Application of FLT3LG protein in preparation of lung adenocarcinoma postoperative prognosis evaluation reagent or kit
阅读说明：本技术 Flt3lg蛋白在制备肺腺癌术后预后评估试剂或者试剂盒中的应用 (Application of FLT3LG protein in preparation of lung adenocarcinoma postoperative prognosis evaluation reagent or kit ) 是由 秦思达 杨成成 于 2019-11-07 设计创作，主要内容包括：本发明属于生物医学技术领域,具体公开了一种FLT3LG蛋白在制备肺腺癌术后预后评估试剂或者试剂盒中的应用。本发明以FLT3LG蛋白作为标志物,通过免疫组织化学的方法检测肺腺癌组织标本,并判断肺腺癌患者的预后；FLT3LG蛋白肺腺癌中高表达,而在癌旁组织中低表达；FLT3LG蛋白在肺腺癌中的表达水平和患者生存期有关；高表达患者预后不好,生存时间短。FLT3LG蛋白表达水平不但与肺腺癌患者总生存期有关,也和患者无瘤生存期有关,FLT3LG蛋白高表达患者较早出现复发或转移；FLT3LG蛋白作为肺腺癌诊断和预后标志物,具有广阔的应用前景和较大的社会效益。(The invention belongs to the technical field of biomedicine, and particularly discloses application of FLT3LG protein in preparation of a lung adenocarcinoma postoperative prognosis evaluation reagent or kit. The invention takes FLT3LG protein as a marker, detects lung adenocarcinoma tissue specimens by an immunohistochemical method, and judges the prognosis of a lung adenocarcinoma patient; FLT3LG protein is highly expressed in lung adenocarcinoma and is low expressed in paracarcinoma tissues; the expression level of FLT3LG protein in lung adenocarcinoma is related to the survival time of patients; the prognosis of the high-expression patient is poor, and the survival time is short. The expression level of the FLT3LG protein is related to the overall survival time of the lung adenocarcinoma patients and the tumor-free survival time of the patients, and the patients with high FLT3LG protein expression have relapse or metastasis early; the FLT3LG protein is used as a lung adenocarcinoma diagnosis and prognosis marker, and has wide application prospect and great social benefit.)
1. An application of FLT3LG protein in preparing a reagent or a kit for prognosis evaluation after lung adenocarcinoma surgery.
2. The use of the FLT3LG protein in the preparation of a reagent or a kit for the postoperative prognostic assessment of lung adenocarcinoma according to claim 1, wherein the FLT3LG protein is used as a molecular marker, and FLT3LG primary antibody, HRP-labeled secondary antibody and an immunohistochemical test reagent are used to analyze the expression level of FLT3LG protein in lung adenocarcinoma tissues, so as to assess the postoperative prognostic survival of a patient according to the expression level of FLT3LG protein.
3. The use of the FLT3LG protein according to claim 2 in the preparation of a reagent or a kit for the postoperative prognostic assessment of lung adenocarcinoma, wherein the primary FLT3LG antibody is a rabbit anti-human FLT3LG monoclonal antibody, and the secondary HRP-labeled antibody is a horseradish peroxidase-labeled mouse anti-rabbit IgG antibody.
4. Use of the FLT3LG protein according to claim 2 in preparing a reagent or a kit for prognosis evaluation after lung adenocarcinoma surgery, wherein the immunohistochemical reagent comprises xylene, ethanol, 3% H2O2Solution, 1% BSA blocking solution, DAB chromogenic reagent and hematoxylin.
5. The use of the FLT3LG protein according to claim 2 in preparing a reagent or a kit for the prognosis evaluation after lung adenocarcinoma surgery, wherein the FLT3LG protein expression level is detected in a biological sample, and the biological sample is formalin-fixed paraffin-embedded lung adenocarcinoma tissue.
6. The use of the FLT3LG protein in the preparation of a reagent or a kit for the prognostic evaluation of lung adenocarcinoma after operation according to claim 1, wherein the total survival and the tumor-free survival of lung adenocarcinoma patients after operation are shortened when the expression level of the FLT3LG protein is increased.
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to an application of FLT3LG protein in preparation of a lung adenocarcinoma postoperative prognosis evaluation reagent or kit.
Background
Lung cancer is the most serious malignant tumor with the highest morbidity and cancer mortality worldwide, and seriously threatens the life health of human beings. In China, lung cancer has replaced liver cancer to become the highest-mortality malignant tumor at present, and accounts for 22.7 percent of the total death number of all malignant tumors, and the morbidity and mortality of the lung cancer still rapidly increase. According to statistics, about 40 ten thousand cases of lung cancer die each year in China, and the lung cancer becomes the malignant tumor with the highest morbidity and mortality in China. Despite recent advances in lung cancer diagnosis and treatment with advances in technology, the survival rate of lung cancer patients is still not ideal, only about 20%, for 5 years after treatment. The 5-year survival rate of lung cancer patients is not ideal, and is related to the fact that most lung cancer patients are in a local advanced stage or have metastasis at the time of diagnosis, so that the lung cancer patients lose timely intervention and treatment opportunities, and the prognosis condition of the lung cancer patients is seriously influenced. Therefore, the key to improving the prognosis survival rate of the lung cancer patients lies in early diagnosis and intervention treatment.
Lung adenocarcinoma is a type of lung cancer, which is a malignant tumor derived from glandular epithelium in lung tissue, and has various growth patterns including acinar, papilla, bronchioloalveolar or parenchymal growth, and the like. Lung adenocarcinoma accounts for about 40% of primary lung tumors, and the proportion of lung adenocarcinoma is increasing year by year, and is likely to occur in women and those who do not smoke. Lung adenocarcinoma is common in peripheral lung cancer, has no obvious symptoms in the early stage, and is still easily ignored when the symptoms of the respiratory system such as cough, hemoptysis, chest pain and the like are developed along with the disease. In the late stage, symptoms are related to invasion and metastasis of tumors, such as stimulation of pleural effusion, pain caused by metastasis to bone joints, corresponding symptoms caused by metastasis to the skull and the like.
Currently, diagnostic treatment strategies for lung adenocarcinoma range from traditional TNM staging to treatment regimens, to individualized targeted therapies based on patient differential genotyping. Such as: the tyrosine kinase inhibitor has good curative effect on lung adenocarcinoma patients with Epidermal Growth Factor Receptor (EGFR) gene mutation, and the mutation is especially common in lung adenocarcinoma patients of Asian and female. Among them, deletion of exon 19 or point mutation of L858R 21 of EGFR gene is a more common therapeutically effective type of mutation. Therefore, the detection of the EGFR gene can effectively predict the targeted treatment effect of the lung adenocarcinoma. In addition, mutations in Anaplastic Lymphoma Kinase (ALK) gene rearrangement, ROS sarcoma carcinogen-receptor tyrosine kinase (ROS1), were better in young non-smoking patients. The mutation rate of the compound is lower than that of EGFR, but the compound is also used as an indispensable index for molecular biological detection of patients with lung adenocarcinoma, and directly guides the treatment scheme of the patients.
However, the treatment of patients with early and middle stage lung adenocarcinoma still takes operation as the main part, and the prediction of postoperative survival of the patients has no good index. At present, tumor serum markers Cyfta21-1, CEA, CA125 and the like which are clinically and frequently used for lung cancer cannot well reflect the postoperative prognosis of a lung cancer patient, and the detection of oncogenes (such as Ras, myc, erb-b2 and the like) or cancer suppressor genes (p53, RB and the like) cannot specifically evaluate the lung adenocarcinoma prognosis. Therefore, there is a need to further explore related proteins that can predict the prognosis of lung adenocarcinoma patients after surgery.
FLT3LG (Fms-related tyrosine kinase 3ligand) Fms-related tyrosine kinase 3ligand is a protein encoded by the FLT3LG gene in humans. It is structurally homologous to Stem Cell Factor (SCF) and colony stimulating factor 1 (CSF-1). In synergy with other growth factors, FLT3LG stimulates the proliferation and differentiation of various blood cell progenitors. For example, it is the major growth factor that stimulates the growth of dendritic cells. In addition, the kit has a certain effect on prognosis evaluation of malignant tumors such as osteosarcoma and the like, but the lung cancer prognosis evaluation is not reported in documents.
Disclosure of Invention
The invention provides an application of FLT3LG protein in preparing a lung adenocarcinoma postoperative prognosis evaluation reagent or a kit, wherein the expression level of FLT3LG protein in lung adenocarcinoma is related to the survival time of a patient and the tumor-free survival time of the patient, the survival time of the patient with high FLT3LG protein expression is short, and the patient has relapse or metastasis early, and the FLT3LG protein is used as a lung adenocarcinoma diagnosis and prognosis marker, so that the kit has wide application prospect and great social benefit.
The invention provides an application of FLT3LG protein in preparation of a lung adenocarcinoma postoperative prognosis evaluation reagent or a kit.
Preferably, in the application, the FLT3LG protein is used as a molecular marker, the FLT3LG primary antibody, the HRP-labeled secondary antibody and an immunohistochemical experiment reagent are used for analyzing the expression level of the FLT3LG protein in lung adenocarcinoma tissues, and the postoperative prognosis survival period of the patient is evaluated according to the expression level of the FLT3LG protein.
Preferably, in the above application, the primary FLT3LG antibody refers to rabbit anti-human FLT3LG monoclonal antibody, and the HRP-labeled secondary antibody refers to horseradish peroxidase-labeled mouse anti-rabbit IgG antibody.
Preferably, in the above application, the immunohistochemical reagent comprises xylene, ethanol, and 3% H2O2Solution, 1% BSA blocking solution, DAB chromogenic reagent and hematoxylin.
Preferably, in the above application, the FLT3LG protein expression level is detected in a biological sample, wherein the biological sample is lung adenocarcinoma tissue embedded in paraffin after formalin fixation.
Preferably, in the above application, the method for detecting the relative expression level of FLT3LG protein in lung adenocarcinoma tissue is as follows:
a) fixing the specimen with formaldehyde, dehydrating with alcohol, and embedding and slicing paraffin after xylene is transparent;
b) baking at 50 deg.C for 2 hr, slicing, dewaxing with xylene, and gradient alcohol hydrating;
c) volume fraction 3% H2O2Incubating for 10min, washing with distilled water and PBS buffer solution for 2 times, each time for 5min
d) Performing antigen retrieval with 0.01M citric acid buffer solution, and heating in microwave oven at 98 deg.C for 10 min;
e) after cooling to room temperature, PBS buffer was added 2 times for 5min each
f) After 1h blocking with volume fraction 1% BSA, primary antibody incubations: rabbit anti-human FLT3LG monoclonal antibody was added and the mixture was refrigerated overnight (at least 12h) at 4 ℃.
g) Re-warming at room temperature for 20min, and buffering with PBS for 5min for 2 times;
h) and (3) secondary antibody incubation: adding corresponding horseradish peroxidase-labeled mouse anti-rabbit IgG antibody, and incubating at 37 ℃ for 30 min;
i) PBS buffer for 5min for 2 times; developing the DAB kit for 1-10 min;
j) fully washing with tap water to stop dyeing; counterstaining with hematoxylin for 10 s;
k) gradient alcohol dehydration, xylene transparency, sealing and air drying.
l) observing results under a microscope, randomly selecting 10 areas, and grading;
m) is divided into 0 to 3 points according to the staining intensity, 1 to 4 points according to the percentage of the positive cells, the multiplication of the staining index and the positive cells is the staining index, the high expression of FLT3LG protein is divided into more than or equal to 4 points, and the low expression is divided into less than 4 points.
Preferably, in the above application, the total survival time and the tumor-free survival time after the operation of the lung adenocarcinoma patient are shortened by the increase of the expression level of FLT3LG protein.
Compared with the prior art, the application of the FLT3LG protein in preparing the reagent or the kit for the prognosis evaluation after lung adenocarcinoma surgery has the following beneficial effects:
the invention aims to provide a novel application of FLT3LG protein, in particular to an application in preparing a reagent or a kit for prognosis evaluation of lung adenocarcinoma.
The invention is widely and deeply researched, and firstly discovers that the relative expression quantity of FLT3LG protein in lung adenocarcinoma tissues is detected by an immunohistochemical method, so that the prognosis recurrence or metastasis and the postoperative survival period of a lung adenocarcinoma patient can be judged, and the detection of the expression quantity by taking the FLT3LG protein as a molecular marker can be used for guiding the prognosis judgment of the lung adenocarcinoma based on the correlation between the relative expression quantity of the FLT3LG protein and the lung adenocarcinoma.
The discovery of the relevance of the FLT3LG protein and the lung adenocarcinoma provides a new method for predicting the recurrence and metastasis of lung adenocarcinoma prognosis and the postoperative survival period, has important value for judging the prognosis of lung adenocarcinoma patients, and when the immunohistochemical score is more than or equal to 4, the lung adenocarcinoma patients are easy to relapse or metastasize after operation, and the survival time is short.
The invention utilizes immunohistochemical technology to detect the relative expression quantity of the FLT3LG protein in lung adenocarcinoma tissues, statistically analyzes clinical pathological data of patients and combines follow-up information to determine the relevance of the relative expression quantity of the FLT3LG protein to the prognosis of lung adenocarcinoma patients after operation. The FLT3LG protein can be used as a protein molecular marker for judging the prognosis of a patient with lung adenocarcinoma, and has certain effects on postoperative adjuvant therapy and disease monitoring.
Drawings
FIG. 1 shows positive results of immunohistochemical staining of FLT3LG protein in lung adenocarcinoma tissue;
FIG. 2 shows the result of negative immunohistochemical staining of FLT3LG protein in lung adenocarcinoma tissue;
FIG. 3 shows positive results of immunohistochemical staining of FLT3LG protein in paracancer lung tissue;
FIG. 4 shows the negative result of the immunohistochemical staining of FLT3LG protein in the paracancer lung tissue;
FIG. 5 is a graph of FLT3LG protein expression versus overall survival in lung adenocarcinoma patients;
FIG. 6 is a graph of FLT3LG protein expression versus tumor-free survival in patients with lung adenocarcinoma;
FIG. 7 is a KM-Plotter database analyzing the relationship between FLT3LG gene expression and prognosis in lung adenocarcinoma;
FIG. 8 is a KM-Plotter database analyzing the relationship between FLT3LG gene expression and prognosis in squamous cell lung carcinoma.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The invention provides application of FLT3LG protein in preparation of a lung adenocarcinoma postoperative prognosis evaluation kit. The term "lung adenocarcinoma surgery" refers to the lung adenocarcinoma patients who have undergone surgical treatment, and the term "prognosis evaluation" refers to the prediction of survival time and recurrence and metastasis of the patients after tumor surgical resection. The amino acid sequence of the FLT3LG protein is shown as SEQ ID NO.1, and the nucleotide sequence of the encoded FLT3LG protein is shown as SEQ ID NO. 1.
To demonstrate the effect of the present invention, we conducted a related study:
first, research method
1. Patient collection: 132 lung cancer surgery patients were collected from 1 month 2010 to 2014 12 months sienna transportation university first subsidiary hospital and were confirmed as lung adenocarcinoma after the surgery. Of these, 69 men and 63 women ranged in age from 32 to 80 years (median age 60.5 years). In the experiment, the non-small cell lung cancer is staged according to the pathological description of the postoperative patient and the 8 th lung cancer TNM stage of the International anticancer Union (UICC) in 2017. Since stage IV patients do not benefit from surgery, the specimens collected are stage I, stage II, and stage III patients, and no stage IV patients.
2. Patient enrollment conditions: (1) the selected patients are not treated by any anti-tumor treatment methods such as chemotherapy, radiotherapy, immunotherapy and the like before operation; (2) the pathological examination of the patient after the operation proves that the lung adenocarcinoma is formed; (3) the patient had no other tumors and had no history of other tumors; (4) the patient's surgical procedure is radical lung cancer (radical lung cancer resection); (5) the patients and their families agree to take the samples of lung adenocarcinoma resection specimen after the operation.
3. Follow-up: overall Survival (OS) refers to the time from the date of lung cancer surgery to the death or loss of visit of the patient. The last follow-up date is 12 months in 2018. Survival for all patients ranged from 1-95 months, with median survival of 43.5 months.
4. Preparation of immunohistochemical experimental liquid
4.1 preparation of gradient alcohol solution: the volume fractions of 70%, 80%, 85%, 90% and 95% alcohol are diluted with absolute ethanol and distilled water.
4.2 preparation of PBS buffer solution: sodium chloride 12g, sodium dihydrogen phosphate anhydrous (Na)2HPO4)1.725g, dipotassium hydrogen phosphate anhydrous (K)2HPO4)0.3g of potassium chloride and 0.3g of distilled water are used for preparing 1200ml of the solution, and the pH value is adjusted to 7.2;
4.3 preparation of citric acid buffer solution/antigen retrieval solution (0.01M): citric acid A solution: 2.1g of citric acid is fully dissolved in 100ml of distillation water; trisodium citrate solution B: 2.9g of trisodium citrate is dissolved in 100ml of distillation water; and sequentially extracting 9ml of citric acid A solution and 41ml of trisodium citrate B solution, adding 450ml of distilled water, and mixing uniformly to obtain 500ml of total amount, so as to prepare the citric acid buffer solution with the pH value of 6.0, and reserving for later use.
4.4 mounting agent: 0.5ml of PBS buffer solution in 4.2 is uniformly mixed with the same volume of glycerol for use, and the mixture is required to be prepared for use.
5. Preparation of specimen and immunohistochemical reaction (a method for detecting the relative expression of FLT3LG protein in lung adenocarcinoma tissue)
5.1 specimen fixation: all isolated lung adenocarcinoma specimens were fixed within 3h, using 40% formaldehyde by volume fraction (commercially available formaldehyde reagents are available).
5.2, preparing a wax block: fresh lung adenocarcinoma specimens (about 1.0 cm. times.1.0 cm. times.0.2 cm in size) from the operating room were fixed with 40% volume fraction formaldehyde at room temperature around 25 ℃ for at least 12 h. Then dehydrated with alcohol and xylene transparent (85% ethanol 5min, 90% ethanol 5min, 95% ethanol 5min, anhydrous ethanol 5min, first xylene soaking 10min, second xylene soaking 10min) and paraffin embedding.
5.3 slicing preparation: cutting into pieces with thickness of 4-6 μm with manual paraffin slicer, spreading in water at 40-45 deg.C, and naturally flattening slightly-wrinkled paraffin tape by water tension and water temperature. Then carefully placed into the slides, then all slides were placed in a 40 ℃ dry box overnight (at least 12h), then placed in a 65 ℃ oven for 1.5h, and finally placed in a 37 ℃ incubator overnight (at least 12h), and the resulting paraffin sections were stored at ambient temperature and taken up for use.
5.4 immunohistochemical Experimental procedures
a) Baking slices: placing the paraffin sections into an oven to be baked for 2 hours at 50 ℃;
b) dewaxing: paraffin sections were placed in xylene for 20min (first time) and xylene for 20min (second time);
c) hydration: conventional hydration, i.e. 10min of absolute ethyl alcohol, 5min of 95% alcohol, 5min of 80% alcohol and 5min of 70% alcohol;
d) and (3) incubation: volume fraction 3% H2O2Incubating for 10min at room temperature to eliminate the activity of endogenous peroxidase in tumor tissues;
e) washing: washing with distilled water, soaking in PBS buffer solution twice for 5min each time;
f) antigen retrieval: placing the slices into 0.01M citric acid antigen repairing buffer solution, heating in microwave oven at 98 deg.C for 10min, taking out, and standing at room temperature for 30 min;
g) washing: washing with PBS buffer solution twice, each for 5 min;
h) after 1h blocking with volume fraction 1% BSA, primary antibody incubations: adding rabbit anti-human FLT3LG monoclonal antibody (primary antibody, Abcame company, cat # ab52648, 1:100 dilution) with corresponding concentration, placing into a wet box, placing gauze and a small amount of water into the box, and placing into a refrigerator at 4 deg.C overnight (at least 12 h);
i) rewarming: taking out the product the next day, standing at room temperature, and rewarming for 20 min;
j) washing: washing with PBS buffer solution twice, each for 5 min;
k) and (3) secondary antibody incubation: adding corresponding horse radish peroxidase-labeled mouse anti-rabbit IgG antibody (secondary antibody, Boaosen, cat # bs-0295M-HRP, 1:500 dilution), and incubating at 37 deg.C for 30 min;
l) rinsing: washing with PBS buffer solution twice, each for 5 min;
m) color development: performing color development by using a DAB kit (comprising solution A and solution B), namely adding 1 drop of the DAB kit solution A and 1 drop of the DAB kit solution B into 1ml of distilled water under the condition of keeping out of the sun, mixing and then dropping into slices;
n) observation: after development for 30 seconds, the staining was observed under a microscope at intervals of about 10 seconds, and the development time was increased if necessary. The dyeing time is 1-10 min.
o) stop staining: fully washing with tap water;
p) counterdyeing: counterstaining with hematoxylin for 10 s;
q) dehydration: dehydrating with 80% ethanol for 5min, 95% ethanol for 5min, and 100% ethanol for 10 min;
r) transparent: xylene 10min (first), xylene 10min (second);
s) sealing sheet: dripping a sealing tablet into the mixture for sealing, and putting the mixture into an air box for air drying;
t) observation: the sections were observed under a microscope, and 10 regions of each section tissue were randomly selected for observation and scored.
u) judging the result:
in this set of experiments, positive results of immunohistochemical staining of tumor cell FLT3LG were shown by the appearance of specific tan particles in the cytoplasm of the cells, see fig. 1; a negative result of the histochemical staining is represented by almost no specific staining in the cytoplasm of the cells, see fig. 2. In each specimen, 10 high-power fields were randomly selected under 400 × field for each index, and the percentage of positive cells was calculated. In positive staining, the colorless score was 0; light yellow is rated 1; the deep yellow color was rated 2 points; the brown-yellow color was rated 3. Setting the non-positive cells to 0 point; the percentage of positive cells is 1 point < 25%; the percentage of the positive cells is 2 minutes between 25 percent and 50 percent; the percentage of the positive cells accounting for 50-75 percent is 3 minutes; the percentage of positive cells greater than 75% was 4 points. Multiplying the color score by the percentage score of the positive cells to obtain a staining index, wherein the staining index is more than or equal to 4, the high expression of the FLT3LG protein in the tumor tissue is judged, and the staining index is less than 4, the low expression of the FLT3LG protein in the tumor tissue is judged.
6. Analysis by statistical methods
Statistical analysis was performed using SPSS 20.0 software. In the experimental results, the counting data were counted using the chi-square test method. Survival analysis adopts a Kaplan-Meier method, the difference significance is tested by using Log-Rank, and a survival curve is drawn by GraphPadprism 5 software. And analyzing the risk factors of the lung adenocarcinoma patient prognosis by using a single-factor and multi-factor Cox regression model. P <0.05 was considered statistically significant.
Second, research results
Comparison of expression levels of FLT3LG protein in lung adenocarcinoma tissue and paracancer lung tissue
FIG. 1 shows positive results of immunohistochemical staining of FLT3LG protein in lung adenocarcinoma tissue; FIG. 2 shows the result of negative immunohistochemical staining of FLT3LG protein in lung adenocarcinoma tissue; FIG. 3 shows the positive result of the immunohistochemical staining of the protein FLT3LG in the paracancer lung tissue, and FIG. 4 shows the negative result of the immunohistochemical staining of the protein FLT3LG in the paracancer lung tissue. In 132 lung adenocarcinoma tissues, the FLT3LG protein was highly expressed by 70 cases (53%), and lowly expressed by 62 cases (47%); in 82 cases of paracancer lung tissues, FLT3LG protein was highly expressed in 25 cases (37.8%) and low in 57 cases (62.2%). As can be seen from table 1, FLT3LG protein expression in lung adenocarcinoma tissue was significantly higher than in paracancerous lung tissue (p < 0.05).
TABLE 1 expression of FLT3LG protein in lung adenocarcinoma tissue and paracancer lung tissue
2. Relationship between FLT3LG protein expression and clinical pathological data in lung adenocarcinoma tissue
Table 2 shows the relationship between FLT3LG protein expression and clinical pathological data and factors, and the expression of FLT3LG protein in lung adenocarcinoma tissue is obviously related to whether lymph node metastasis occurs and TNM stage of tumor (P <0.05), but is not related to sex, age, smoking, tumor differentiation degree, tumor size, tumor position, tumor type and the like (P > 0.05).
TABLE 2 relationship between FLT3LG protein expression and clinical pathological data and factors
Correlation of FLT3LG expression with prognosis of patients with lung adenocarcinoma
We performed survival analysis on FLT3LG high-expression and low-expression groups in lung adenocarcinoma by using Kaplan-Meier method, and found that the total survival time (OS) of FLT3LG high-expression patients (FLT3LG +) low-expression patients (FLT3LG-) is obviously shortened (FIG. 5), and the difference of the two in Log rank test has statistical significance (P < 0.001). Analyzing the relationship between the expression of FLT3LG and the recurrence and metastasis of the lung adenocarcinoma patients, finding that the patients with FLT3LG and high-expression lung adenocarcinoma (FLT3LG +) have lower expression (FLT3LG-), and the tumor-free survival period (DFS) is obviously shortened (figure 6), the difference is significant (P is 0.001), and the patients with FLT3LG and low expression are more likely to have recurrence and metastasis.
4. Single-factor and multi-factor Cox regression analysis of influence factors on overall survival time of lung adenocarcinoma patients
As shown in Table 3, in the single-factor analysis, the tumor size, the lymph node metastasis condition, the TNM stage and the FLT3LG protein expression condition have statistical significance (p is less than 0.05) on the overall survival prognosis of the lung adenocarcinoma patients, and the factors are all the factors influencing the overall survival of the lung adenocarcinoma patients. Table 4 shows that the expression of FLT3LG protein, the size of tumor, and lymph node metastasis are independent risk factors for the overall prognosis survival of lung adenocarcinoma (p <0.05) by selecting the tumor size, the lymph node metastasis, and the FLT3LG protein expression at the same time (since the TNM stage is determined by the tumor size and the lymph node metastasis, this factor is eliminated in the multifactorial analysis).
TABLE 3 Cox Single factor analysis Total Life time influencing factors
TABLE 4 Cox multifactor analysis Total Life time independent influence factors
5. Single-factor and multi-factor Cox regression analysis of influence factors on tumor-free survival of lung adenocarcinoma patients
As shown in Table 5, in the single-factor analysis, the tumor size, the lymph node metastasis, the TNM stage and the FLT3LG protein expression have statistical significance (p is less than 0.05) on the tumor-free survival of the lung adenocarcinoma patients, and all the factors are the tumor-free survival influencing factors of the lung adenocarcinoma patients. Table 6 shows that the tumor size, lymph node metastasis and FLT3LG protein expression were selected simultaneously, and the FLT3LG protein expression, tumor size and lymph node metastasis were found to be independent risk factors for the tumor-free survival of lung adenocarcinoma (p <0.05) using Cox multifactorial survival analysis.
TABLE 5 Cox Single factor analysis of tumor-free survival influencing factors
TABLE 6 Cox multifactor analysis Total Life time independent influence factors
KM-Plotter database validation results
Data analysis was performed by KM-Plotter database (http:// kmplot. com/analysis /), and in public database, further validated the relationship of FLT3LG gene expression to lung adenocarcinoma. The results are shown in FIGS. 7 and 8. Among them, 720 lung adenocarcinoma population analyzed the relationship between FLT3LG gene expression and patient prognosis, it can be seen that the survival time of 388 patients with lower expression of 332 patients with high expression of FLT3LG gene was shortened, the difference was statistically significant (P <0.001), and in this database, the relative risk coefficient of FLT3LG high expression was 1.6 (95% confidence interval was 1.27-2.02). While analyzing the relationship between FLT3LG gene expression and prognosis in 524 lung squamous cell carcinomas, 251 lung squamous cell carcinomas with high expression and 273 lung squamous cell carcinomas with low expression of FLT3LG showed no significant difference in prognosis (P ═ 0.23). The expression level of the FLT3LG gene can be used for pertinently predicting the postoperative prognostic survival time of the lung adenocarcinoma patient, and is consistent with the prediction of the postoperative prognostic survival time of the lung adenocarcinoma patient by the FLT3LG protein expression of our experimental results.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> first subsidiary Hospital of medical college of Western-Ann transportation university
Application of <120> FLT3LG protein in preparation of lung adenocarcinoma postoperative prognosis evaluation reagent or kit
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gccccgcagc cccctctgct cctcctactg ctgctgcccg tgggcctcct gctgctggcc 600
gctgcctggt gcctgcactg gcagaggacg cggcggagga caccccgccc tggggagcag 660
gtgccccccg tccccagtcc ccaggacctg ctgcttgtgg agcactga 708