Application of reagent for detecting SENP7 protein expression level in preparation of lung adenocarcinoma diagnosis or prognosis product

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses application of a reagent for detecting the expression level of SENP7 protein in preparation of lung adenocarcinoma diagnosis or prognosis products, and the amino acid sequence of the SENP7 protein is shown as SEQ ID NO.1. A research result shows that the expression of the SENP7 protein in lung adenocarcinoma tissues is obviously higher than that in para-carcinoma tissues, and the expression level of the SENP7 protein is closely related to TNM staging and total lifetime of a patient, so that the SENP7 protein can be used as an important biomarker and is used for developing products such as chips, kits or test paper for diagnosing lung adenocarcinoma and evaluating prognosis of the patient, and the application prospect is wide. The kit has the advantages of high sensitivity, good specificity and simplicity and convenience in operation, and a new target spot is provided for treatment of lung adenocarcinoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of reagents for detecting SENP7 protein expression levels in the preparation of diagnostic or prognostic products for lung adenocarcinoma. Background Technology

[0002] Lung cancer is one of the most common malignant tumors. Statistics show that it ranks first in both incidence and mortality worldwide, seriously threatening human life and health. However, the etiology of lung cancer is not yet fully understood. Extensive data indicates that risk factors for lung cancer include smoking (including secondhand smoke), asbestos, radon, arsenic, ionizing radiation, halogenated alkenes, polycyclic aromatic compounds, and nickel. Current treatments for lung cancer include surgery and drug therapy. Surgical treatment involves surgical removal of the lung; however, excessive removal of lung tissue can negatively impact postoperative survival. Drug therapy mainly includes chemotherapy and targeted therapy. Long-term chemotherapy can easily lead to drug resistance, reducing treatment effectiveness. Targeted therapy offers a wide variety of drugs, but requires testing for tumor cell gene targets. Therefore, research on the diagnosis and prognosis of lung adenocarcinoma is essential. This research will help in the early detection and diagnosis of lung adenocarcinoma, promote early treatment, increase the chances of successful treatment, and thus reduce mortality.

[0003] Molecular biomarkers have unique advantages in cancer diagnosis and prognosis. Molecular subtyping based on driver genes can be directly linked to specific targeted drugs, while providing more accurate biological prognostic information, achieving precise stratification, and predicting the efficacy of specific therapies and dynamically monitoring disease evolution. However, there are still relatively few molecular biomarkers for lung adenocarcinoma. Therefore, it is necessary to explore more high-performance molecular biomarkers for lung adenocarcinoma. Summary of the Invention

[0004] This invention provides the application of reagents for detecting SENP7 protein expression levels in the preparation of diagnostic or prognostic products for lung adenocarcinoma. The research results of this invention demonstrate that SENP7 protein can serve as a molecular marker for the accurate diagnosis of lung adenocarcinoma, exhibiting high sensitivity, good specificity, simplicity, safety, effectiveness, and patient acceptance, with standardized diagnostic criteria. It can be used to develop products such as chips, kits, or test strips for diagnosing lung adenocarcinoma and assessing patient prognosis, providing a new target for the treatment of lung adenocarcinoma.

[0005] This invention provides the application of a reagent for detecting the expression level of SENP7 protein in the preparation of diagnostic or prognostic products for lung adenocarcinoma, wherein the amino acid sequence of the SENP7 protein is shown in SEQ ID NO.1; SEQ ID NO.1:

[0006] The results of this invention show that SENP7 protein can serve as a molecular marker for the accurate diagnosis of lung adenocarcinoma. It has high sensitivity, good specificity, is simple and easy to use, the diagnostic process is safe and effective, it is easily accepted by patients, and the diagnostic criteria are standardized. It can be used to develop products such as chips, kits or test strips for diagnosing lung adenocarcinoma and assessing patient prognosis, providing a new target for the treatment of lung adenocarcinoma.

[0007] Furthermore, the product is a chip, a reagent kit, or a test strip.

[0008] Furthermore, the reagent kit is SENP7 Gene detection kit or SENP7 protein detection kit.

[0009] Furthermore, the SENP7 protein detection kit includes a detection reagent for detecting a marker for the SENP7 protein; the marker is any one of a radioactive isotope, a nucleotide chromophore, an enzyme, a fluorescent molecule, a chemiluminescent moiety, and a bioluminescent moiety; the marker is used to label the SENP7 protein.

[0010] Furthermore, the SENP7 protein detection kit includes antigen retrieval solution, hydrogen peroxide solution, PBST solution, SENP7 primary antibody, and HRP-labeled goat anti-rabbit secondary antibody.

[0011] Furthermore, the methods for detecting the markers include radiometric methods, immunological methods, fluorescence methods, flow cytometry, latex turbidimetry, biochemical methods, enzymatic methods, hybridization methods, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, chromatography, chemiluminescence methods, magnetoelectric methods, or photoelectric conversion methods.

[0012] Furthermore, the chip includes a gene chip and a protein chip.

[0013] Furthermore, the gene chip includes nucleotide probes for detecting the transcriptional level of the SENP7 gene; the protein chip includes a specific binding reagent for the SENP7 protein.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses immunohistochemical staining to detect samples and has determined that SENP7 protein can be used as a molecular marker for diagnosing lung adenocarcinoma (AUC 0.879, 95% confidence interval 0.830 to 0.929, p < 0.001). It has high sensitivity and specificity (when the immunohistochemical score H-Score of SENP7 protein is 4.5, the sensitivity is 81.9% and the specificity is 88.6%; when the H-Score is greater than 4.5, the patient is diagnosed with lung adenocarcinoma with an accuracy of 85.2%). It is also simple and easy to perform, the diagnostic process is safe and effective, it is easily accepted by patients, the diagnostic criteria are uniform, and it is less affected by personal subjective factors.

[0015] This invention provides the application of reagents for detecting SENP7 protein expression levels in the preparation of diagnostic or prognostic products for lung adenocarcinoma. The results of this invention show that SENP7 protein expression in lung adenocarcinoma tissues is significantly higher than in adjacent normal tissues, and can serve as a biomarker for the diagnosis of lung adenocarcinoma. Furthermore, SENP7 protein expression is closely related to TNM stage and survival in lung adenocarcinoma patients, and is of great significance for prognostic assessment of lung adenocarcinoma. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The figure shows the comparison results of SENP7 expression in lung adenocarcinoma and adjacent normal tissues. *** indicates p<0.001.

[0018] Figure 2 The correlation between SENP7 expression level and prognosis in patients with lung adenocarcinoma was p < 0.001. Figure 3 ROC analysis results for SENP7 to predict the occurrence of lung adenocarcinoma. Detailed Implementation

[0019] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific 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. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0020] Example 1: Expression analysis of SENP7 protein in lung adenocarcinoma and adjacent tissues.

[0021] 1. Source of tissue samples: The 180 samples from 94 patients with lung adenocarcinoma were obtained from the tissue sample bank of Shanghai Xinchao Biotechnology Co., Ltd. The samples included 94 cancerous tissues and 86 matched adjacent normal tissues located 1.5 cm from the cancer. Surgery occurred between January 2004 and June 2009, with a final follow-up date of August 2014. All cases were pathologically confirmed as lung adenocarcinoma, with ages ranging from 20 to 84 years, and a median age of 60 years. The case data of the 94 patients are shown in Table 1.

[0022] Table 1. Clinicopathological data of 94 patients with lung adenocarcinoma 2. Tissue chip fabrication The tissue microarray was fabricated by Shanghai Xinchao Biotechnology Co., Ltd. All donor tissue blocks underwent routine pathological sectioning and HE staining, followed by secondary diagnosis by pathologists who marked typical pathological sites on the HE sections. Using a Beecher Instruments Inc. tissue microarray fabrication instrument, 1.5 mm diameter holes were drilled in blank recipient tissue blocks. Then, based on the markings on the HE sections, target tissue cores were obtained from the corresponding locations on the donor tissue blocks and placed into the array wells of the recipient blocks. This process was repeated to create a matching array of lung adenocarcinoma and adjacent normal tissue (HLugA180Su06). Serial sections (4 μm thickness) were prepared using a Leica microtome (Germany). The sections were mounted on imported glass slides treated to prevent detachment, yielding the tissue microarray.

[0023] 3. Immunohistochemical staining Immunohistochemical experiments were performed on the prepared tissue microarrays using a two-step immunohistochemistry kit from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0024] The two-step immunohistochemistry kit contains antigen retrieval solution, hydrogen peroxide solution, and HRP-labeled goat anti-rabbit secondary antibody.

[0025] The tissue microarray was placed on a slide oven and baked at 65°C for 2 hours. Dewaxing was then performed in a fume hood. After dewaxing, the microarray was rinsed in tap water for 5 minutes, then placed in an immunohistochemistry cassette containing ddH2O and washed on a shaker for 5 minutes. The ddH2O in the cassette was discarded, and antigen retrieval solution was added. The temperature of the constant-temperature water bath was adjusted to 99°C. The cassette containing the tissue microarray was placed in the water bath, and the real-time temperature in the cassette was measured with a thermometer. Timing was started when the temperature reached 95°C, and antigen retrieval was performed for 18 minutes.

[0026] After antigen retrieval, the immunohistochemistry cassette was removed from the water bath. The tissue microarray was allowed to return to room temperature (25°C). The antigen retrieval solution in the cassette was discarded, and PBST solution was added. The microarray was washed three times, 5 min each time, on a shaker. The tissue microarray was then removed from the cassette, and the surrounding liquid was wiped clean with absorbent paper. An immunohistochemical pen was used to draw a circle around the tissue, enclosing it within the circle. The microarray was then placed in a humidified chamber, and 3% hydrogen peroxide solution was added to the tissue. The microarray was incubated in the dark for 10 min to remove peroxidase. After peroxidase removal, PBST solution was added to the cassette, and the tissue microarray was placed in it. The microarray was washed three times, 10 min each time, on a shaker. The tissue microarray was removed from the cassette, and any remaining liquid was shaken off. The microarray was then placed in a humidified chamber, and blocking solution was added to the tissue. The humidified chamber was then closed and incubated at room temperature for 1.5 h.

[0027] After blocking, the blocking solution was removed from the tissue, and the tissue chip was placed in a humidified chamber. SENP7 primary antibody (Invitrogen, catalog number PA5-99191) was prepared at a ratio of 1:200 using antibody dilution buffer (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.). The prepared primary antibody solution was added dropwise to the tissue, the chamber was capped, and incubated overnight at 4°C. After primary antibody incubation, the humidified chamber was removed from the 4°C chamber and allowed to return to room temperature (25°C) for 30 minutes. After returning to room temperature, PBST solution was added to the immunohistochemistry cassette, the tissue chip was placed inside, and the cassette was washed three times for 10 minutes each time on a shaker.

[0028] After removing the PBST solution from the tissue, place the tissue chip in a humidified chamber and add HRP-labeled goat anti-rabbit secondary antibody (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) to the tissue. Cover the chamber and incubate at room temperature (25℃) for 1 h. After the secondary antibody incubation is complete, add PBST solution to the immunohistochemistry cassette, place the tissue chip in it, and wash three times on a shaker for 10 min each time.

[0029] After removing the PBST solution from the tissue, place the tissue microarray under a microscope. Prepare DAB chromogenic solution at a ratio of 1:20 and add it to the tissue microarray for staining. When the specific staining is obvious and the background is clean, place the tissue microarray in tap water to stop the staining. Immerse the tissue microarray in hematoxylin staining solution for 2.5 min, rinse in tap water for 5 min, dip the tissue microarray briefly in hydrochloric acid alcohol, remove it quickly, and rinse in tap water for 5 min. Immerse the tissue microarray in dilute ammonia solution for inversion blueing for 1.5 min, rinse in tap water for 5 min, and dehydrate the tissue. After dehydration, remove the slide from the clearing solution, add neutral resin to the tissue, and then cover with a coverslip to complete the immunohistochemical staining.

[0030] 4. Quantitative analysis Immunohistochemical staining results were photographed, and ImageJ software was used to convert the positive area and staining intensity within each section into corresponding H-Score values, achieving semi-quantitative analysis of tissue staining. The H-Score for histochemical staining ranged from a minimum of 0 to a maximum of 8, with a median of 4. Patients were grouped according to their H-Score scores: those above the median were classified as high-expression, and those below or equal to the median were classified as low-expression.

[0031] 5. Statistical Analysis The expression of SENP7 protein in lung adenocarcinoma and adjacent normal tissues was analyzed using a t-test. The correlation between SENP7 protein expression and clinical indicators in lung adenocarcinoma patients was analyzed using a chi-square test. The correlation between SENP7 protein expression and prognosis in lung adenocarcinoma patients was analyzed using Kaplan-Meier survival analysis and log-rank statistical tests for univariate survival. p < 0.05 was considered statistically significant.

[0032] 6. Test Results 6.1 Expression analysis of SENP7 protein in lung adenocarcinoma and adjacent normal tissues Immunohistochemical analysis results as follows Figure 1 As shown, the expression of SENP7 protein in lung adenocarcinoma tissue was significantly higher than that in adjacent normal tissue (p<0.001).

[0033] 6.2 Correlation between SENP7 protein and clinical indicators in patients with lung adenocarcinoma Patients with lung adenocarcinoma were grouped according to factors such as age, sex, or TNM stage. The correlation between different factors and SENP7 protein expression was analyzed using the chi-square test. The results are shown in Table 2. The expression level of SENP7 protein in lung adenocarcinoma was not significantly related to the patient's age and sex (p>0.05), but was closely related to the patient's TNM stage (p<0.001).

[0034] Table 2. Correlation between SENP7 protein expression and clinical indicators in lung adenocarcinoma patients. 6.3 Follow-up of patients with lung adenocarcinoma The surgeries were performed between January 2004 and June 2009, with a final follow-up date of August 2014. Univariate analysis of survival using Kalplan-Meier survival analysis and log-rank statistical tests showed that... Figure 2 As shown, lung adenocarcinoma patients with low SENP7 expression in cancer tissue had a longer overall survival (p<0.001), and their 5-year survival rate was significantly higher than that of lung adenocarcinoma patients with high SENP7 expression.

[0035] The above results indicate that the detection of SENP7 is of great significance for the diagnosis and prognosis of lung adenocarcinoma.

[0036] Example 2: Validation of SENP7 protein in the diagnosis of lung adenocarcinoma.

[0037] 1. Source of tissue samples The cancer tissue samples from 105 patients with lung adenocarcinoma and the adjacent normal tissue samples from 105 patients were obtained from the tissue sample bank of Shanghai Xinchao Biotechnology Co., Ltd.

[0038] 2. Tissue chip fabrication The tissue microarrays were fabricated by Shanghai Chipover Biotechnology Co., Ltd. Using the TMAGrand Master tissue microarray fabrication instrument, target tissue cores were obtained by drilling holes in the recipient paraffin blocks and placing them into the array wells of the recipient paraffin blocks. The hole diameter was 1.5 mm. This process was repeated to finally fabricate the dot array blocks HLugA060PG02 and HLugA150CS03 of lung adenocarcinoma tissue and adjacent normal tissue. Paraffin sections were continuously sectioned at a thickness of 4 μm using a Leica paraffin microtome. The sections were then mounted on imported glass slides treated to prevent detachment, thus obtaining the tissue microarrays.

[0039] 3. Immunohistochemical staining and quantitative analysis The operating steps are the same as in Example 1.

[0040] 4. Statistical Analysis The diagnostic efficacy of SENP7 protein expression intensity value for lung adenocarcinoma was analyzed using ROC curve analysis.

[0041] 5. Test Results AUC, or the area under the ROC curve, is the most commonly used parameter for evaluating the characteristics of the ROC curve and an important indicator of experimental accuracy. Figure 3The area under the curve (AUC) was 0.879, with a 95% confidence interval of 0.830 to 0.929 (p < 0.001), indicating that SENP7 protein can serve as a biomarker for diagnosing lung adenocarcinoma. When the immunohistochemical score (H-Score) of SENP7 protein was 4.5, the sensitivity was 81.9% and the specificity was 88.6%. In individual testing, a SENP7 protein immunohistochemical score (H-Score) greater than 4.5 was used to diagnose lung adenocarcinoma, with an accuracy of 85.2%.

[0042] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments.

[0043] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a reagent for detecting SENP7 protein expression levels in the preparation of diagnostic or prognostic products for lung adenocarcinoma, characterized in that, The amino acid sequence of the SENP7 protein is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The product is a chip, reagent kit, or test strip.

3. The application according to claim 2, characterized in that, The kit is SENP7 Gene detection kit or SENP7 protein detection kit.

4. The application according to claim 3, characterized in that, The SENP7 protein detection kit includes a detection reagent for detecting a marker for the SENP7 protein; the marker is any one of a radioactive isotope, a nucleotide chromophore, an enzyme, a fluorescent molecule, a chemiluminescent moiety, and a bioluminescent moiety; the marker is used to label the SENP7 protein.

5. The application according to claim 4, characterized in that, The SENP7 protein detection kit includes antigen retrieval solution, hydrogen peroxide solution, PBST solution, SENP7 primary antibody, and HRP-labeled goat anti-rabbit secondary antibody.

6. The application as described in claim 4, characterized in that, The methods for detecting the markers include radiometric methods, immunological methods, fluorescence methods, flow cytometry, latex turbidimetry, biochemical methods, enzymatic methods, hybridization methods, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, chromatography, chemiluminescence methods, magnetoelectric methods, or photoelectric conversion methods.

7. The application according to claim 2, characterized in that, The chips include gene chips and protein chips.

8. The application according to claim 7, characterized in that, The gene chip includes nucleotide probes for detecting the transcriptional level of the SENP7 gene; the protein chip includes a specific binding reagent for the SENP7 protein.

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