Application of FGFBP2 as lung squamous cell carcinoma diagnosis marker

Abstract

The invention belongs to the technical field of molecular markers, and particularly relates to application of FGFBP2 as a lung squamous cell carcinoma diagnosis marker. The invention provides a biomarker for lung squamous cell carcinoma diagnosis, the biomarker is FGFBP2 gene or FGFBP2 protein, the content of the marker in serum of a lung squamous cell carcinoma patient is remarkably increased, the expression level of the marker in lung squamous cell carcinoma tissue is remarkably higher than that of a normal person, and the difference has statistical significance. By detecting the expression level of FGFBP2 in human serum, lung squamous cell carcinoma patients and normal people can be effectively distinguished, and the FGFBP2 can be used for diagnosing lung squamous cell carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of molecular biomarker technology, specifically relating to the application of FGFBP2 as a diagnostic biomarker for squamous cell carcinoma of the lung. Background Technology

[0002] Lung squamous cell carcinoma (LUSC) is the second most prevalent subtype of non-small cell lung cancer (NSCLC) after lung adenocarcinoma (LUAD), accounting for approximately 25-30% of all lung cancer cases. The prognosis of LUSC patients is closely related to the cancer stage at diagnosis. When LUSC is detected early, surgery is the most effective and potentially curative option. However, for advanced LUSC with distant metastases, the 5-year survival rate is typically less than 10%. Currently, among serum biomarkers, carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC-Ag) have single-item positive rates of 35.71% and 69.05%, respectively, still exhibiting low specificity and poor sensitivity. Therefore, there is an urgent need to identify novel biomarkers to improve the early diagnosis rate of LUSC patients. Summary of the Invention

[0003] In view of the problems and shortcomings of the existing technology, the present invention provides the application of FGFBP2 as a diagnostic marker for squamous cell carcinoma of the lung.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides the application of a reagent for detecting the expression level of the FGFBP2 gene or FGFBP2 protein in the preparation of products for the diagnosis of lung squamous cell carcinoma.

[0005] According to the above application, preferably, the product is used to detect the expression level of FGFBP2 gene or FGFBP2 protein in a sample by RT-PCR, real-time quantitative PCR, in situ hybridization, Northern Blot, immunohistochemistry, enzyme-linked immunosorbent assay, protein chip, immunoblotting or microfluidic immunoassay.

[0006] According to the above applications, preferably, the product contains an antigen or antibody that specifically binds to the FGFBP2 protein, or contains primers that specifically amplify the FGFBP2 gene, or contains a probe that specifically detects the FGFBP2 gene.

[0007] More preferably, the antibody is a monoclonal antibody, a polyclonal antibody, or a single-domain antibody.

[0008] According to the above application, preferably, the sample is serum, plasma, interstitial fluid, urine, cells or tissue.

[0009] More preferably, the sample is serum.

[0010] According to the above applications, preferably, the product is a protein chip, a formulation, or a reagent kit.

[0011] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention is the first to discover that the content of FGFBP2 protein in the serum of patients with squamous cell carcinoma of the lung is significantly increased, and the expression level in squamous cell carcinoma tissue of the lung is significantly higher than that of normal people, and the difference is statistically significant. Therefore, FGFBP2 protein can be used as a biomarker for the diagnosis of squamous cell carcinoma of the lung. The AUC value of the ROC curve of FGFBP2 protein for diagnosing and differentiating squamous cell carcinoma of the lung from healthy controls is 0.726, the specificity is 50.67%, and the sensitivity is 99.53%. The existing serum auxiliary diagnostic biomarker for squamous cell carcinoma of the lung, squamous cell carcinoma antigen (SCC), has a sensitivity of 30-60% for diagnosing and differentiating squamous cell carcinoma of the lung from healthy controls. Therefore, compared with the existing serum auxiliary diagnostic biomarker for squamous cell carcinoma of the lung, FGFBP2 has higher sensitivity, and the detection of FGFBP2 protein content in serum can be used for the diagnosis of squamous cell carcinoma of the lung. Moreover, in clinical applications, it can be used for non-invasive screening or in combination with other biomarkers.

[0012] (2) This invention uses serum as the test sample, which has the advantages of easy collection, processing and preservation. The detection of FGFBP2 protein in serum can be used alone as a biomarker for non-invasive diagnosis of squamous cell carcinoma of the lung. The detection of FGFBP2 protein in serum is performed by ELISA, which takes about 3 hours and is easy to operate. Attached Figure Description

[0013] Figure 1 Single-cell sequencing was used to analyze the expression of FGFBP2 mRNA in different lung tissues. Figure 2 To analyze the expression of FGFBP2 mRNA in different cell types of LUSC tissue using single-cell sequencing; Figure 3 To detect the expression of FGFBP2 in LUSC and normal lung tissues by immunohistochemistry; where A is a representative immunohistochemical staining image of FGFBP2 in normal lung tissue and squamous cell carcinoma tissue; B is the expression score of FGFBP2 in 90 cases of squamous cell carcinoma tissue and paired normal tissues; Figure 4 To detect the levels of FGFBP2 in the serum of healthy individuals and patients with LUSC using the ELISA method; Figure 5 ROC curve analysis of FGFBP2 in patients with squamous cell carcinoma of the lung. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Example 1: Study on the expression of FGFBP2 in lung squamous cell carcinoma cells 1. Using single-cell sequencing, compare the differences in cell subsets and gene expression between lung squamous cell carcinoma tissue and normal tissue. (1) Test materials Five cases of normal lung tissue from non-smokers, three cases of normal lung tissue from smokers, five cases of lung tissue from smokers with chronic obstructive pulmonary disease, and three cases of lung tissue from patients with squamous cell carcinoma of the lung were selected from the Department of Thoracic Surgery at Henan Provincial People's Hospital. The lung tissue from patients with squamous cell carcinoma of the lung was confirmed by pathological diagnosis, and the normal lung tissue was the normal tissue adjacent to the cancer that was surgically removed.

[0016] (2) Test methods 1) Cut ≥0.2g of fresh tissue, rinse the surface of blood or impurities with PBS (without calcium and magnesium ions) or physiological saline, and immediately place it into a pre-cooled EP tube containing Miltenyi preservation solution. Seal the centrifuge tube with sealing film and store at 4℃. 2) Under aseptic conditions, wash lung tissue twice with pre-cooled RPMI 1640 + 0.04% BSA medium, and then use surgical scissors to thoroughly mince the tissue into pieces of approximately 0.5 mm. 3 Place the small pieces into freshly prepared enzymatic digestion solution (Trypsin and Collagenase, Type I), and incubate at 37°C for 30 minutes, inverting and mixing once every 5-10 minutes. 3) Filter the digested cell suspension through a BD 40μm cell sieve 1-2 times, centrifuge at 300g for 5 min at 4℃, resuspend the pellet in an appropriate amount of culture medium, add an equal volume of erythrocyte lysis buffer (MACS, catalog number 130-094-183), mix well, let stand at 4℃ for 10 min, centrifuge the cell suspension at 300g for 5 min, discard the supernatant, wash the pellet once with culture medium, centrifuge at 300g for 5 min, discard the supernatant, resuspend the cell pellet in 100μL of culture medium to obtain a single-cell suspension, and detect cell concentration and viability using a Luna cell counter. 4) Single-cell sequencing was performed using the 10X Genomics platform. The single-cell suspension prepared in step 3) was adjusted to a cell concentration of 700-1200 cells / μL. The sequencing and library construction were carried out according to the instructions of the 10×Genomics Chromium Next GEM SingleCell 3ʹ Reagent Kits v3.1 (catalog number: 1000268). 5) The constructed library was sequenced using the Illumina Nova 6000 PE150 platform. The raw data was analyzed using the 10xgenomics official software Cell Ranger (version 7.0.1) and compared with the reference genome (human: GRCh38). The high-throughput single-cell transcriptome data was quantified by identifying barcode markers that distinguish cells in the sequence and UMI markers of different mRNA molecules in each cell, and high-quality control statistics such as cell number, median gene count, and sequencing saturation were obtained.

[0017] 6) Based on the initial quality control by Cell Ranger, the data was further standardized, dimensionality reduced and clustered using the Seurat (version 4.0.0) software package. After marker gene identification, cell types were identified using the SingleR (version 1.4.1) package.

[0018] (3) Test results We used the FindMarkers function in the Seurat (v3.1.1) software package to perform differential expression analysis on normal lung tissue from non-smokers, normal lung tissue from smokers, lung tissue from smokers with COPD, and lung squamous cell carcinoma tissue. We used the Wilcoxon rank-sum test and screened for significantly differentially expressed genes based on a corrected p-value less than 0.05 and a mean log2 fold change greater than 0.5. We statistically analyzed the expression levels of FGFBP2 mRNA in normal lung tissue from non-smokers, normal lung tissue from smokers, lung tissue from smokers with COPD, and lung tissue from patients with lung squamous cell carcinoma. The results are as follows: Figure 1As shown. By Figure 1 It is known that FGFBP2 mRNA is significantly highly expressed only in the lung tissue of patients with squamous cell carcinoma of the lung, while it is lowly expressed in normal lung tissue of non-smokers, normal lung tissue of smokers, and lung tissue of smokers with chronic obstructive pulmonary disease.

[0019] Further statistical analysis was performed on single-cell sequencing data of FGFBP2 expression levels in different cell types (ciliated cells, lung squamous cell carcinoma cells, type II alveolar epithelial cells, type I alveolar epithelial cells, bronchiolar secretory cells, goblet cells, and basal cells) in lung tissue from patients with squamous cell carcinoma of the lung. The results are as follows: Figure 2 As shown. By Figure 2 It is known that FGFBP2 mRNA is highly expressed in lung squamous cell carcinoma cells, but not in other cell types.

[0020] 2. Immunohistochemistry was used to detect the expression of FGFBP2 in LUSC and normal lung tissue. To verify whether FGFBP2 expression is increased in lung squamous cell carcinoma, immunohistochemical staining was used to detect the expression of FGFBP2 in normal lung tissue and lung squamous cell carcinoma tissue.

[0021] (1) Test materials Lung tissues and their paired normal tissues from 90 patients with squamous cell carcinoma of the lung were selected. The tissue microarray was purchased from Shanghai Chipchao Biotechnology Co., Ltd., product number HLugS180Su02.

[0022] (2) Test methods Immunohistochemical staining was performed using an immunochromatographic kit (catalog number: SD3102, Henan Sainuo Biotechnology Co., Ltd., Henan Province), and the specific method is as follows: 1) The paraffin sections were baked at 60℃ for 1.5 hours, and immediately after baking, they were placed in xylene I for 20 minutes to dewax, and then placed in xylene II for 20 minutes to dewax. 2) The sections were placed in 100% ethanol I for 5 min, 100% ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min in sequence, and then rinsed with running water for 10 min. 3) Microwave retrieval: Place the slice in 0.01M sodium citrate buffer, microwave on medium-high for about 3 minutes until boiling, cool to room temperature for 90 seconds, then heat to boiling again. Repeat this heating process 5 times, then cool to room temperature. 4) Rinse the sections in PBS 3 times, 5 min each time, to remove excess water. Then draw a hydrophobic frame around the tissue and add endogenous peroxidase blocking agent into the hydrophobic frame until the tissue is completely covered. Insulate at room temperature for 10 min. 5) Remove the endogenous peroxidase blocking agent, add FGFBP2 antibody (catalog number: HPA039180, Sigma, 1:200) directly to the hydrophobic frame to cover the tissue, and incubate the section in a humidified chamber at 4 °C overnight; 6) Remove the humidified chamber and allow it to return to room temperature. Rinse the tissue three times in PBS, 10 min each time. Add secondary antibody to the tissue and incubate at 37°C for 30 min. Remove the secondary antibody and rinse the sections three times in PBS, 10 min each time. 7) Add freshly prepared DAB staining solution to the tissue, observe it under a microscope, remove the DAB after 2 minutes of staining, and place the section in distilled water for 5 minutes. 8) Counterstaining: Add hematoxylin to the tissue, stain at room temperature for 4 min, rinse with running water for 5 min, differentiate with 1% hydrochloric acid alcohol for 3 s, rinse with running water for 5 min to return to blue; 9) The sections were placed in 70% ethanol for 5 min, 80% ethanol for 5 min, 90% ethanol for 5 min, 95% ethanol I for 5 min, 95% ethanol II for 5 min, 100% ethanol I for 5 min, and 100% ethanol II for 5 min in sequence. After that, they were placed in xylene I for 20 min and xylene II for 20 min. After drying, they were mounted with neutral resin and dried at room temperature. After that, the sections were observed and images were acquired.

[0023] (3) Test results Immunohistochemical staining results as follows Figure 3 As shown, where Figure 3 A shows representative immunohistochemical staining of FGFBP2 in normal lung tissue and squamous cell carcinoma of the lung. Figure 3 B represents the score for FGFBP2 expression levels in 90 lung squamous cell carcinoma tissues and paired normal tissues, calculated by... Figure 3 A and Figure 3 As shown in B, FGFBP2 is significantly highly expressed in lung squamous cell carcinoma tissue.

[0024] Example 2: ELISA detection of serum FGFBP2 expression level The levels of FGFBP2 in the serum of healthy individuals and patients with squamous cell carcinoma of the lung were detected by enzyme-linked immunosorbent assay (ELISA).

[0025] 1. Experimental Samples: A total of 75 healthy individuals and 217 patients with squamous cell carcinoma of the lung were collected from the Department of Laboratory Medicine of Henan Provincial People's Hospital. The serum from healthy individuals was designated as the Normal group (control group), and the serum from patients with squamous cell carcinoma of the lung was designated as the LUSC group.

[0026] The inclusion and exclusion criteria for sample collection are as follows: 1.1 Inclusion criteria: 1.1.1 Lung squamous cell carcinoma patient group (LUSC group): ① The diagnosis was confirmed by histopathology as squamous cell carcinoma of the lung; ② Age 18 and above, gender not limited; ③ Sign an informed consent form, indicating willingness to cooperate with the blood sample collection and data use in this study.

[0027] 1.1.2 Healthy control group (Normal group): ① Chest CT scans over the past year have shown no malignant pulmonary nodules; ② No infections have occurred recently; ③ Sign an informed consent form, indicating willingness to allow the use of their serum samples.

[0028] 1.2 Exclusion criteria (applicable to LUSC and Normal groups): ① Those with other malignant tumors or a history of cancer; ② Recent acute infection, fever, or other conditions that may affect serum indicators; ③ Comorbid severe systemic diseases such as liver and kidney, or underlying diseases that affect the interpretation of serum indicators; ④ Pregnant or breastfeeding women; ⑤ Individuals with mental disorders or who cannot understand the research content; ⑥ Those who have not signed the informed consent form.

[0029] Serum collection: Collect 3 mL of blood using a coagulation tube. After collection, let it stand at 4°C for 4 hours. Remove the blood sample and centrifuge at 1500 rpm for 15 min to separate the serum. Collect the supernatant serum and store it for later use.

[0030] 2. Experimental methods: The expression levels of FGFBP2 protein in serum samples from the Normal and LUSC groups were detected by ELISA using the Human Fibroblast Growth Factor Binding Protein 2 (FGFBP2) ELISA Kit (catalog number: abx351604, Abbexa, Cambridge, UK). The procedure was performed according to the kit instructions. The specific steps for ELISA detection are as follows: (1) Set up standard wells, test sample wells and blank control wells on the pre-coated plate respectively, and record their positions. Make duplicate wells for each standard and perform gradient dilution for the sample wells. (2) Add 100 µL of standard, control or sample to each corresponding well, seal the microplate with sealing film, and incubate at 37°C for 90 min; (3) Remove the sealing film and discard the liquid in the hole. No plate washing is required in this step. (4) Add 100 µL of working solution of reagent A to each well, reseal the plate, and incubate at 37°C for 30 min; (5) Remove the sealing film and discard the liquid. Wash the plate 3 times on a plate shaker with 1X washing buffer at 50 rpm for 5 min each time; (6) Add 100 µL of working solution of reagent B to each well, seal the plate, and incubate at 37°C for 30 min; (7) Discard the liquid, add washing buffer, and wash the plate 5 times as described in the previous step; (8) Add 90 µL of TMB substrate solution to each well, seal the plate, and incubate at 37°C for 15 min, avoiding light exposure; (9) Add 50 µL of stop solution to each well and immediately read the absorbance at 450 nm.

[0031] 3. Experimental Results: The expression levels of FGFBP2 protein in serum samples from the Normal and LUSC groups are as follows: Figure 4 As shown. By Figure 4 It was found that the level of FGFBP2 was significantly increased in the serum of patients with squamous cell carcinoma of the lung (P<0.0001). In 217 patients with LUSC, the protein was detected in 99.54% of the serum samples, with concentrations ranging from 0.47 ng / mL to 807.66 ng / mL.

[0032] Furthermore, SPSS software was used to analyze the expression levels of FGFBP2 protein in serum samples from the Normal and LUSC groups using receiver operating characteristic (ROC) curves. The results are as follows: Figure 5 As shown.

[0033] Depend on Figure 5 The results show that the area under the ROC curve (AUC) of FGFBP2 protein in differentiating LUSC patients from healthy controls is 0.726, indicating some diagnostic value. At a cutoff value of 11.82 ng / mL, the sensitivity reaches 99.53% and the specificity reaches 50.67%. These results suggest that serum FGFBP2, as a diagnostic marker, has better sensitivity and is helpful for the non-invasive diagnosis or screening of squamous cell carcinoma of the lung.

Claims

1. Application of reagents for detecting the expression level of FGFBP2 gene or FGFBP2 protein in the preparation of products for the diagnosis of lung squamous cell carcinoma.

2. The application according to claim 1, characterized in that, The product is used to detect the expression level of FGFBP2 gene or FGFBP2 protein in samples by RT-PCR, real-time quantitative PCR, in situ hybridization, Northern Blot, immunohistochemistry, enzyme-linked immunosorbent assay, protein chip, immunoblotting or microfluidic immunoassay.

3. The application according to claim 2, characterized in that, The product contains an antigen or antibody that specifically binds to the FGFBP2 protein, or primers that specifically amplify the FGFBP2 gene, or probes that specifically detect the FGFBP2 gene.

4. The application according to claim 3, characterized in that, The antibody is a monoclonal antibody, a polyclonal antibody, or a single-domain antibody.

5. The application according to claim 2, characterized in that, The sample may be serum, plasma, interstitial fluid, urine, cells, or tissue.

6. The application according to claim 5, characterized in that, The sample was serum.

7. The application according to any one of claims 1 to 6, characterized in that, The product is a protein chip, formulation, or reagent kit.

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