Application of LRSAM1 protein in preparation of lung adenocarcinoma diagnosis or prognosis product

Abstract

The invention relates to a tumor marker, in particular to application of LRSAM1 protein in preparation of lung adenocarcinoma diagnosis or prognosis products, and the amino acid sequence of the LRSAM1 protein is as shown in SEQ ID NO.1. The research result shows that the expression of the LRSAM1 protein in the lung adenocarcinoma tissue is obviously higher than that of the LRSAM1 protein in the adjacent para-carcinoma tissue, and the LRSAM1 protein can be used as a biomarker for diagnosing the lung adenocarcinoma. In addition, expression of the LRSAM1 protein is closely related to pathological grading, T staging, N staging, M staging and TNM staging. According to follow-up visit of lung adenocarcinoma patients, the LRSAM1 protein is of great significance to prognosis of lung adenocarcinoma, and a new target spot is provided for treatment of lung adenocarcinoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor markers, and specifically relates to use of LRSAM1 protein in preparing a lung adenocarcinoma diagnosis or prognosis product. Background Art

[0002] Lung cancer is a common malignant tumor that mainly originates from lung tissue cells. There are two pathological types, including non-small cell lung cancer and small cell lung cancer, of which non-small cell lung cancer accounts for about 80% of all lung cancers. Lung adenocarcinoma originates from the bronchial mucosal epithelium and mucous glands and is a type of non-small cell lung cancer. Early lung adenocarcinoma usually has no obvious symptoms. Some patients may experience symptoms such as coughing, sputum, chest pain, and shortness of breath, but these symptoms are often non-specific and easily overlooked. Due to the lack of obvious early symptoms of lung adenocarcinoma, 75% of patients have already developed to the middle and late stages when diagnosed, and the failure to intervene and treat early leads to a poor prognosis for lung cancer. Therefore, it is necessary to further explore new diagnostic and prognostic markers for lung adenocarcinoma to provide new indicators for individualized precision treatment. Summary of the invention

[0003] In order to solve the defect that lung cancer cannot be discovered and treated as early as possible in the prior art, the present invention provides a new use of LRSAM1 protein, specifically providing the use of LRSAM1 protein in preparing a lung adenocarcinoma diagnosis or prognosis product. The present invention uses an immunohistochemical method to detect the protein expression intensity value of LRSAM1 protein, which is of great significance for the prognosis of lung adenocarcinoma and provides a new target for the treatment of lung adenocarcinoma.

[0004] The present invention provides a use of a detection reagent for LRSAM1 protein in preparing a lung adenocarcinoma diagnosis and / or a diagnostic product, wherein the amino acid sequence of the LRSAM1 protein is shown in SEQ ID NO.1.

[0005] SEQ ID NO.1:

[0006] .

[0007] Preferably, the product includes a chip, a test kit, a test paper or a nucleic acid membrane strip.

[0008] Preferably, the chip includes a gene chip and a protein chip.

[0009] Preferably, the gene chip comprises nucleotide probes for detecting the transcription level of the LRSAM1 gene; and the protein core comprises a specific binding agent for the LRSAM1 protein.

[0010] Preferably, the kit comprises a LRSAM1 gene detection kit and a LRSAM1 protein detection kit.

[0011] Preferably, the diagnostic and / or prognostic sample is lung adenocarcinoma and / or adjacent cancer tissue.

[0012] Preferably, the diagnostic and / or prognostic samples are from patients with lung adenocarcinoma, suspected patients with lung adenocarcinoma, people susceptible to lung adenocarcinoma, people at high risk of lung adenocarcinoma or healthy people.

[0013] Preferably, the detection reagent is a reagent capable of detecting a marker, wherein the marker is any one of a radioactive isotope, a nucleotide chromophore, an enzyme, a fluorescent molecule, a chemiluminescent part, a magnetic particle and a bioluminescent part; and the marker is used to label the LRSAM1 protein.

[0014] Preferably, the radioactive isotopes include 32P, 35S and 125I.

[0015] Preferably, the nucleotide chromophore comprises pyrene, perylene and naphthylamide.

[0016] Preferably, the enzymes include horseradish peroxidase and alkaline phosphatase.

[0017] Preferably, the fluorescent molecules include fluorescein isothiocyanate, rhodamine, Cy3 and Cy5.

[0018] Preferably, the chemiluminescent moiety comprises acridinium esters, luminol and luminol derivatives.

[0019] Preferably, the bioluminescent moiety comprises biotin and digoxigenin.

[0020] Preferably, the method for detecting the marker is a radiometric method, an immunological method, a fluorescent method, a flow cytometry, a latex turbidimetry, a biochemical method, an enzymatic method, a hybridization method, a gas chromatography-mass spectrometry method, a liquid chromatography-mass spectrometry method, a chromatography method, a chemiluminescence method, a magnetoelectric method or a photoelectric conversion method.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides the use of LRSAM1 protein in preparing a product for diagnosis or prognosis of lung adenocarcinoma. The research results of the present invention show that the expression of LRSAM1 protein in lung adenocarcinoma tissue is significantly higher than that in adjacent paracancerous tissue, and can be used as a biomarker for diagnosis of lung adenocarcinoma. In addition, the expression of LRSAM1 protein is closely related to pathological grade, T stage, N stage, M stage and TNM stage.

[0023] The present invention uses immunohistochemistry to detect samples and determines that LRSAM1 protein can be used as a molecular marker for diagnosing lung adenocarcinoma. The method is simple and easy to perform, the diagnostic process is safe and effective, and it is easily accepted by patients. The diagnostic criteria are unified and less affected by personal subjective factors.

[0024] The research results of the present invention show that the expression of LRSAM1 protein is closely related to pathological grading, T staging, N staging, M staging and TNM staging. According to the follow-up of lung adenocarcinoma patients, it is known that LRSAM1 protein is of great significance for the prognosis of lung adenocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The diagram represents the expression of LRSAM1 protein in lung adenocarcinoma tissue and adjacent para-cancerous tissue, wherein A represents the expression of LRSAM1 protein in lung adenocarcinoma tissue; B represents the expression of LRSAM1 protein in adjacent para-cancerous tissue.

[0026] Figure 2 The results of quantitative analysis of the expression level of LRSAM1 protein in lung adenocarcinoma tissues and adjacent paracancerous tissues.

[0027] Figure 3 The correlation between LRSAM1 protein expression and clinical indicators of patients with lung adenocarcinoma. A represents the correlation between LRSAM1 protein expression and gender; B represents the correlation between LRSAM1 protein expression and age.

[0028] Figure 4 The correlation between LRSAM1 protein expression and clinical indicators of patients with lung adenocarcinoma. A represents the correlation between LRSAM1 protein expression and T stage; B represents the correlation between LRSAM1 protein expression and N stage; C represents the correlation between LRSAM1 protein expression and M stage; D represents the correlation between LRSAM1 protein expression and TNM stage; E represents the correlation between LRSAM1 protein expression and pathological grade.

[0029] Figure 5 To analyze the correlation between LRSAM1 protein expression and prognosis of lung adenocarcinoma.

[0030] Figure 6 It is the ROC curve diagram of the protein expression intensity value of LRSAM1 protein. DETAILED DESCRIPTION

[0031] The present invention is described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0032] The inventive concept of the present invention is as follows:

[0033] Lung cancer is a common malignant tumor. It is often easily ignored because of its mild early symptoms. Most patients have already developed to the middle or late stage when diagnosed. Failure to intervene and treat early leads to a poor prognosis of lung cancer. Therefore, it is necessary to further explore new diagnostic and prognostic markers for lung adenocarcinoma to provide new indicators for individualized precision treatment.

[0034] Based on this, the present invention provides the use of a detection reagent for LRSAM1 protein in the preparation of a lung adenocarcinoma diagnosis and / or prognosis product, and the amino acid sequence of the LRSAM1 protein is shown in SEQ ID NO. 1. In the present invention, the protein expression intensity value of the LRSAM1 protein in the sample to be tested is detected by immunohistochemistry; if the protein expression intensity value of the LRSAM1 protein in the sample to be tested is greater than 78.36, the patient is judged to be a patient with lung adenocarcinoma or at high risk of lung adenocarcinoma, which is of great significance for the prognosis of lung adenocarcinoma and provides a new target for the treatment of lung adenocarcinoma.

[0035] Example 1

[0036] Use of LRSAM1 protein in preparing lung adenocarcinoma diagnosis or prognosis products

[0037] Experiment 1: Analysis of LRSAM1 protein expression in lung adenocarcinoma and adjacent tissues.

[0038] S1. Source of tissue samples, the method is as follows:

[0039] 80 samples of lung adenocarcinoma patients were collected from the tissue sample bank of Shanghai Xinchao Biotechnology Co., Ltd. The surgery time was from January 2010 to December 2012, and the final follow-up time was October 2022. All cases were pathologically confirmed as lung adenocarcinoma. There were 28 males and 52 females, aged 30 to 82 years, with a median age of 60 years. Each case contained cancer tissue and matching adjacent tissue 1.5 cm away from the cancer. The patient case data are shown in Table 1.

[0040] Table 1 Clinical pathological data of 80 patients with lung adenocarcinoma

[0041]

[0042] S2. Tissue chip production, the method is as follows:

[0043] The tissue chip was made by Shanghai Xinchao Biotechnology Co., Ltd. All donor tissue wax blocks were routinely sliced ​​and stained with HE. The pathologists made a secondary diagnosis and marked the typical pathological sites on the HE sections. The tissue chip maker Beecher Instruments. Inc was used to punch holes in the blank wax block of the recipient wax block. The hole diameter was 1.5 mm. Then, according to the marking range on the HE sheet, the target tissue core was obtained at the corresponding position of the donor tissue wax block and placed in the array hole of the recipient wax block. The above steps were repeated to finally make a 160-point array block HCol-Ade160Sur-01 that matched lung adenocarcinoma tissue and adjacent tissue. The slicer was used to slice continuously at a thickness of 5 μm, and the slices were mounted on imported slides that had been treated with anti-slipping to make tissue chips. The slicer was purchased from Leica, Germany.

[0044] S3. Immunohistochemistry experiment, the method is as follows:

[0045] The EnVision two-step method was used to perform immunohistochemistry experiments on the prepared tissue chips.

[0046] (1) The paraffin sections were baked in an oven at 67°C for 2 hours and rinsed three times with phosphate buffered saline (PBS), pH 7.4, for 3 minutes each time.

[0047] (2) Use pH 6.0 citrate buffer to heat in microwave until boiling, then place the dewaxed and hydrated tissue sections on a high-temperature plastic slice rack, put them into the boiling buffer, and microwave them at medium speed for 10 minutes. After that, take out the microwave box and let it cool naturally under running water. Take out the slides from the buffer, rinse them twice with distilled water, and then rinse them three times with PBS, each time for 3 minutes.

[0048] (3) Add 1 drop of 3% H2O2 to each slice and incubate at room temperature for 10 minutes to block the activity of endogenous peroxidase. Rinse with PBS three times, 3 minutes each time.

[0049] (4) Remove the PBS solution, add 1 drop of LRSAM1 antibody to each section at a dilution ratio of 1:100, and incubate at room temperature for 2 hours. Rinse with PBS three times, 3 minutes each time.

[0050] (5) Add 1 drop of HRP-labeled goat anti-rabbit secondary antibody to each section and incubate at room temperature for 60 minutes. Rinse with PBS three times, 5 minutes each time.

[0051] (6) Add 1 drop of DAB coloring solution to each slice and observe the color development under a microscope. When the staining result turns brown, stop. Rinse with PBS three times, 5 minutes each time.

[0052] S4. Quantitative analysis, the method is as follows:

[0053] The staining results were scanned and photographed, and the number of positive cells and their staining intensity in each slice were converted into corresponding H-Score values ​​to achieve the purpose of semi-quantitative tissue staining. The lowest H-Score of histochemistry score was 24.36, the highest was 152.75, and the median was 96.15. The H-Score score was used for grouping, with those above the median being the high expression group and those below the median being the low expression group.

[0054] S5. Statistical analysis, the method is as follows:

[0055] The expression of LRSAM1 protein in lung adenocarcinoma and adjacent tissues was analyzed by t-test. The correlation between LRSAM1 protein expression and clinical indicators of lung adenocarcinoma patients was analyzed by Spearman's correlation analysis. The correlation between LRSAM1 protein and the prognosis of lung adenocarcinoma patients was analyzed by Kaplan-Meier survival analysis and log-rank statistical test for univariate analysis of survival. p<0.05 was statistically significant.

[0056] S6. Experimental results are as follows:

[0057] (1) Analysis of LRSAM1 protein expression in lung adenocarcinoma and adjacent tissues.

[0058] Immunohistochemical analysis results showed that LARSAM1 protein was expressed in the cytoplasm of lung adenocarcinoma and adjacent tissues. Figure 1 As shown in Figure 2, t-test analysis showed that the expression of LARSAM1 protein in lung adenocarcinoma was significantly higher than that in adjacent tissues, p<0.001. Figure 2 .

[0059] (2) Correlation between LRSAM1 protein and clinical indicators of patients with lung adenocarcinoma.

[0060] Lung adenocarcinoma patients were grouped according to age, gender, T stage, N stage, M stage, TNM stage or pathological grade, and the correlation between different factors and LARSAM1 protein expression was tested by Pearson correlation analysis. The results are shown in Table 2. Figure 3 and Figure 4 As shown, the expression of LARSAM1 protein in lung adenocarcinoma had no significant statistical relationship with the patient's gender or age, p>0.05; it was closely related to the patient's T stage, N stage, M stage, TNMT stage and pathological grade, p<0.05.

[0061] Table 2 Correlation between LRSAM1 protein expression and clinical indicators in patients with lung adenocarcinoma

[0062]

[0063]

[0064] (3) Follow-up of patients with lung adenocarcinoma was as follows: surgery was performed from January 2010 to December 2012, and the final follow-up was October 2022. The univariate analysis of survival using the Kalplan-Meier survival analysis method and the log-rank statistical test showed that, Figure 5 As shown in the results, lung adenocarcinoma patients with low expression of LRSAM1 in cancer tissues 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 expression of LRSAM1. This result suggests that the detection of LRSAM1 is of great significance for the prognosis of lung adenocarcinoma.

[0065] Experiment 2: Validation of LRSAM1 protein in the diagnosis of lung adenocarcinoma.

[0066] S1. Sources of tissue samples are as follows:

[0067] 80 samples from lung adenocarcinoma patients and 80 normal alveolar samples were obtained from the tissue sample bank of Shanghai Weiao Biotechnology Co., Ltd., including 60 male samples and 100 female samples, aged 30 to 75 years old, with a median age of 59 years old.

[0068] S2. Tissue chip production, as follows:

[0069] The tissue chip was made by Shanghai Viao Biotechnology Co., Ltd. The target tissue core was punched in the recipient wax block using the tissue chip making instrument TMAGrandMaster and placed in the array hole of the recipient wax block. The hole diameter was 1.5 mm. The above steps were repeated to finally make a 160-point array block ZL-LUC1601 of lung adenocarcinoma tissue and normal alveolar tissue. The slices were sliced ​​continuously with a thickness of 5 μm using a microtome, and the slices were mounted on imported slides that had been treated to prevent detachment to make tissue chips.

[0070] S3. Immunohistochemistry experiments, as follows:

[0071] The EnVision two-step method was used to perform immunohistochemistry experiments on the prepared tissue chips.

[0072] (1) The paraffin sections were baked in an oven at 67°C for 2 hours and rinsed three times with PBS (pH 7.4), each time for 3 minutes.

[0073] (2) Use a pH=6.0 citrate buffer to heat in a microwave until boiling, then place the dewaxed and hydrated tissue sections on a high-temperature plastic slice rack, put them into the boiling buffer, and microwave them at medium speed for 10 minutes. After that, take out the microwave box and let it cool naturally under running water. Take out the slides from the buffer, rinse them twice with distilled water, and then rinse them three times with PBS, each time for 3 minutes.

[0074] (3) Add 1 drop of 3% H2O2 to each slice and incubate at room temperature for 10 minutes to block the activity of endogenous peroxidase. Rinse with PBS three times, 3 minutes each time.

[0075] (4) Remove the PBS solution, add 1 drop of LRSAM1 antibody to each section at a dilution ratio of 1:100, and incubate at room temperature for 2 hours. Rinse with PBS three times, 3 minutes each time.

[0076] (5) Add 1 drop of HRP-labeled goat anti-rabbit secondary antibody to each section and incubate at room temperature for 60 minutes. Rinse with PBS three times, 5 minutes each time.

[0077] (6) Add 1 drop of DAB coloring solution to each slice and observe the color development under a microscope. When the staining result turns brown, stop. Rinse with PBS three times, 5 minutes each time.

[0078] S4. Quantitative analysis is as follows:

[0079] The staining results were scanned and photographed, and the number of positive samples and their staining intensity in each slice were converted into corresponding H-Score values ​​to achieve the purpose of semi-quantitative tissue staining.

[0080] S5. Statistical analysis is as follows:

[0081] The ROC curve was used to analyze the diagnostic efficacy of the protein expression intensity value of LRSAM1 protein for lung adenocarcinoma.

[0082] S6. Experimental results are as follows:

[0083] AUC is the area under the ROC curve. It is the most commonly used parameter to evaluate the characteristics of the ROC curve and is also an important indicator of test accuracy. If AUC is below 0.7, it means that the diagnostic accuracy is low; if AUC is above 0.7, it can meet the requirements of clinical diagnosis. Figure 6 It can be seen that the area under the curve AUC is 0.867, the 95% confidence interval is 0.805-0.930, p<0.0001, and the AUC of LRSAM1 protein is 0.867, which is greater than 0.7, indicating that LRSAM1 protein can be used as a biomarker for diagnosing lung adenocarcinoma. When the protein expression intensity value of LRSAM1 protein is 78.36, the sensitivity is 77.5% and the specificity is 91.2%. When performing individual testing, when the protein expression intensity value of LRSAM1 protein is greater than 78.36, it is judged as a lung adenocarcinoma patient, and the false positive rate is 8.8%.

[0084] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0085] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these 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 these modifications and variations.

Claims

1. Use of a detection reagent for LRSAM1 protein in the preparation of a lung adenocarcinoma diagnosis and / or prognosis product, characterized in that: The amino acid sequence of the LRSAM1 protein is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The products include chips, test kits, test strips or nucleic acid membrane strips.

3. The use according to claim 2, characterized in that The chips include gene chips and protein chips.

4. The use according to claim 3, characterized in that The gene chip includes a nucleotide probe for detecting the transcription level of the LRSAM1 gene; The protein chip includes a specific binding agent for LRSAM1 protein.

5. The use according to claim 2, characterized in that The kit comprises a LRSAM1 gene detection kit and a LRSAM1 protein detection kit.

6. The use according to claim 1, characterized in that The sample for diagnosis and / or prognosis is lung adenocarcinoma and / or adjacent tissue.

7. The use according to claim 6, characterized in that The samples come from patients with lung adenocarcinoma, suspected patients with lung adenocarcinoma, people susceptible to lung adenocarcinoma, people at high risk of lung adenocarcinoma, or healthy people.

8. The use according to claim 1, characterized in that The detection reagent is a reagent capable of detecting a marker; The label is any one of a radioisotope, a nucleotide chromophore, an enzyme, a fluorescent molecule, a chemiluminescent moiety, and a bioluminescent moiety; The marker is used to label the LRSAM1 protein.

9. The use according to claim 8, characterized in that The method for detecting the marker is a radiation method, an immunological method, a fluorescence method, a flow fluorescence method, a latex turbidimetry method, a biochemical method, an enzyme method, a hybridization method, a gas chromatography-mass spectrometry method, a liquid chromatography-mass spectrometry method, a chromatography method, a chemiluminescence method, a magnetoelectric method or a photoelectric conversion method.

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