CN111500733B - Molecular marker for early diagnosis of non-small cell lung cancer in peripheral blood mononuclear cells - Google Patents
Molecular marker for early diagnosis of non-small cell lung cancer in peripheral blood mononuclear cells Download PDFInfo
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Abstract
The invention discloses a molecular marker for early diagnosis of non-small cell lung cancer in peripheral blood mononuclear cells. The invention provides an application of a substance for detecting the expression level of a CD226 gene in PBMC of a person to be detected, a substance for detecting the expression level of a CD247 gene in PBMC of the person to be detected and a substance for detecting the smoking quantity condition of the person to be detected in the preparation of products with the functions of 1) and/or 2): 1) detecting or assisting to detect whether the patient to be detected has the non-small cell lung cancer; 2) distinguishing or assisting in distinguishing whether the patient to be detected is a patient with non-small cell lung cancer or benign nodules of lung; the smoking amount condition is whether the smoking amount is more than 40 bags per year. The experiment of the invention proves that the diagnosis combination consisting of CD226, CD247 and the smoking amount (more than 40 bags per year) of the patient can effectively distinguish the non-small cell lung cancer patient and the lung benign nodule patient, and is expected to become a molecular marker for early diagnosis of the non-small cell lung cancer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a molecular marker for early diagnosis of non-small cell lung cancer in peripheral blood mononuclear cells.
Background
The global cancer statistics in 2018 show that lung cancer is still the first malignancy with morbidity (11.6%) and mortality (18.4%). As the industrial development process is accelerated, the air pollution is increased and the number of smokers is increased, the incidence rate and the death rate of the lung cancer are increased year by year, and the situation is very severe. In all cases, Non-Small Cell Lung Cancer (NSCLC) is present in approximately 85% of all Lung Cancer cases, and mainly includes squamous carcinoma, adenocarcinoma, large Cell carcinoma, etc. The non-small cell lung cancer is high in malignancy, the 5-year survival rate of patients is low, and 70% of patients belong to advanced lung cancer at the initial diagnosis. The lung cancer is also the most common malignant tumor in China, and because the population base is large, the number of new cases and death cases of the lung cancer in China is far higher than that of other countries, and the disease burden is always very heavy. The incidence of lung cancer in the united states has continued to decline over the last few years, primarily due to multi-stage lung cancer prevention and screening programs. The current main screening means is low-dose spiral CT, but the defects of radiation exposure, anxiety caused by patients and high cost exist, and the screening result is high in false positive, so that over-diagnosis is caused. The invasive technique based on tissue biopsy can cause adverse reactions such as pneumothorax, hemorrhage and the like, and the false negative result is easily generated due to the limitation of tissue biopsy material taking and the heterogeneity of tumors. Therefore, the discovery of early tumor markers and the realization of preoperative noninvasive early diagnosis are imminent.
Liquid biopsy taking body fluid as a detection object is a novel noninvasive molecular pathology detection method, and comprehensive information of tumor gene or protein expression in a patient body is obtained by detecting marker information released by tumor cells into the body fluid. The main test items for liquid biopsy include: tumor marker molecules, blood cells, Circulating Tumor Cells (CTCs), Circulating Tumor DNA (ctDNA), and exosomes (exosomes). The liquid biopsy specimen mainly comprises blood, urine, saliva, milk, pleural fluid, cerebrospinal fluid and the like. Because the sample is body fluid, the material taking way is simple and convenient, the sampling and analysis at any time can be realized, the characteristics of no hysteresis and good repeatability are realized, and a plurality of short plates for histopathological examination are made up.
Although circulating extracellular gene assays show promise for early detection of cancer, these assays have several drawbacks that limit their clinical utility. Including (1) low recovery of mirnas, mrnas, etc. in plasma or serum; (2) after hemolyzing blood cells such as erythrocytes, a large amount of nucleic acid can be released into blood plasma or blood serum, and a non-specific result is generated; (3) the source of variability of circulating extracellular plasma genes may lead to inconsistent or even inconsistent diagnostic results for the same type of cancer between reports. Since miRNAs are associated with proteins (e.g., argonaute, 3 lipoproteins, etc.) in the blood, the problem becomes more complicated or contained in cell fragments designated as exosomes, microparticles, microvesicles or extracellular vesicles. Furthermore, from 2008 to 2013, a total of 154 circulating extracellular miRNA expression profiles were identified among 26 different types of tumors. However, no single diagnostic feature appears between the various reports. Despite the diagnostic efficacy of existing non-small cell lung cancer diagnostic markers, several tasks remain to be solved, additional external validation is required to determine a standard collection protocol and confirm gene signatures and their accuracy, a larger scale prospective cohort study of lung nodule patients is required to more fully determine the effects of potential confounding effects or diseases, and to assess the overall clinical feasibility and utility of this approach.
Tumor cells, after they are recognized by the immune system, may evade immune surveillance by developing resistance to T cell-induced apoptosis or local expression of immune regulatory molecules and cytokines. In addition, suppression and evasion of the immune system may occur as early as in premalignant events in the development and progression of cancer. Peripheral Blood Mononuclear Cells (PBMCs) are the first line of defense of the immune system against cancer and consist of monocytes, T cells, B cells and natural killer cells. Once cancer immunogenicity or immune evasion has occurred, differences in the expression of certain molecules in PBMCs may occur, and such changes may precede pre-neoplastic lesions. It has been shown that differential molecular expression of PBMC is found in many types of patients with early stage tumors, and this differential change is independent of substantial tumor burden, and can avoid the disadvantages of some current extracellular biomarkers, their expression is involved in the recognition of cancer by immune system and the escape of tumor cells from immune system, and the immune escape of cancer and tumor cells recognized by immune system occurs in early stage tumor, therefore, their differential expression can reflect the state of early stage tumor, and the molecular change of PBMC can be determined to be the replacement window of the state of cancer development. Furthermore, the analysis of biomarkers in PBMCs can overcome some of the obstacles of body fluid based detection, as it is a cell based and easy to implement detection method. PBMC can generate a large amount of high-quality RNA, the expression of genes can be reliably determined by qRT-PCR, so that the differential molecular expression in the PBMC can be used as a molecular marker which theoretically has higher diagnostic value, and the RNA differential expression profile in the PBMC can provide a novel biomarker for early discovery of malignant tumors.
Therefore, the PBMC can be used as an ideal model for screening molecular markers for early diagnosis of lung cancer in blood.
Disclosure of Invention
An object of the present invention is to provide a substance for detecting the expression level of a CD226 gene in PBMC of a specimen, a substance for detecting the expression level of a CD247 gene in PBMC of the specimen, and the use of a substance for detecting the smoking amount of the specimen.
The invention provides an application of a substance for detecting the expression level of a CD226 gene in PBMC of a person to be detected, a substance for detecting the expression level of a CD247 gene in PBMC of the person to be detected and a substance for detecting the smoking quantity condition of the person to be detected in the preparation of a product with at least one function of 1) to 3):
1) detecting or assisting to detect whether the patient to be detected has the non-small cell lung cancer;
2) diagnosing or assisting to diagnose whether the patient to be detected has the non-small cell lung cancer;
3) distinguishing or assisting in distinguishing whether the patient to be detected is a patient with non-small cell lung cancer or benign nodules of lung;
the smoking amount condition is whether the smoking amount is more than 40 bags per year.
Another object of the present invention is to provide a substance for detecting the expression level of CD226 gene in PBMC of a subject and use of the substance for detecting the expression level of CD247 gene in PBMC of the subject.
The invention provides a substance for detecting the expression level of CD226 gene in PBMC of a person to be detected and application of the substance for detecting the expression level of CD247 gene in PBMC of the person to be detected in preparation of a product with at least one function of 1) -3):
1) detecting or assisting to detect whether the patient to be detected has the non-small cell lung cancer;
2) diagnosing or assisting to diagnose whether the patient to be detected has the non-small cell lung cancer;
3) distinguishing or assisting in distinguishing whether the patient to be detected is a patient with non-small cell lung cancer or benign nodules of lung;
the invention also provides a substance for detecting the expression level of CD226 gene in PBMC of a person to be detected, a substance for detecting the expression level of CD247 gene in PBMC of the person to be detected, a substance for detecting the smoking quantity of the person to be detected and application of the readable vector B described in formula 2 in preparation of a product with at least one function of 1) to 3):
1) detecting or assisting to detect whether the patient to be detected has the non-small cell lung cancer;
2) diagnosing or assisting to diagnose whether the patient to be detected has the non-small cell lung cancer;
3) distinguishing or assisting in distinguishing whether the patient to be detected is a patient with non-small cell lung cancer or benign nodules of lung;
the formula 2: p ═ exp (U)/[1+ exp (U) ], wherein U ═ 4.1047+2.6575 × n +3.6105 × log (CD226) -1.7212 × log (CD 247);
wherein, P is the detection value of the person to be detected, and log (CD226) is the log value of the CD226 gene expression (specifically relative expression) in PBMC of the person to be detected; log (CD247) is the log value of the expression level (specifically relative expression level) of the CD247 gene in the PBMC of the patient to be detected;
exp (U) is the exp value of U;
n is 1 or 0, if the smoking quantity of the person to be detected is more than 40 bags per year, n is 1; if the smoking quantity of the person to be detected is less than or equal to 40 bags per year, n is 0;
the invention also provides a substance for detecting the expression level of the CD226 gene in the PBMC of a person to be detected, a substance for detecting the expression level of the CD247 gene in the PBMC of the person to be detected and application of the readable vector A described in the formula 1 in preparing a product with at least one function of 1) to 3):
1) detecting or assisting to detect whether the patient to be detected has the non-small cell lung cancer;
2) diagnosing or assisting to diagnose whether the patient to be detected has the non-small cell lung cancer;
3) distinguishing or assisting in distinguishing whether the patient to be detected is a patient with non-small cell lung cancer or benign nodules of lung;
the formula 1 is P ═ exp (U)/[1+ exp (U) ], wherein U ═ 4.1198+2.8282 × log (CD226) -1.2212 × log (CD 247);
p is a detection value of a person to be detected, and log (CD226) is a log value of CD226 gene expression (specifically relative expression) in PBMC of the person to be detected; log (CD247) is the log value of the expression level (specifically relative expression level) of the CD247 gene in the PBMC of the patient to be detected;
exp (U) is the exp value of U.
In the application, the substance for detecting the expression quantity of the CD226 gene in the PBMC of the patient to be detected comprises a primer pair for amplifying the CD226 gene;
the substance for detecting the expression quantity of the CD247 gene in the PBMC of the patient to be detected comprises a primer pair for amplifying the CD247 gene.
A final object of the invention is to provide a product.
The product comprises a substance for detecting the expression level of the CD226 gene in PBMC of a subject and a substance for detecting the expression level of the CD247 gene in the PBMC of the subject;
the product has at least one of the following functions 1) to 3): 1) detecting or assisting to detect whether the patient to be detected has the non-small cell lung cancer; 2) diagnosing or assisting to diagnose whether the patient to be detected has the non-small cell lung cancer; 3) and distinguishing or assisting to distinguish whether the patient to be detected is the patient with non-small cell lung cancer or benign nodules of lung.
The product also comprises a substance for detecting the smoking amount of the person to be detected;
the smoking amount condition is whether the smoking amount is more than 40 bags per year.
The product also comprises a readable carrier B recording the formula 2 or a readable carrier A recording the formula 1;
the readable carrier B is also recorded with a judgment standard B:
if the P value of the to-be-detected person is greater than or equal to 0.8886, the to-be-detected person suffers from or is candidate to suffer from non-small cell lung cancer; or the subject is or is candidate for non-small cell lung cancer other than benign nodules of the lung;
if the P value of the subject to be tested is less than 0.8886, the subject to be tested does not suffer from or is not candidate for suffering from non-small cell lung cancer; or the subject is or is candidate for a benign nodule in the lung rather than a non-small cell lung cancer;
or the like, or, alternatively,
the readable carrier A is also recorded with a judgment standard A: and if the P value of the to-be-detected person is greater than or equal to 0.9043, the to-be-detected person suffers from or is candidate to suffer from non-small cell lung cancer, or the to-be-detected person is or is candidate to suffer from non-small cell lung cancer instead of benign nodules of the lung.
If the P value of the subject to be tested is less than 0.9043, the subject to be tested does not suffer from or is not candidate for suffering from non-small cell lung cancer; or the subject is or is candidate for a benign nodule in the lung rather than a non-small cell lung cancer.
The diagnosis is an early diagnosis.
The invention detects the differential expression of four genes related to the immune regulation, namely CD86, CD226, CD247 and ICAM-1, by qRT-PCR technology: the expression level of the gene in blood PBMC of the experimental group of patients (105 non-small cell lung cancer patients and 20 benign nodule lung patients) is detected, and the result shows that: the expression level of CD86(P ═ 0.0037), CD226(P ═ 0.0033), CD247(P ═ 0.0004) and ICAM-1(P ═ 0.0026) in PBMC of the patient with non-small cell lung cancer is significantly higher than that of the control group of benign tubercle patients; the expression levels of four genes in the patients in the validation group (101 cases of non-small cell lung cancer and 21 cases of benign nodules in lung) were detected, and the results showed that the expression levels in the PBMCs of non-small cell lung cancer were higher than those in the control group, namely CD86(P ═ 0.0085), CD226(P ═ 0.0107), CD247(P ═ 0.0028) and ICAM-1(P ═ 0.0008), consistent with those in the experimental group. By integrating the above results, the clinical diagnosis value of the diagnostic combination consisting of CD226, CD247 and the patient smoking amount (more than 40 bags/year) was found to be the highest by stepwise logistic regression analysis, the AUC value was 0.9079, the sensitivity was 0.7579 and the specificity was 0.9. The combination can effectively distinguish the non-small cell lung cancer patients and the lung benign nodule patients, and is expected to become a molecular marker for early diagnosis of the non-small cell lung cancer.
Drawings
FIG. 1 shows the results of qRT-PCR in the experimental group; note: p < 0.05, P < 0.01.
FIG. 2 is a ROC curve of the differentially expressed genes in the experimental group.
FIG. 3 shows the results of qRT-PCR assay in the validation set; note: p < 0.05, P < 0.01.
FIG. 4 is a ROC curve for the validation set of differentially expressed genes.
FIG. 5 is a ROC curve for the best diagnostic combination.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The materials and methods used in the following examples are as follows:
1. collection of samples
1.1 blood sample origin
The subject group cooperates with the thoracic surgery of the general hospital of the liberation military of Chinese people, all lung cancer patients and benign tuberculous pulmonary patients admitted to the hospital to be treated are selected as research objects, and the patients are fully informed and signed with informed consent before being admitted to the hospital to be treated. Then, a preoperative blood sample of a patient is collected, and the blood sample is separated into three parts of blood plasma, PBMC and RBC and is subpackaged into an RNase-free EP tube for freezing storage. The detailed medical record data of the patient including sex, age, smoking amount, smoking age, infectious virus infection, cancer history and other information is recorded and maintained during specimen collection.
1.2 sample inclusion criteria and grouping basis
According to the clinical data of the patients, all blood samples of patients with the past cancer history or infectious virus infection (such as infectious hepatitis, syphilis, HIV and the like) are removed. PBMC specimens from patients with non-small cell lung adenocarcinoma, non-small cell lung squamous carcinoma and benign nodules of the lung (including alveolar epithelial hyperplasia, chronic granuloma, fibroplasia, polyp, etc.) without any surgery, radiotherapy, chemotherapy or drug treatment were used as subjects and randomly divided into two groups: experimental and validation groups. And detecting and verifying the differential expression genes and the proteins.
2. qRT-PCR detection method in PBMC
2.1 isolation of mononuclear lymphocytes (PBMC)
1) Adding PBS into the blood sample after plasma separation to make up the blood sample to the original volume, and uniformly mixing;
2) slowly adding the uniformly mixed blood into a 15mL centrifuge tube which is prepared in advance and is filled with an equal volume of lymphocyte separation fluid along the tube wall;
3) centrifuging at room temperature for 15 minutes at 400 g;
4) after the centrifugation is finished, the solution in the tube is clearly divided into three layers. The upper layer is PBS solution, the middle is lymphocyte separation liquid, and the bottom of the tube is erythrocyte layer. A thin and compact white membrane is arranged between the upper layer and the middle layer, namely a PBMC layer. Carefully sucking the leucocyte layer cells into a 15mL centrifuge tube, adding PBS to the 15mL scale, and repeatedly reversing and uniformly mixing;
5) centrifuging at room temperature for 5 minutes at 400 g;
6) settling the centrifuged PBMC at the bottom of the tube, discarding the supernatant, adding 1mL of erythrocyte lysate to resuspend the cells, and transferring the cells to a 1.5mL centrifuge tube;
7) centrifuging at 2000rpm for 5 min at room temperature;
8) discarding the supernatant, adding 1mL of PBS and re-suspending the cells;
9) centrifuging at 2000rpm for 5 min at room temperature;
10) discarding the supernatant, and collecting the white precipitate as PBMC, and freezing and storing at-80 deg.C;
2.2 extraction and purification of Total RNA in PBMC
1) Taking a unfrozen PBMC sample, adding 1mL of Trizol reagent, and uniformly blowing and stirring until no obvious cell mass exists in the tube;
2) standing for 5 minutes at room temperature;
3) adding 0.2mL of chloroform, and carrying out vortex oscillation and uniform mixing for 15 seconds;
4) standing for 2-3 minutes at room temperature;
5) centrifuging at 12000 Xg for 15 min at 4 deg.C in a low-temperature high-speed centrifuge;
6) after centrifugation, the mixed liquid in the tube is divided into three obvious layers, wherein the upper layer is a colorless transparent water phase containing RNA, the middle layer is a white protein layer, and the lower layer is a red organic phase. Carefully pipette about 400. mu.L of the upper aqueous phase into a 1.5mL centrifuge tube;
7) adding equal volume of absolute ethyl alcohol, blowing, beating and uniformly mixing;
8) repeatedly inverting until the precipitate which may be generated after adding ethanol is fully dissolved;
9) adding 400 mu L of upper aqueous phase into a purification column with a collecting pipe;
10) centrifuging at 12000 Xg for 15 s at room temperature, discarding the filtrate in the collection tube, and putting the purification column into the collection tube again;
11) repeating the two steps once;
12) adding 350 μ L of Wash Buffer I on a purification column film, centrifuging at room temperature of 12000 Xg for 15 seconds, discarding the filtrate and the collection tube, and replacing the collection tube with a new one;
13) add 80. mu.L PureLinkDNase mix (Table 1) on the purification column membrane, incubated for 15 minutes at room temperature;
table 1 shows the composition table of PureLinkDNase mix
14) Adding 350 μ L of Wash Buffer I on the film, centrifuging at 12000 Xg for 15 s at room temperature, discarding liquid and collecting tube, and replacing with new collecting tube;
15) adding 500 mu L of Wash Buffer II containing ethanol into a centrifugal column;
16) centrifuging at 12000 Xg for 15 s at room temperature, and discarding the liquid in the collecting tube;
17) repeating the two steps once;
18) centrifuging at 12000 Xg for 1 min at room temperature to dry RNA on the cell, discarding the collection tube and replacing the recovery tube;
19) carefully add 30. mu.L RNase-Free-Water to the center of the purification column membrane;
20) standing for 1 minute at room temperature;
21) the RNA was collected at 12000 Xg at room temperature by centrifugation for 1 minute.
2.3 reverse transcription of PBMC RNA samples
1) Uniformly diluting the extracted PBMC RNA sample to a proper concentration;
2) preparing 2 × reverse transcription mixed solution (table 2), and standing on ice for later use;
TABLE 2 reverse transcription reaction System
3) After preparing the reverse transcription mixed solution, adding the mixed solution into 8 connecting tubes respectively, and then adding 15 mu L of RNA samples respectively to prepare a reaction system of 30 mu L. Slightly blowing, uniformly mixing, tightly covering, and centrifuging for a short time;
4) placed in a 96-well PCR instrument and set the reaction conditions as follows (table 3):
table 3 shows the reverse transcription PCR conditions
2.4qRT-PCR
2.4.1 primer design
Primers were designed for real-time fluorescent quantitative PCR based on the gene sequences of CD86, CD226, CD247, ICAM-1 and GAPDH, and the specific primer names and sequences are shown in Table 4:
table 4 shows the sequence information of the primers used in the PCR reaction
The sequence from top to bottom in column 2 of the above table is sequence 1-sequence 10.
2.4.2qRT-PCR reaction System and conditions
Adding appropriate amount of RNase-free-water according to the primer synthesis instruction to prepare 10 μ M primer solution, using THUNDERBIRD from TOYOBOTMThe SYBR qPCR Mix Without ROX kit was formulated with the following reaction system according to the instructions (table 5):
TABLE 5 qRT-PCR reaction System
Adding the prepared qPCR reaction mixed solution into a 96-hole PCR plate, adding 18 mu L of mixed solution into each reaction hole, adding 2 mu LcDNA to prepare a 20 mu L reaction system, and putting the reaction system into a real-time fluorescence quantitative PCR instrument for reaction, wherein the PCR reaction conditions are as follows (Table 6):
table 6 shows the conditions of qRT-PCR reaction
Each sample was plated with 3 duplicate wells using GAPDH as the reference gene. And after the qPCR reaction is finished, taking the average Ct value of three duplicate wells of the target gene in each sample as a final result.
The calculation formula of the relative expression quantity of each target gene is 2^ -delta Ct, wherein the delta Ct is Ct (target gene) -Ct (reference gene).
2.5 statistical analysis method
The statistical analysis of the qRT-PCR data is completed by using an SAS system, and the relative expression quantity of the target gene of each sample for statistical analysis is measured to be the average value of three biological repeated experiments. Adopting a two-tailed t test to meet normal distribution; if the distribution is not in accordance with the normal distribution, the difference between groups can be considered to have statistical significance by using Kruskal-Wallis nonparametric test if P is less than 0.05. Pearson correlation analysis is used for evaluating the relation between the expression quantity of the target gene in the plasma of a non-small cell lung cancer patient and a benign sarcoidosis patient of the lung and the demographic and clinical characteristics, AUC is used for evaluating the sensitivity and specificity of the target gene as a lung cancer diagnosis marker, and stepwise logistic regression analysis is used for constructing an optimal diagnosis combination model.
Example 1 discovery of molecular markers for early diagnosis of non-Small cell Lung cancer
1. Detection of expression levels of CD86, CD226, CD247 and ICAM-1 in PBMCs of experimental patients
1) Clinical data from study Subjects in the Experimental group
The study of this experimental group contained 105 patients with non-small cell lung cancer (NSCLC group) and 20 patients with BENIGN nodules in the lung (BENIGN group). The NSCLC group (91.4% stage I, 4.8% stage II, 2.9% stage III, 0% stage IV, 0.9% unknown) included 87 persons with adenocarcinoma (82.9%), 18 persons with squamous cell carcinoma (17.1%). Specific clinical information is shown in Table 7, and it can be seen that there is no difference in the age, sex and smoking amount between the NSCLC group and BENIGN group samples (P values are all greater than 0.05).
Table 7 is a summary of clinical data of the subjects of the experimental group
Note: the P value is calculated from the Chi-square test or Fisher's exact test for categorical variables and the T test or Wilcoxon rank sum test for continuous variables.
2) qRT-PCR detection result of gene expression quantity
The relative expression amounts (2^ -Delta Ct) of the four genes CD86, CD226, CD247 and ICAM-1 in blood PBMCs of 105 non-small cell lung cancer patients and 20 benign nodule patients in lung of an experimental group were detected by using qRT-PCR technology (the method and the primers are described above), and t-test of two independent samples was carried out.
As shown in fig. 1, CD86(P ═ 0.0037), CD226(P ═ 0.0033), CD247(P ═ 0.0004) and ICAM-1(P ═ 0.0026) were all tested to express higher than in the group of patients with benign nodules in the lung.
3) ROC analysis of differentially expressed genes
The relative expression amounts of the four genes obtained in 2) above were plotted in GraphPad Prim 6, and the probability of these four PBMC genes as non-small cell lung cancer diagnostic molecular markers was evaluated by AUC values (area under the curve), the greater the AUC value, the higher the sensitivity and specificity of the gene as a non-small cell lung cancer diagnostic molecular marker.
As a result, as shown in FIG. 2, the AUC values of the four genes, CD86, CD226, CD247 and ICAM-1, were 0.72, 0.61, 0.81 and 0.71, respectively.
2. Test results for expression amounts of CD86, CD226, CD247 and ICAM-1 in PBMCs of patients in the group of validation
In order to verify the diagnostic sensitivity and specificity of CD86, CD226, CD247 and ICAM-1 as molecular markers of lung cancer, the expression levels of four genes, CD86, CD226, CD247 and ICAM-1, were also detected in PBMC samples of patients of another verification group by using qRT-PCR.
1) Validating clinical data of a group of subjects
The study of this validation set contained 101 patients in the non-small cell lung cancer patient group (NSCLC group) and 21 patients with BENIGN nodules in the lung (BENIGN group). The NSCLC group (90.1% stage I, 6.9% stage II, 1.0% stage III, 2.0% stage IV, unknown 0%) includes 80 persons with adenocarcinoma (79.2%), 21 persons with squamous cell carcinoma (20.8%). Specific clinical information is shown in Table 8, and it can be seen that there is no difference between the test group and the control group in terms of age, sex and smoking amount (P value is greater than 0.05).
Table 8 is a summary of clinical data for the validation set of subjects
Note: the P value is calculated from the Chi-square test or Fisher's exact test for categorical variables and the T test or Wilcoxon rank sum test for continuous variables.
2) qRT-PCR detection results
The relative expression amounts (2^ -Delta Ct) of four genes including CD86, CD226, CD247 and ICAM-1 in blood PBMCs of 101 non-small cell lung cancer patients and 21 benign nodule patients in lungs in a verification group are detected by using qRT-PCR technology, and t test of two independent samples is carried out.
The results are shown in fig. 3, which shows that CD86(P ═ 0.0085), CD226(P ═ 0.0107), CD247(P ═ 0.0028) and ICAM-1(P ═ 0.0008) were also significantly higher in expression in the validation group of non-small cell lung cancer patients than in the lung benign nodule patient group.
3) ROC analysis of differentially expressed genes in non-small cell lung cancer patients
According to the relative expression amounts of the four genes in the verification group, respective ROC curves are drawn by utilizing GraphPad Prim 6 software, the capability of the four genes as molecular markers for diagnosing the non-small cell lung cancer is evaluated through AUC values, and the higher the AUC value is, the higher the diagnosis efficiency of the genes as the molecular markers on the non-small cell lung cancer is.
As a result, as shown in FIG. 4, the AUC values of the four genes of CD86, CD226, CD247 and ICAM-1 were 0.68, 0.58, 0.71 and 0.72, respectively.
3. Molecular marker determination and method for early diagnosis of non-small cell lung cancer
1) CD226 and CD247 gene expression level as non-small cell lung cancer diagnosis marker
Comparing the expression levels of the four genes in the two independent samples of the experimental group and the verification group, the expression levels of CD86, CD226, CD247 and ICAM-1 in NSCLC are all higher than those of benign tubercle patients in lungs, the results of the two experiments are consistent, and the expression difference of the four genes in PBMCs of NSCLC patients and benign tubercle patients has statistical significance (P value is less than 0.05). The two sets of data were then combined together to perform stepwise logistic regression analysis to obtain a combination of early diagnostic markers for NSCLC with high diagnostic sensitivity and specificity.
The gene expression is logarithmically transformed, and clinical characteristics (age, sex, smoking number and the like) of the patient with non-small cell lung cancer and the patient with benign nodules of lung are compared by chi-square test on classification variables and T test on continuous variables or Wilcoxon rank-sum test. The best marker combination for lung cancer diagnosis was selected using stepwise logistic regression analysis in which the clinical characteristics of all the subjects mentioned above and the logistic regression results with only the expression level of the gene of interest as univariate were considered.
When the gene expression level alone is first analyzed as a diagnostic marker for non-small cell lung cancer using stepwise logistic regression method, the best diagnostic combination is the combination of CD226 and CD247 expression levels. AUC values were 0.8863 (fig. 5A), with 95% confidence intervals: 0.8257-0.9469.
The mathematical modeling formula is as follows: p ═ exp (U)/[1+ exp (U) ], wherein U ═ 4.1198+2.8282 × log (CD226) -1.2212 × log (CD247) (formula 1).
In the formula, P is the detection value of a sample, and log (CD226) is the log value of the relative expression quantity of the CD226 gene in PBMC of the sample; log (CD247) is the log value of the relative expression quantity of the CD247 gene in the PBMC of the patient to be detected;
exp (U) is the exp value of U; exp is an exponential function with the natural constant (2.71828) as the base;
here, the diagnostic threshold P is calculated as 0.9043 with the john index (correct index) as a reference.
Detecting the expression level of CD226 and CD247 in PBMC of a patient, and if the P value calculated by the formula is greater than or equal to 0.9043, the patient is considered to have non-small cell lung cancer, and the model diagnosis sensitivity is 0.69474 and specificity is 1.
Therefore, the P value of the patient to be tested can be calculated by substituting the relative expression amounts of CD226 and CD247 in PBMC of the patient to be tested into the formula 1, and whether the patient to be tested is non-small cell lung cancer or benign nodules of lung can be distinguished according to the following standard A or whether the patient to be tested is suffering from non-small cell lung cancer can be judged according to the following standard A;
and (3) judging the standard A:
if the P value of the to-be-detected person is greater than or equal to 0.9043, the to-be-detected person suffers from or is candidate to suffer from non-small cell lung cancer; or the subject is or is candidate for non-small cell lung cancer other than benign nodules of the lung.
If the P value of the subject is less than 0.9043, the subject is or is candidate for a benign nodule in the lung rather than a non-small cell lung cancer.
2) CD226 and CD247 gene expression level combined with clinical data as non-small cell lung cancer diagnosis marker
When the expression levels of the CD226 and CD247 genes are combined with clinical data to serve as a non-small cell lung cancer diagnostic marker, the optimal diagnostic combination is found to be the diagnostic combination consisting of the expression levels of the CD226 and CD247 and the smoking amount (through whether the expression is more than 40 packets/year and whether 2.6575 is used in the formula or not), the AUC value reaches 0.9079 (figure 5B), and the 95% confidence interval is (0.8423, 0.9735).
The mathematical modeling formula is as follows: p ═ exp (U)/[1+ exp (U) ], wherein U ═ 4.1047+2.6575 × n +3.6105 × log (CD226) -1.7212 × log (CD247) (formula 2).
In formula 2, P is the detection value of the subject to be detected, and log (CD226) is the log value of the relative expression quantity of the CD226 gene in PBMC of the subject to be detected; log (CD247) is the log value of the relative expression quantity of the CD247 gene in the PBMC of the patient to be detected;
exp (U) is the exp value of U; exp is an exponential function with the natural constant (2.71828) as the base;
n is 1 or 0, and is used for measuring whether a coefficient 2.6575 is adopted in the formula, and if the smoking quantity of the tester is more than 40 bags/year, n is 1; if the smoking amount of the examiner is less than or equal to 40 bags/year, n is 0;
the diagnostic threshold P of this model was calculated as 0.8886 based on the john index.
Given the expression and smoking of CD226 and CD247 in PBMCs of a patient, if the P value calculated by the formula is greater than or equal to 0.8886, the patient is considered to have non-small cell lung cancer, and the model diagnosis sensitivity is 0.7579 and specificity is 0.9.
Therefore, the P value of the patient to be tested can be calculated by substituting the relative expression amounts of CD226 and CD247 in PBMC of the patient to be tested and the smoking amount of the patient to be tested into the formula 2, and whether the patient to be tested has non-small cell lung cancer or not is judged according to the following standard B, or whether the patient to be tested is non-small cell lung cancer or benign nodules of the lung is distinguished according to the following standard B;
and (4) judging the standard B:
if the P value of the to-be-detected person is greater than or equal to 0.8886, the to-be-detected person suffers from or is candidate to suffer from non-small cell lung cancer; or the subject is or is candidate for non-small cell lung cancer other than benign nodules of the lung;
if the P value of the subject to be tested is less than 0.8886, the subject does not suffer from or is not candidate for suffering from non-small cell lung cancer; the subject is or is candidate for a benign nodule in the lung rather than a non-small cell lung cancer.
Therefore, the kit for determining whether a subject has non-small cell lung cancer may include: a reagent for detecting the expression level of CD226 and CD247 in PBMC of a subject;
or detecting the annual smoking amount of the person to be detected;
or a readable carrier describing the following formula 1 or formula 2;
the readable carrier is also recorded with the judgment standard A or B.
Example 2, use of expression levels of CD226 and CD247 in detecting whether a subject has non-small cell lung cancer
Firstly, detecting the expression quantity of CD226 and CD247 in PBMC of a patient to be detected
1. PBMC of a person to be detected is separated and detected: 32 non-small cell lung cancers (pathologically diagnosed at stage I) and 17 benign nodules in the lung (which had been clinically diagnosed) were used as the subjects to be examined, in the same manner as 2.1 of the one of example 1;
2. extraction and purification of total RNA in PBMC: the method is the same as 2.2 of the first embodiment 1;
3. reverse transcription of PBMC RNA samples: the method is the same as 2.3 of the first embodiment 1;
4、qRT-PCR：
using cDNA reverse transcribed from the above 3 as a template, and performing qRT-PCR with the primers CD226-F/CD226-R and CD247-F/CD247-R in Table 4, respectively; GAPDH was used as the reference gene.
The relative expression levels of CD226 and CD247 were obtained.
5. Calculating the P value
The relative expression amounts of CD226 and CD247 in PBMC of the specimen were substituted into the following formula 1 to obtain the P value of the specimen.
The mathematical modeling formula is as follows: p ═ exp (U)/[1+ exp (U) ], wherein U ═ 4.1198+2.8282 × log (CD226) -1.2212 × log (CD247) (formula 1).
In the formula, P is the detection value of a sample, and log (CD226) is the log value of the relative expression quantity of the CD226 gene in PBMC of the sample; log (CD247) is the log value of the relative expression quantity of the CD247 gene in the PBMC of the patient to be detected;
exp (U) is the exp value of U; exp is an exponential function with the natural constant (2.71828) as the base;
judging whether the patient to be detected has non-small cell lung cancer according to the following standard A, or distinguishing whether the patient to be detected is non-small cell lung cancer or benign nodules of the lung according to the following standard A;
and (3) judging the standard A:
if the P value of the to-be-detected person is greater than or equal to 0.9043, the to-be-detected person suffers from or is candidate to suffer from non-small cell lung cancer; or the subject is or is candidate for non-small cell lung cancer other than benign nodules of the lung.
If the P value of the subject is less than 0.9043, the subject is or is candidate for a benign nodule in the lung rather than a non-small cell lung cancer.
The results are given in table 9 below:
table 9 shows the results of the tests on the patients to be examined
In the above table, column 1 is the patient number, column 2 is the patient age, column 3 is the patient sex, column 4 is the relative expression level of CD247 gene in patient PBMC, column 5 is the relative expression level of CD226 gene in patient PBMC, column 6 is the U value in formula 1, column 7 is the P value in formula 1, column 8 is the pathological diagnosis result of the case: NSCLC is non-small cell lung cancer; column 9 indicates whether the result of the P value judgment of the present invention agrees with the cytological diagnosis result.
As can be seen from table 9, it is,
the diagnosis result of 28 of 32 cases of non-small cell lung cancer is consistent with the diagnosis result of a cell line, only 4 cases are inconsistent, and the positive detection rate of the method for the non-small cell lung cancer is 87.5 percent;
the positive detection rate of the method for the benign tubercle patient in the lung is 47% in 8 of 17 benign tubercle patients in the lung, and the pathological diagnosis result is inconsistent with the result of the method for the invention in 9 benign tubercle patients in the lung, which may be due to the fact that the pathological diagnosis result is the result of collecting samples, the follow-up research should be carried out on the benign tubercle patients at high risk, and whether the benign patients are converted into non-small cell lung cancer or not, and the pathological diagnosis false positive may exist.
Example 3 expression levels of CD226 and CD247 and the amount of smoking in the test subject
Firstly, detecting the expression quantity of CD226 and CD247 in PBMC of a patient to be detected
1. PBMC of a person to be detected is separated and detected: 32 non-small cell lung cancers (pathologically diagnosed as stage I) and 17 benign nodules in the lung (which had been clinically diagnosed) were used as the subjects to be examined, in the same manner as 2.1 of the first example 1;
2. extraction and purification of total RNA in PBMC: the method is the same as 2.2 of the first embodiment 1;
3. reverse transcription of PBMC RNA samples: the method is the same as 2.3 of the first embodiment 1;
4、qRT-PCR：
using cDNA reverse transcribed from the above 3 as a template, and performing qRT-PCR with the primers CD226-F/CD226-R and CD247-F/CD247-R in Table 4, respectively; GAPDH was used as the reference gene.
The relative expression levels of CD226 and CD247 were obtained.
5. Counting whether the annual smoking amount of the person to be detected is more than 40 bags;
6. calculating the P value
The P value of the specimen was obtained by substituting the relative expression amounts of CD226 and CD247 in PBMC of the specimen into the following formula 2.
P ═ exp (U)/[1+ exp (U) ], wherein U ═ 4.1047+2.6575 × n +3.6105 × log (CD226) -1.7212 × log (CD247) (formula 2).
In formula 2, P is the detection value of the subject to be detected, and log (CD226) is the log value of the relative expression quantity of the CD226 gene in PBMC of the subject to be detected; log (CD247) is the log value of the relative expression quantity of the CD247 gene in the PBMC of the patient to be detected;
exp (U) is the exp value of U; exp is an exponential function with the natural constant (2.71828) as the base;
n is 1 or 0, and is used for measuring whether a coefficient 2.6575 is adopted in the formula, and if the smoking quantity of the tester is more than 40 bags/year, n is 1; if the smoking amount of the examiner is less than or equal to 40 bags/year, n is 0;
judging whether the patient to be detected has non-small cell lung cancer according to the following standard B, or distinguishing whether the patient to be detected has non-small cell lung cancer or benign nodules of the lung according to the following standard B;
and (4) judging the standard B:
if the P value of the to-be-detected person is greater than or equal to 0.8886, the to-be-detected person suffers from or is candidate to suffer from non-small cell lung cancer; or the subject is or is candidate for non-small cell lung cancer other than benign nodules of the lung;
if the P value of the subject is less than 0.8886, the subject is or is candidate for a benign nodule in the lung rather than a non-small cell lung cancer.
The results are given in table 10 below:
table 10 shows the results of the examination of the patients
In the above table, the 1 st column indicates the patient number, the 2 nd column indicates the patient age, the 3 rd column indicates the patient sex, the 4 th column indicates the smoking amount, the 5 th column indicates the n value of the smoking amount coefficient, the 6 th column indicates the relative expression level of the CD247 gene in the PBMC of the patient, the 7 th column indicates the relative expression level of the CD226 gene in the PBMC of the patient, the 8 th column indicates the U value in the formula 2, the 9 th column indicates the P value in the formula 2, and the 10 th column indicates the pathological diagnosis result that NSCLC is non-small cell lung cancer; the 12 th column indicates whether the result of the p-value judgment of the present invention coincides with the result of the pathological diagnosis.
As can be seen from the table 10, it is,
the diagnosis result of 31 of 32 cases of non-small cell lung cancer is consistent with the cell line diagnosis result, only 1 case is inconsistent, and the positive detection rate of the method for the non-small cell lung cancer is 96.7 percent;
the results of diagnosis by the method of the invention are consistent with the cell line diagnosis results in 10 of 17 benign pulmonary nodule patients, the threshold value of 3 patients is adopted, the results of diagnosis by the method of the invention are inconsistent with the cell line diagnosis results in 4 patients, and the positive detection rate of the invention for the benign pulmonary nodule patients is 58.9%.
Since the pathological diagnosis results are the results of collecting samples, follow-up studies should be performed on these high-risk benign nodule patients, and if there is a transition from benign patients to non-small cell lung cancer, false positive pathological diagnosis may exist.
SEQUENCE LISTING
<110> military medical research institute of military science institute of people's liberation force of China
<120> molecular marker for early diagnosis of non-small cell lung cancer in peripheral blood mononuclear cells
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Claims (4)
1. The application of a substance for detecting the expression level of CD226 gene in PBMC of a person to be detected and a substance for detecting the expression level of CD247 gene in PBMC of the person to be detected in the preparation of a product with at least one function of 1) -3):
1) detecting or assisting to detect whether the patient to be detected has the non-small cell lung cancer;
2) diagnosing or assisting to diagnose whether the patient to be detected has the non-small cell lung cancer;
3) and distinguishing or assisting to distinguish whether the patient to be detected is the patient with non-small cell lung cancer or benign nodules of lung.
2. Use of a substance for detecting the expression level of CD226 gene in PBMC of a subject to be examined, a substance for detecting the expression level of CD247 gene in PBMC of said subject to be examined, and a readable vector B described in formula 2 for preparing a product having at least one function of 1) to 3):
1) detecting or assisting to detect whether the patient to be detected has the non-small cell lung cancer;
2) diagnosing or assisting to diagnose whether the patient to be detected has the non-small cell lung cancer;
3) distinguishing or assisting in distinguishing whether the patient to be detected is a patient with non-small cell lung cancer or benign nodules of lung;
the formula 2: p ═ exp (U)/[1+ exp (U) ], wherein U ═ 4.1047+2.6575 × n +3.6105 × log (CD226) -1.7212 × log (CD 247);
wherein, P is the detection value of a person to be detected, and log (CD226) is the log value of the expression quantity of the CD226 gene in PBMC of the person to be detected; log (CD247) is the log value of the expression quantity of the CD247 gene in the PBMC of the patient to be detected;
exp (U) is the exp value of U;
n is 1 or 0, if the smoking quantity of the person to be detected is more than 40 bags per year, n is 1; if the smoking amount of the person to be detected is less than or equal to 40 bags per year, n is 0.
3. The application of a substance for detecting the expression level of CD226 gene in PBMC of a subject to be detected, a substance for detecting the expression level of CD247 gene in PBMC of the subject to be detected and a readable vector A describing formula 1 in the preparation of a product having at least one function of 1) to 3):
1) detecting or assisting to detect whether the patient to be detected has the non-small cell lung cancer;
2) diagnosing or assisting to diagnose whether the patient to be detected has the non-small cell lung cancer;
3) distinguishing or assisting in distinguishing whether the patient to be detected is a patient with non-small cell lung cancer or benign nodules of lung;
the formula 1 is P ═ exp (U)/[1+ exp (U) ], wherein U ═ 4.1198+2.8282 × log (CD226) -1.2212 × log (CD 247);
p is a detection value of a person to be detected, and log (CD226) is a log value of CD226 gene expression quantity in PBMC of the person to be detected; log (CD247) is the log value of the expression quantity of the CD247 gene in the PBMC of the patient to be detected;
exp (U) is the exp value of U.
4. Use according to any one of claims 1 to 3, characterized in that:
the substance for detecting the expression quantity of the CD226 gene in the PBMC of the patient to be detected comprises a primer pair for amplifying the CD226 gene;
the substance for detecting the expression quantity of the CD247 gene in the PBMC of the patient to be detected comprises a primer pair for amplifying the CD247 gene.
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