for early lung cancer diagnosis and distant metastasis prediction based on platelet LncRNA-STON2 and a kit prepared from the marker, and belongs to the field of medical treatment and health. The marker is selected from plasma platelets LncRNA-STON 2; the kit for preparing the marker comprises: a reverse transcription reaction system, a fluorescent quantitative PCR reaction system and an internal reference system; compared with healthy donors, the expression level of LncRNA-STON2 in plasma platelets of lung cancer patients is remarkably increased for the first time, and ROC curve analysis shows that AUC is 0.613, sensitivity is 61.5% and specificity is 59.9%. LncRNA-STON2 expression was significantly elevated in early lung cancer patients compared to healthy donors. ROC curve analysis showed AUC of 0.713, sensitivity of 69.2, and specificity of 71.9%.
Description
Technical Field
The invention relates to a marker for early lung cancer diagnosis and distant metastasis prediction based on platelet LncRNA-STON2 and a kit prepared from the marker, and belongs to the field of medical treatment and health.
Background
The lung cancer is a malignant tumor with the highest mortality and morbidity in all countries in the world, has hidden onset and lacks specificity in early clinical manifestation, most patients are in local late stage or have distant metastasis when diagnosed, the prognosis is poor, and the 5-year survival rate is only 2%. Therefore, there is an urgent need for a more sensitive diagnostic marker, which provides objective basis for early diagnosis, early treatment and prognosis of lung cancer with distant metastasis.
Platelets are anucleated blood cells 2-4 μm in diameter, derived from megakaryocytes in the bone marrow, with a short half-life of only 7-10 days, second in number to the red blood cell line in the human blood circulation. In addition to its hemostatic effect, platelets also play an important role in tumor progression. Firstly, the platelet can produce angiopoietin-1 and the like, promote the proliferation of vascular endothelial cells and the generation of new blood vessels, thereby providing a proper microenvironment for the implantation and growth of tumor cells; at the same time, tumor cells can also activate circulating resting platelets, which can reduce local tumor cell apoptosis and induce epithelial-mesenchymal transition (EMT) in tumor cells through direct physical action and release of TGF- β. Second, activated platelets act as a bridge for tumor cell adhesion to vascular endothelial cells, mediating tumor cell adhesion to and across the vessel wall into the blood circulation. In blood, Circulating Tumor Cells (CTC) cause platelet aggregation to form a platelet-tumor cell complex, which not only helps tumor cells to shield mechanical injury caused by blood stream splicing force, but also can protect tumor cells from escaping from the attack of immune system, so that platelets participate in the whole process of tumor occurrence and development. The circulating platelet interacts with tumor cells in the whole life cycle to obtain tumor-related biomolecules; on the other hand, the protein, nucleic acid, vesicle, particle and the like which are free in circulation can be continuously taken up and enriched. As such, platelets, as educated, have significantly altered proteomic and RNA expression profiles and are therefore referred to as "tumor-educated platelets (TEPs)"; meanwhile, thanks to the closed membrane structure, the platelet can isolate the bioactive molecules in circulation, and the obtained biological information related to the tumor can be completely stored, so that the platelet becomes a large amount of concentrated biological repositories of the bioactive molecules from the tumor. For these reasons, platelet-carried substances have great potential as novel tumor biomarkers.
The existing clinical diagnosis technologies such as X-ray, B-ultrasonic and the like can not be found in early clinical stage, but the examination of CT, MRI, PET and the like can find smaller tumor tissues, but the specificity is low, and the diagnosis can be clearly determined by the aid of pathological biopsy. The use of endoscopes such as bronchofiberscopes can improve the diagnosis rate of non-small cell lung cancer, but all the endoscopes are interventional examination, have great damage and pain to patients, have high technical requirements on operators, can not take biopsy from all parts in the lung, and are not suitable for large-scale crowd screening and general investigation. The liquid biopsy is used as a novel detection means, has the advantages of high specificity, no wound, rapidness and the like, and is the trend and direction of the development of cancer examination.
Disclosure of Invention
In order to solve the problems and the current situation, the invention provides a kit for early diagnosis of lung cancer, which is convenient and quick to use and has high specificity.
A marker for early diagnosis of lung cancer and early warning of lung cancer metastasis, wherein the marker is selected from plasma platelets LncRNA-STON 2; the LncRNA-STON2 has the sequence shown in SEQ No. 1.
The reagent kit for early diagnosis of lung cancer and early warning of lung cancer metastasis prepared by adopting the marker comprises: a reverse transcription reaction system, a fluorescent quantitative PCR reaction system and an internal reference system;
the fluorescent quantitative PCR reaction system comprises: SYBR Green mixed liquor, a forward primer, a reverse primer and pure water;
the forward primer sequence of the fluorescent quantitative PCR reaction system is shown as SEQ No. 2;
the reverse primer sequence of the fluorescent quantitative PCR reaction system is shown as SEQ No. 3.
The SYBR Green mixed solution comprises: PCR buffer solution, SYBR Green I dye, ROX, dNTPs and Mg2+。
The internal reference system comprises a forward primer and a reverse primer of the internal reference ACTB.
The sequence of the forward primer of the internal reference ACTB is shown as SEQ No. 4;
the reverse primer sequence of the internal reference ACTB is shown as SEQ No. 5.
The reverse transcription reaction system comprises polyadenylic acid polymerase, reverse transcriptase mixed liquor, reverse transcription buffer solution and double distilled water without nuclease.
The preparation method of the kit comprises the following steps:
(1) extracting platelet RNA;
(2) extracting total RNA;
(3) genomic DNA removal reaction
Reaction system: 2. mu.L of 5 XgDNA Eraser Buffer, 1. mu.L of gDNA Eraser, 7. mu.L of TotalRNA. Reaction conditions are as follows: keeping at 42 ℃ for 2 minutes or at room temperature for 5 minutes to 30 minutes and keeping at 4 ℃;
(4) reverse transcription reaction
Reaction system: 10. mu.L of the reaction solution of step 1, 1. mu.L of PrimeScript RT Enzyme Mix I, 1. mu.L of RT Primer Mix, 4. mu.L of 5 XPrimeScript Buffer 2(for Real Time), 4. mu.L of RNase Free dH 2O; reaction conditions are as follows: 15 minutes at 37 ℃, 5 seconds at 85 ℃ and keeping at 4 ℃;
(5) preparation before fluorescent quantitative PCR amplification
Preparing a primer solution: centrifuging at 4000rpm for 30-60 s before opening the cover; then slowly opening the cover of the tube, adding a proper amount of ultrapure filtered water for dissolving to ensure that the final concentration of the ultrapure filtered water is 10 mu M; after covering the cover, fully shaking and uniformly mixing;
(6) fluorescent quantitative PCR amplification reaction
An amplification system: 12.5. mu.L SYBR Green Master Mix fluorescent dye, 0.5. mu.L forward primer, 0.5. mu.L reverse primer, 1. mu.L cDNA solution, 10.5. mu.L ribozyme-free water; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 minutes, denaturation at 95 ℃ for 15 seconds, renaturation at 60 ℃ for 1 minute, and detection of the dissolution curve at 97 ℃ for 1 minute, 65 ℃ for 2 minutes and 97 ℃ for 40 cycles;
(7) reaction of internal reference system
Taking ACTB gene as an internal reference gene; the specific reaction system is as follows: 2.5. mu.L SYBR Green Master Mix fluorescent dye, 0.5. mu.L forward primer, 0.5. mu.L reverse primer, 1. mu.L cDNA solution, 10.5. mu.L ribozyme-free water; reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 minutes, denaturation at 95 ℃ for 15 seconds, renaturation at 60 ℃ for 1 minute, and detection of the dissolution curve at 97 ℃ for 1 minute, 65 ℃ for 2 minutes and 97 ℃ for 40 cycles;
(8) data analysis
Drawing a fluorescence amplification curve and a dissolution curve to obtain a Ct value, and analyzing a fluorescence quantitative PCR result by using a delta Ct method, wherein the delta Ct value represents the relative expression quantity of the target RNA, and the smaller the delta Ct value is, the stronger the target RNA expression is;
Δ Ct is the target RNA Ct value — internal reference RNA Ct value.
The kit is applied to early diagnosis of lung cancer and early warning of lung cancer metastasis.
The invention has the beneficial effects
1. The expression level of LncRNA-STON2 in plasma platelets of lung cancer patients is remarkably increased
Compared with healthy donors, the expression level of LncRNA-STON2 in plasma platelets of lung cancer patients is obviously increased for the first time, and ROC curve analysis shows that AUC is 0.613, sensitivity is 61.5% and specificity is 59.9%.
2. A significant increase in LncRNA-STON2 expression in plasma platelets in early lung cancer patients compared to healthy donors, a significant increase in LncRNA-STON2 expression in early lung cancer patients. ROC curve analysis showed AUC of 0.713, sensitivity of 69.2, and specificity of 71.9%.
Expression level of LncRNA-STON2 in distant metastatic lung cancer patients was lower than that in non-metastatic lung cancer patients.
LncRNA-STON2 expression is reduced in patients with distant metastatic lung cancer compared to patients with non-distant metastatic lung cancer.
Expression level of LncRNA-STON2 in lymph node metastatic lung cancer patients is lower than that in non-lymph node metastatic lung cancer patients.
LncRNA-STON2 expression was reduced in lymph node metastatic lung cancer patients compared to non-lymph node metastatic lung cancer patients.
Drawings
FIG. 1 expression levels of plasma platelets LncRNA-STON2 in lung cancer patients;
FIG. 2 expression level of LncRNA-STON2 in early lung cancer;
FIG. 3 ROC curve of LncRNA-STON2 diagnosis of lung cancer patients;
FIG. 4 ROC curve of LncRNA-STON2 diagnosis of early lung cancer patients;
FIG. 5 is a graph of the expression levels of platelets LncRNA-STON2 in plasma for distant metastasis of lung cancer;
FIG. 6 is a graph showing the expression level of platelets LncRNA-STON2 in plasma in lymph nodes metastasizing from lung cancer.
Detailed Description
The following examples are intended to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the practice are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The implementation case is as follows: application of LncRNA-STON2 in diagnosis of early lung cancer
1. Design of experiments
The experiment is designed to collect plasma samples of patients diagnosed as primary treatment of lung cancer and healthy volunteers in Shandong province tumor hospitals, separate and enrich platelets and obtain platelet RNA. The expression of LncRNA-STON2 was detected by qPCR method, and its expression was statistically analyzed for differences between healthy volunteers and lung cancer, and its diagnostic efficacy was analyzed.
2. Patients and samples involved in the experiment
130 healthy controls and 172 lung cancer patients admitted to the eastern affiliated eastern tumor hospital of the university of eastern Shandong, collected from 1 month to 12 months of 2017 and subjected to TNM classification based on postoperative pathology examination. Clinical data included in this study included mainly age, sex, AJCC tumor stage (8 th edition), alcohol consumption, history of smoking, and pathological type of lung cancer. The number of patients with early stage lung cancer is 32, and the number of healthy controls is 130. All patients received fiberbronchoscopy, percutaneous aspiration or surgical biopsy. About 2ml of peripheral blood sample of a subject is collected before chemotherapy, radiotherapy and surgical treatment, placed in a vacuum tube containing EDTA anticoagulant, mixed evenly, centrifuged at 120g for 10 minutes at room temperature, and the supernatant is taken and repeated twice. Then 360g, 20 minutes. Platelet samples were then collected in centrifuge tubes and stored at-80 ℃ until use.
3. Determination of the level of platelets LncRNA-STON2 in plasma
The expression level of platelet LncRNA-STON2 in plasma of lung cancer patients and healthy volunteers was analyzed using qPCR method, as follows:
3.1 extraction of platelet RNA
Centrifuging 120g of two milliliters of blood added with EDTA anticoagulant for 10 minutes conventionally, sucking the upper plasma into an EP tube, centrifuging 120g for 10 minutes again, sucking the upper plasma into a new EP tube, centrifuging 360g for 20 minutes, discarding the upper plasma, retaining platelet sediment, and storing the sediment at-80 ℃ for later use.
3.2 extraction of Total RNA
Adding 0.5ml of TRIzol Reagent into platelet sediment obtained by a low-speed centrifugation method, repeatedly beating the platelet sediment and placing the platelet sediment for 10 to 20 minutes to ensure that the platelets are fully cracked; adding 100 mu L of trichloromethane, shaking and mixing evenly, and incubating for 10 minutes. Centrifuge at 12000g for 15 min at 4 deg.C (the mixture separates into a lower red phenol-chloroform liquid phase, a white floc in the middle layer, a colorless aqueous phase in the upper layer, and RNA distributed in the aqueous phase). Transfer the upper aqueous phase to a fresh RNase-free EP tube with a pipette (take care not to suck in the middle layer). After adding 250. mu.L of isopropanol and incubating for 10 minutes, centrifugation was carried out at 12000g at 4 ℃ for 10 minutes to allow white RNA precipitates to appear, and the supernatant liquid was removed. Adding 750 μ L75% ethanol, shaking, mixing, centrifuging at 4 deg.C 7500g for 5 min, washing RNA, and removing upper layer liquid. Air-drying the RNA tube for 5-10 minutes, adding 15-20 mu L of RNase-free water to dissolve the RNA, repeatedly blowing and beating by using a pipette, and incubating in a water bath at 55-60 ℃ for 5-10 minutes to fully dissolve the RNA. The RNA samples were stored at-80 ℃ until use. The ASP-3700 analysis software was used to examine sample concentration and purity.
3.3 reverse transcription reaction and qPCR
(1) Genomic DNA removal reaction: reaction system: mu.L of 5 XgDNA Eraser Buffer, 1. mu.L gDNA Eraser, 7. mu.L TotalRNA. Reaction conditions are as follows: held at 42 ℃ for 2 minutes (or room temperature for 5 minutes to 30 minutes) and 4 ℃.
(2) Reverse transcription reaction: reaction system: mu.L of the reaction mixture from step (1), 1. mu.L of PrimeScript RT Enzyme Mix I, 1. mu.L of RT Primer Mix, 4. mu.L of 5 XPimeScript Buffer 2(for Real Time), 4. mu.L of RNase Free dH 2O. Reaction conditions are as follows: 15 minutes at 37 ℃, 5 seconds at 85 ℃ and 4 ℃. According to the sample quantity, the mixed reaction solution is prepared according to the reaction system shown in the table below, and is shaken and uniformly mixed.
TABLE 1 reverse transcription reaction System
In the biological safety cabinet, the prepared reaction solution is respectively added into the holes of the corresponding PCR amplification plates, attention is paid to avoid pollution, and then a transparent film is attached. Note that 1 secondary well was made for each sample and the experiment was repeated 3 times. Carefully centrifuge and mix well. The reaction was carried out on the computer, using a LightCycler 480 quantitative PCR instrument for detection, according to the procedure set forth in the following table.
TABLE 2 PCR reaction conditions
3.4 reaction of internal reference System
Taking an ACTB gene as an internal reference gene; the specific reaction system is as follows: 2.5. mu.L SYBR Green Master Mix fluorescent dye, 0.5. mu.L forward primer, 0.5. mu.L reverse primer, 1. mu.L cDNA solution, 10.5. mu.L ribozyme-free water; reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 minutes, denaturation at 95 ℃ for 15 seconds, renaturation at 60 ℃ for 1 minute, and detection of the dissolution curve at 97 ℃ for 1 minute, 65 ℃ for 2 minutes and 97 ℃ for 40 cycles;
4. mathematical statistical analysis
Statistical analysis was performed using SPSS 22.0 and GraphPad Prism 6.0 software. Comparing the measured data, if the measured data obeys normal distribution, using parameter test and expressing the measured data as the mean +/-standard deviation; if not, non-parametric tests were used to test for Mann-Whitney and expressed as median, interquartile range. The comparison between the three groups used DKruskoal-Wallis H test. Diagnostic combinations were established using binary logistic regression, and differentially expressed LncRNA-STON2 was determined by the receiver operating characteristic curve (ROC curve) and the area under the curve (AUC). The difference is statistically significant when p is less than 0.05.
5. Summary of case characteristics of lung cancer patients and relationship to LncRNA-STON2 expression levels
The results in Table 3 analyzed the clinical characteristics of lung cancer patients, including age, gender, smoking and drinking status, histological type and tumor size. LncRNA-STON 2-expressing water was independent of age, sex, smoking, alcohol consumption, tumor size, metastasis and histological type.
TABLE 3 summary of lung cancer case characteristics and relationship to LncRNA-STON2 expression levels
6. Plasma platelet LncRNA-STON2 level difference between healthy volunteers and lung cancer patients
The LncRNA-STON2 levels of plasma platelets from lung cancer patients and healthy donors were quantified using qPCR to determine whether LncRNA-STON2 could be a biomarker for diagnosing lung cancer. The results showed that the expression level of LncRNA-STON2 was significantly increased in all lung cancer patients, and the difference was statistically significant (p ═ 0.0008), as shown in fig. 1. Similarly, the level of LncRNA-STON2 in plasma platelets from early lung cancer patients and healthy donors was quantified using qPCR to determine whether LncRNA-STON2 could be used as a biomarker for diagnosing early lung cancer. The results showed that the expression level of LncRNA-STON2 was significantly increased in all patients with early lung cancer, and the difference was statistically significant (p ═ 0.0001) as shown in fig. 2.
7. ROC curve analysis of diagnostic efficacy of plasma platelets LncRNA-STON2 on Lung cancer
The invention further utilizes ROC curve analysis to detect the diagnosis efficiency of the LncRNA-STON2 on the lung cancer, and the ROC curve analysis shows that the AUC is 0.613, the sensitivity is 61.5 percent and the specificity is 59.9 percent. See fig. 3.
8. Diagnostic efficacy of plasma platelets LncRNA-STON2 on early lung cancer
The invention further utilizes ROC curve analysis to detect the diagnosis efficiency of the LncRNA-STON2 on early lung cancer, and the ROC curve analysis shows that the AUC is 0.713, the sensitivity is 69.2 percent and the specificity is 71.9 percent. See fig. 4.
9. Differential levels of plasma platelets LncRNA-STON2 in distant metastases and non-metastases of lung cancer patients
The qPCR assay results showed that the plasma platelet LncRNA expression levels were reduced in distant metastatic lung cancer patients compared to non-distant metastatic lung cancer patients, with statistical differences (P <0.0376), see figure 5.
10. Differential levels of plasma platelets LncRNA-STON2 in lymph node metastatic and non-metastatic lung cancer patients
The qPCR results showed that the expression level of plasma platelet LncRNA was decreased in lymph node metastatic lung cancer patients compared with non lymph node metastatic lung cancer patients, and the difference was statistically significant (P <0.0366), as shown in FIG. 6.
Sequence listing
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Claims (1)
1. The application of the kit in the preparation of products for early diagnosis of lung cancer and early warning of lymph node metastasis is characterized in that the kit comprises: a reverse transcription reaction system, a fluorescent quantitative PCR reaction system and an internal reference system; the fluorescent quantitative PCR reaction system comprises a forward primer and a reverse primer aiming at plasma platelet LncRNA-STON 2;
the sequence of the LncRNA-STON2 is shown as SEQ No. 1;
the fluorescent quantitative PCR reaction system comprises: SYBR Green mixed liquor, a forward primer, a reverse primer and pure water;
the forward primer sequence of the fluorescent quantitative PCR reaction system is shown as SEQ No. 2;
the reverse primer sequence of the fluorescent quantitative PCR reaction system is shown as SEQ No. 3;
the SYBR Green mixed solution comprises: PCR buffer solution, SYBR Green I dye, ROX, dNTPs and Mg2+；
The internal reference system comprises a forward primer and a reverse primer of the internal reference ACTB;
the sequence of the forward primer of the internal reference ACTB is shown as SEQ No. 4;
the reverse primer sequence of the internal reference ACTB is shown as SEQ No. 5;
the reverse transcription reaction system comprises polyadenylic acid polymerase, reverse transcriptase mixed liquor, reverse transcription buffer solution and double distilled water without nuclease.
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