A biomarker for lung cancer diagnosis and application thereof

Abstract

The application belongs to the technical field of biomedical detection, and particularly relates to a biomarker for lung cancer diagnosis and application thereof. A new lung cancer biomarker anti-FDX1 autoantibody is found for the first time, and the expression level of the plasma anti-FDX1 autoantibody can be detected to distinguish lung cancer patients from normal people, and the experimental results also show that the anti-FDX1 autoantibody can be used as a detection index for different clinical characteristics of lung cancer. Further, the anti-FDX1 autoantibody is combined with carcinoembryonic antigen to construct a new lung cancer detection model as a marker, and experimental data show that the combined model can significantly improve the lung cancer detection efficiency, which has important significance for lung cancer diagnosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical detection, and particularly relates to a biomarker for lung cancer diagnosis and application thereof. BACKGROUND

[0002] Lung cancer is the most common primary malignant tumor of the lung, which can spread to the surrounding or even the whole body, and its mortality is higher than that of most cancer types. It is generally believed that lung cancer is related to smoking, but studies have shown that about one-fourth of lung cancer patients worldwide occur in non-smoking populations, which may be caused by environmental and genetic factors.

[0003] The concept of "copper death" was proposed by Tsvetkov, which is a new type of cell death mode with copper dependence, different from common death modes such as apoptosis, pyroptosis, and ferroptosis, and is a current research hotspot. Copper is a static cofactor, which plays an important role in maintaining the homeostasis of copper ions in organisms. When its concentration exceeds the threshold for maintaining the homeostasis of the body, it will have toxic side effects on cells. Studies have found that copper death related genes (CRGs) are abnormally expressed in tumors such as pancreatic cancer, liver cancer, breast cancer, and lung cancer, and are related to tumor occurrence and immune infiltration.

[0004] Copper death related protein FDX1 (ferredoxin) is a reductase, which is an upstream regulator of protein thioacylation modification. On the one hand, it can participate in the regulation of protein thioacylation. On the other hand, FDX1 reduces Cu 2+ to more toxic Cu + , leading to inhibition of Fe-S cluster protein synthesis and induction of cell copper death. Abnormal expression of copper death related proteins can induce the body to produce corresponding autoantibodies. Autoantibodies are antibodies against self organs, tissues, cells and their components, which have the characteristics of easy detection and diagnosis before biopsy, and can be used as biomarkers of diseases. Therefore, in order to reduce the mortality rate of lung cancer and improve the 5-year survival rate, it is urgent to explore lung cancer biomarkers with strong specificity, which will provide new ideas for clinical detection and screening of lung cancer. SUMMARY

[0005] One of the purposes of the present application is to provide a biomarker for lung cancer diagnosis.

[0006] To achieve the above purpose, the present application adopts the following technical solutions:

[0007] A biomarker for lung cancer diagnosis, the biomarker is anti-FDX1 autoantibody.

[0008] Further, the biomarker is blood or plasma anti-FDX1 autoantibody.

[0009] The second object of the present application is to provide an application of a biomarker for lung cancer diagnosis.

[0010] To achieve the above object, the present application adopts the following technical solutions:

[0011] The application of the above-mentioned biomarker for lung cancer diagnosis in the preparation of an in vitro lung cancer diagnosis product.

[0012] Further, the in vitro lung cancer diagnosis product is a reagent for detecting the level of the lung cancer marker.

[0013] Further, the in vitro lung cancer diagnosis product is a kit for detecting the level of the lung cancer marker.

[0014] The third object of the present application is to provide the application of the above-mentioned biomarker for lung cancer diagnosis in the construction of a lung cancer diagnosis model to improve the lung cancer detection efficiency.

[0015] To achieve the above object, the present application adopts the following technical solutions:

[0016] The application of the above-mentioned biomarker for lung cancer diagnosis in the construction of a lung cancer diagnosis model.

[0017] Further, the biomarker anti-FDX1 autoantibody and carcinoembryonic antigen are combined as markers for the construction of a lung cancer diagnosis model.

[0018] Further, the construction of the lung cancer diagnosis model includes obtaining the concentrations of the biomarkers anti-FDX1 autoantibody and carcinoembryonic antigen from a sample, substituting the detected concentrations into a Logistic regression model for analysis, and diagnosing lung cancer according to the analysis results.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The present application first discovers a new lung cancer biomarker anti-FDX1 autoantibody, which can be used to distinguish lung cancer patients from normal people by detecting the expression level of plasma anti-FDX1 autoantibody, and the experimental results also show that anti-FDX1 autoantibody can be used as a detection index for different clinical features of lung cancer. Further, the present application combines anti-FDX1 autoantibody and carcinoembryonic antigen as markers to construct a new lung cancer detection model, and the experimental data show that the combined model can significantly improve the lung cancer detection efficiency, which is of great significance for the diagnosis of lung cancer. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure for the differential expression of anti-FDX1 autoantibody in small samples and the detection efficiency for lung cancer;

[0022] Figure 2 Figure for the differential expression of anti-FDX1 autoantibodies in large samples and the detection efficiency of lung cancer;

[0023] Figure 3 Figure for the expression of anti-FDX1 autoantibodies in lung cancer patients with different clinical characteristics in large samples;

[0024] Figure 4 Figure for the detection efficiency of anti-FDX1 autoantibodies in lung cancer patients with different clinical characteristics in large samples;

[0025] Figure 5 Figure for the detection efficiency of anti-FDX1 autoantibodies and carcinoembryonic antigen CEA as markers for lung cancer. DETAILED DESCRIPTION

[0026] The application will be further described in detail below in conjunction with specific examples. Unless otherwise specified, the equipment and reagents used in each example can be obtained from commercial channels.

[0027] Some materials and reagents involved in the following examples are briefly introduced as follows:

[0028] Sample source: All samples were collected from a certain third-grade class-A hospital in Henan Province, and the approval of the Ethics Committee of Zhengzhou University was obtained. All research subjects have signed the informed consent form.

[0029] FDX1 protein required for the experiment was purchased from Wuhan Huamei Biological Engineering Co., Ltd.; single-component TMB color developing solution was purchased from Beijing Solaybao Co., Ltd.

[0030] I. Examples

[0031] Example 1

[0032] Example 1 explores the expression of anti-FDX1 autoantibodies in large samples and small samples and the detection efficiency of lung cancer. The specific process is as follows:

[0033] 1. Collection and processing of plasma samples

[0034] 1.1, Collecting information of research subjects

[0035] A total of 828 subjects were included in the study, including 414 lung cancer patients (LC) and 414 healthy controls (NC). The study sample was further divided into a small sample detection group (LC = 87; NC = 87) and a large sample validation group (LC = 327; NC = 327). The inclusion criteria for LC included: (1) clear histopathological results and histopathological type (including adenocarcinoma, squamous cell carcinoma); (2) no history of other malignancies; (3) complete medical history; (4) no autoimmune disease; (5) all new cases; (6) patients over the age of 18.

[0036] The inclusion criteria for NC samples included: (1) no history of lung or other malignancies; (2) no autoimmune disease; (3) age over 18 years. The clinical information of the samples used in the experiment is shown in Table 1.

[0037] Table 1

[0038]

[0039] 1.2, Collection and processing of plasma samples

[0040] 3-4 mL of venous blood was collected from the elbow of all study subjects in a 5 mL vacuum blood collection tube containing EDTA-K2 anticoagulant. The blood collection tube was inverted several times to mix the blood and anticoagulant thoroughly. Then, the blood was centrifuged in a regular speed centrifuge (3000 rpm / min) for 5 minutes. After centrifugation, the upper plasma was aspirated into a 1.5 mL centrifuge tube and labeled with the sample number on the top and side of the tube. The tube was stored at -80°C and the blood collection date and storage location were recorded. Before use, the plasma sample was removed and thawed in a 4°C refrigerator to avoid repeated freezing and thawing.

[0041] 2. ELISA method for detecting the expression of anti-FDX1 autoantibodies in the test sample

[0042] The specific process of anti-FDX1 autoantibody detection is as follows:

[0043] (1) Coating antigen protein: dilute FDX1 protein to 0.125 μg / mL using coating solution (weigh 1.5 g sodium carbonate, 2.9 g sodium bicarbonate, 800 mL deionized water (pH = 9.6), then add pure water to 1 L), shake well, and use a syringe to evenly distribute the diluted FDX1 protein (50 μL per well) into the 96-well enzyme-linked immunosorbent plate. The FDX1 protein coating solution should fully cover the bottom of each well of the 96-well enzyme-linked immunosorbent plate, and the plate should be covered with a film and incubated overnight at 4°C.

[0044] (2) Sealing: The next day, shake off the FDX1 protein coating liquid in the enzyme-labeled plate, and dry it on the blotting paper. Add 100 μL of 2% BSA sealing liquid (dissolve 2 g of bovine serum albumin BSA in 100 mL of 1x phosphate buffer, and stir until completely dissolved) to each well, and cover it with a film. Incubate at 37°C for 2 h. The 1x phosphate buffer is prepared by taking 100 mL of 10x PBST and diluting it to 1 L with deionized water;

[0045] (3) Washing the plate: After sealing, remove the enzyme-labeled plate, shake off the sealing liquid, and dry it on the blotting paper. Use the automatic plate washing machine to wash the enzyme-labeled plate 3 times, then remove the enzyme-labeled plate and dry it on the blotting paper until there is no liquid in the bottom of each well.

[0046] (4) Primary antibody incubation: Dilute the antibody diluent (1% BSA, dissolve 1 g of BSA in 100 mL of 1x phosphate buffer, and stir until completely dissolved) to 1:100 for the test plasma. Add 50 μL of diluted test plasma to the sealed 96-well enzyme-labeled plate, cover it with a film, and incubate at 37°C for 1 h.

[0047] (5) Washing the plate: Remove the enzyme-labeled plate, shake off the primary antibody, and dry it on the blotting paper until there is no liquid in the bottom of each well. Use the automatic plate washing machine to wash the enzyme-labeled plate 5 times, then remove the enzyme-labeled plate and dry it on the blotting paper until there is no liquid in the bottom of each well.

[0048] (6) Secondary antibody incubation: Dilute the secondary antibody (1% BSA, dissolve 1 g of BSA in 100 mL of 1x phosphate buffer, and stir until completely dissolved) to a dilution of 7500:1 for the horseradish peroxidase-labeled goat anti-human IgG. Add 50 μL of the diluted secondary antibody to the enzyme-labeled plate, cover it with a film, and incubate at 37°C for 1 h.

[0049] (7) Washing the plate: Remove the enzyme-labeled plate, shake off the secondary antibody, and dry it on the blotting paper until there is no liquid in the bottom of each well. Use the automatic plate washing machine to wash the enzyme-labeled plate 5 times, then remove the enzyme-labeled plate and dry it on the blotting paper until there is no liquid in the bottom of each well.

[0050] (8) Color development: Use the gun to quickly add 50 μL of color developing liquid to the enzyme-labeled plate, and develop it at room temperature in the dark.

[0051] (9) Termination: During the color development process, the color development condition is observed at all times, and after sufficient color development, the termination solution (25 μL per well) is added to the enzyme-labeled plate by the gun; the preparation process of the termination solution: 20 mL of concentrated sulfuric acid is slowly added to a beaker containing 100 mL of deionized water, and a glass rod is continuously stirred. After completely adding, deionized water is slowly added to make up to 200 mL, and after stirring uniformly, it is transferred to a glass bottle and stored at room temperature;

[0052] (10) Detection: The enzyme-labeled instrument is turned on for preheating 15 minutes in advance, and the detection wavelength parameters are set as OD450 and OD620, respectively. The enzyme-labeled plate is immediately placed on the enzyme-labeled instrument for OD value detection after the termination reaction is completed, wherein OD620 is the background value, and the difference between OD450 and OD620 is taken as the absorbance value of the sample to be tested;

[0053] (11) Result analysis: Mann-Whitney U test is used to analyze the difference in the expression level of anti-FDX1 autoantibody; chi-square test is used to analyze the difference in the positive rate; and the AUC, specificity, sensitivity and 95% confidence interval (95% CI) of anti-FDX1 autoantibody are calculated through the ROC curve. When AUC>0.5 and P<0.05, it is considered that the difference has statistical significance, and the SBI value corresponding to the maximum Youden's Index YI is selected as the cutoff value. The results are shown in Figures 1-2 .

[0054] Figure 1 Figure 1 shows the differential expression and detection efficiency of anti-FDX1 autoantibody in small samples. Figure 2 Figure 2 shows the differential expression and detection efficiency of anti-FDX1 autoantibody in large samples. Figures 1-2 It can be seen that the anti-FDX1 autoantibody is highly expressed in lung cancer patients and has high detection efficiency for lung cancer. The AUC values of anti-FDX1 autoantibody in small samples and large samples are 0.921 and 0.806, respectively, and the sensitivity and specificity of small samples are 90.8% and 85.1%, respectively, and the sensitivity and specificity of large samples are 77.7% and 70.0%, respectively. The above results show that the anti-FDX1 autoantibody can be used to distinguish lung cancer patients from normal people.

[0055] Example 2

[0056] Example 2 explores the expression of anti-FDX1 autoantibody in patients with different clinical characteristics of lung cancer in large samples, and the detection process of anti-FDX1 autoantibody is the same as that in Example 1, and the results are shown in Figure 3 .

[0057] Figure 3Figure of anti-FDX1 autoantibody expression in lung cancer patients with different clinical characteristics. From Figure 3 It can be seen that the expression levels of anti-FDX1 autoantibody in plasma of lung cancer patients in early stage and in late stage, in lymph node metastasis positive and in lymph node metastasis negative, in distant metastasis positive and in distant metastasis negative were not different, but the expression was higher in lung cancer patients with smoking history and drinking history.

[0058] Example 3

[0059] Example 3 detected the detection efficiency of anti-FDX1 autoantibody in lung cancer patients with different clinical characteristics in a large sample, and the results are shown in Figure 4

[0060] Figure 4 Figure of detection efficiency of anti-FDX1 autoantibody in lung cancer patients with different clinical characteristics. From Figure 4 It can be seen that the AUC of anti-FDX1 autoantibody for early stage lung cancer and late stage lung cancer was 0.822 (0.781-0.862), 0.817 (0.760-0.874), respectively; for lymph node metastasis positive (LM+) and lymph node metastasis negative (LM-) patients, the AUC was 0.762 (0.672-0.852), 0.837 (0.802-0.871), respectively; for distant metastasis positive (DM+) and distant metastasis negative (DM-), the AUC was 0.762 (0.672-0.852), 0.837 (0.802-0.871), respectively; for drinking patients, the AUC was 0.740 (0.675-0.806), and for non-drinking patients, the AUC was 0.827 (0.790-0.803); for smoking patients, the AUC was 0.780 (0.727-0.832), and for non-smoking patients, the AUC was 0.819 (0.779-0.858). The above results show that anti-FDX1 autoantibody can be used as a detection index for lung cancer with different clinical characteristics.

[0061] Example 4

[0062] Example 4 explored the detection efficiency of anti-FDX1 autoantibody and carcinoembryonic antigen as a marker for lung cancer, and the specific process was as follows:

[0063] Select 104 lung cancer patients and 199 healthy control samples with complete information of anti-FDX1 autoantibody and carcinoembryonic antigen (CEA), and use "forward entry method" to include anti-FDX1 autoantibody index and carcinoembryonic antigen CEA level into Logistic regression model. The combined model is P = e x / (1+e x ​)x = -7.628 + 13.092 x (anti-FDX1 autoantibody) + 0.95 x carcinoembryonic antigen CEA, the experimental data are obtained, and the results are as follows Figure 5 and Table 2.

[0064] Table 2

[0065]

[0066] Figure 5 Figure 1 is a diagram of the detection efficiency of lung cancer by anti-FDX1 autoantibody and carcinoembryonic antigen as markers. Figure 5 It can be known that, compared with single indicators (FDX1: AUC = 0.806; CEA: AUC = 0.511), the combined model (FDX1 + CEA) can significantly improve the detection efficiency of lung cancer (AUC = 0.884). And Table 2 also shows that the combined model can significantly improve the specificity and diagnostic accuracy of lung cancer detection.

[0067] In conclusion, the present application first discovers a new lung cancer biomarker anti-FDX1 autoantibody, which can be used to distinguish lung cancer patients from normal people by detecting the expression level of plasma anti-FDX1 autoantibody, and the experimental results also show that anti-FDX1 autoantibody can be used as a detection indicator for lung cancer with different clinical characteristics. Further, the present application constructs a new lung cancer detection model by combining anti-FDX1 autoantibody and carcinoembryonic antigen as markers, and the experimental data show that the combined model can significantly improve the detection efficiency of lung cancer, which has important significance for the diagnosis of lung cancer.

[0068] The above is only the preferred embodiment of the present application, which is not limited to the above examples. For those skilled in the art, various modifications and changes can be made under the principle of the present application. Any modification, improvement, etc. shall be considered within the protection scope of the present application.

Claims

1. Use of a reagent for detecting a biomarker, the biomarker being anti-FDX1 autoantibody, in the preparation of an in vitro diagnostic product for lung cancer.

2. Use according to claim 1, characterized in that, The biomarker is plasma anti-FDX1 autoantibody.

3. Use according to claim 1, characterized in that, The reagent is a reagent for detecting the biomarker in a sample by enzyme-linked immunosorbent assay.

4. Use according to claim 1, characterized in that, The product, when used for diagnosing lung cancer, has a probability calculation formula for predicting lung cancer as follows: P = -7.628 + 13.092 x (anti-FDX1 autoantibody) + 0.95 x carcinoembryonic antigen CEA, wherein P represents probability, anti-FDX1 autoantibody represents the expression amount of anti-FDX1 autoantibody, and carcinoembryonic antigen CEA represents the expression amount of carcinoembryonic antigen CEA.

5. The use according to claim 1, characterized in that, The product is a kit.

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