Detection method of protein eEF1A1 interacting with biomarker AQP1 of nephroblastoma and application thereof

Abstract

The invention discloses a method for detecting a protein eEF1A1 interacting with a biomarker AQP1 of nephroblastoma, which comprises the following steps: carrying out in-vitro culture by using a nephroblastoma cell line Wit-49, extracting total protein by using a cell lysis solution, carrying out co-immunoprecipitation Co-IP by using an anti-AQP1 antibody and an anti-eEF1A1 antibody, and analyzing and detecting the co-precipitation condition of the AQP1 and the eEF1A1 by using Western blot to obtain the protein eEF1A1 interacting with the biomarker AQP1 of nephroblastoma. And verifying the interaction between the two. By combining the detection of AQP1 and eEF1A1, the sensitivity and specificity of early diagnosis can be improved, especially for the diagnosis of early patients with nephroblastoma, in addition, the targeted treatment strategy for eEF1A1 provides a new direction for the treatment of nephroblastoma, and especially, under the condition that effective intervention cannot be performed through an existing treatment scheme, the treatment effect is obviously improved. The targeted treatment means provided by the invention may provide a new treatment choice for patients; targeted therapy avoids systemic toxic and side effects of traditional chemotherapy, and eEF1A1 is inhibited in a targeted manner, so that damage to normal tissues is reduced, adverse reactions of patients are reduced, and treatment acceptability is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a detection method and application of a protein eEF1A1 that interacts with a nephroblastoma biomarker AQP1. Background Art

[0002] Wilms tumor (WT) is a common renal tumor in children, and its pathogenesis is not fully understood. Existing studies have shown that AQP1 (aquaporin 1) plays an important role in the occurrence and development of Wilms tumor.

[0003] AQP1 is considered to be one of the potential biomarkers of Wilms tumor. It participates in the proliferation and metastasis of tumor cells by regulating the balance of intracellular water. However, the function of AQP1 and its mechanism of action in Wilms tumor have not been fully revealed.

[0004] Recent studies have found that AQP1 may regulate its biological functions by interacting with certain proteins. eEF1A1 (eukaryotic translation elongation factor 1A1), as a key factor involved in protein synthesis, may play an important role in the growth and spread of tumor cells.

[0005] Existing technologies for the study of AQP1 are mostly limited to its potential as a biomarker, while there are fewer studies on it as a therapeutic target. Although the high expression of AQP1 in nephroblastoma may provide new opportunities for targeted therapy, existing technologies have failed to fully explore how to target AQP1 or its interacting proteins (such as eEF1A1) for therapeutic intervention.

[0006] Existing technology relies on the expression level of AQP1 in nephroblastoma tissue for diagnosis, but this method may be affected by tumor tissue heterogeneity, difficulty in sample acquisition, and cross-expression of AQP1 in other types of tumors. Therefore, a single AQP1 biomarker may have problems with insufficient sensitivity and low specificity, making it difficult to play a full role in the early diagnosis of nephroblastoma.

[0007] Although existing technologies have revealed the expression pattern and potential role of AQP1 in nephroblastoma, they have not explored in depth the interaction between AQP1 and other key proteins. AQP1 may regulate the proliferation, migration and invasion of tumor cells by interacting with other tumor-related proteins (such as eEF1A1). However, existing studies are limited to the individual functions of AQP1 and ignore its interaction with other proteins, thus failing to fully reveal the mechanism of action of AQP1 in nephroblastoma.

[0008] In addition, in the prior art, although the high expression of AQP1 in nephroblastoma has important biological significance, there are few studies on it as a target for therapeutic intervention. Existing treatment options mainly rely on traditional chemotherapy and radiotherapy, and have failed to fully explore how to target AQP1 and its interacting proteins (such as eEF1A1) for intervention, and have failed to provide new targeted treatment strategies. Summary of the invention

[0009] The purpose of the present invention is to provide a detection method and application of the protein eEF1A1 that interacts with the nephroblastoma biomarker AQP1.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is as follows: a method for detecting the protein eEF1A1 that interacts with the nephroblastoma biomarker AQP1, comprising the following steps:

[0011] S1. Cell culture and preparation: The Wilms tumor cell line Wit-49 was used for in vitro culture. After the cells grew to the logarithmic growth phase, the cells were collected for subsequent experiments.

[0012] S2, immunoprecipitation Co-IP experiment, total protein was extracted by cell lysate, and immunoprecipitation Co-IP was performed using anti-AQP1 antibody and anti-eEF1A1 antibody. The precipitate was analyzed by Western blot to detect the co-precipitation of AQP1 and eEF1A1 to verify the interaction between the two;

[0013] S3. The direct interaction between AQP1 and eEF1A1 was confirmed experimentally, and the stability of the interaction was verified by Western blot at different time points or under drug treatment conditions.

[0014] S4. Analysis of AQP1-specific interacting proteins, protein molecular docking of AQP1 and eEF1A1, and CO-IP verification of AQP1 and interacting protein EEF1A1;

[0015] S5. Finally, the sensitivity and specificity of combined detection of AQP1 and eEF1A1 in the early diagnosis of nephroblastoma were determined by combining combined biomarker detection.

[0016] Preferably, the protein molecular docking of AQP1 and eEF1A1 is performed using DISCOVERYSTUDIO software and the Zdock module to study protein-protein docking.

[0017] Preferably, the specific steps of S5 are as follows:

[0018] Sample collection: Collect blood, urine, or tumor tissue samples from patients with Wilms tumor for testing;

[0019] Immunohistochemistry (IHC): double-labeling of AQP1 and eEF1A1 was performed on tissue sections to observe their expression patterns in nephroblastoma tissues and to evaluate their value in early diagnosis in combination with histopathological analysis.

[0020] ELISA method, using ELISA kits to detect the expression levels of AQP1 and eEF1A1 in blood or urine samples, respectively, combined with clinical patient data for statistical analysis to determine the sensitivity and specificity of combined detection of the two in the early diagnosis of nephroblastoma.

[0021] An application of a protein eEF1A1 that interacts with a Wilms tumor biomarker AQP1, including the implementation of a targeted therapy strategy, comprises the following steps:

[0022] Design siRNA interference sequences. According to the gene sequence of eEF1A1, specific siRNA sequences were designed to interfere with the expression of eEF1A1 by transfecting Wilms tumor cell lines;

[0023] Cell function experiments, including cell proliferation, migration and invasion experiments, were performed to evaluate the inhibitory effects of siRNA interference or small molecule inhibitors on the proliferation, migration and invasion of Wilms tumor cells;

[0024] Mouse tumor model: Wilms tumor cells transfected with eEF1A1 siRNA were injected into mice to observe tumor growth. The expression levels of AQP1 and eEF1A1 in tumor tissues were detected by immunohistochemistry and Western blot to evaluate the effect of targeted therapy.

[0025] Preferably, the cell proliferation assay is a CCK-8 assay.

[0026] Preferably, the migration experiment is a scratch experiment.

[0027] Preferably, the invasion assay is a Transwell assay.

[0028] Compared with the prior art, the advantages of the present invention are:

[0029] By combining the detection of AQP1 and eEF1A1, the sensitivity and specificity of early diagnosis can be improved, especially for the diagnosis of early patients with nephroblastoma. In addition, the targeted treatment strategy for eEF1A1 provides a new direction for the treatment of nephroblastoma, especially when the existing treatment options cannot be used for effective intervention. The targeted treatment provided by the present invention may provide patients with new treatment options.

[0030] Targeted therapy avoids the systemic toxic side effects of traditional chemotherapy. By inhibiting eEF1A1, it reduces damage to normal tissues, reduces adverse reactions in patients, and improves the acceptability of treatment.

[0031] It has significant advantages in the early diagnosis and treatment of nephroblastoma, can effectively improve the accuracy of diagnosis, and provide stronger support for targeted treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 This is a chart showing the enrichment of AQP1 in Wilms tumor cells verified by Western blot of the present invention.

[0034] Figure 2 is an analysis chart of the AQP1-specific interacting proteins of the present invention;

[0035] Figure 3 is a diagram of the protein molecule docking of AQP1 and eEF1A1 of the present invention;

[0036] Figure 4 This is a CO-IP verification diagram of AQP1 and the interacting protein EEF1A1 of the present invention;

[0037] Figure 5 It is a data graph of the sensitivity, specificity and combined detection results of the ELISA detection method of the present invention;

[0038] Figure 6 is a bar graph of the CCK-8 experimental results of the present invention;

[0039] Figure 7 is a bar graph of the invasion experiment results of the present invention;

[0040] Figure 8 is a bar graph of the migration experiment results of the present invention;

[0041] Fig. 9 is a bar graph showing changes in tumor weight in mice of the present invention;

[0042] Fig.10 It is a bar graph showing the changes in tumor volume of mice of the present invention. DETAILED DESCRIPTION

[0043] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0044] The present invention provides a method for detecting the protein eEF1A1 that interacts with the nephroblastoma biomarker AQP1, comprising the following steps:

[0045] Cell culture and preparation, Wilms tumor cell line (Wit-49) was used for in vitro culture, and after the cells grew to the logarithmic growth phase, the cells were collected for subsequent experiments;

[0046] Co-immunoprecipitation (Co-IP) experiment: total protein was extracted from cell lysate, and co-immunoprecipitation (Co-IP) was performed using anti-AQP1 antibody and anti-eEF1A1 antibody. The precipitate was analyzed by Western blot to detect the co-precipitation of AQP1 and eEF1A1 to verify the interaction between the two.

[0047] like Figure 1 As shown, the direct interaction between AQP1 and eEF1A1 was experimentally confirmed, and the stability of the interaction was verified by immunoblotting (Western blot) at different time points or drug treatment conditions;

[0048] Mass spectrometry results were used to analyze AQP1-specific interacting proteins. Figure 2 As shown;

[0049] Protein molecule docking of AQP1 and eEF1A1, such as Figure 3 As shown, the DISCOVERYSTUDIO software and the Zdock module were used to study protein-protein docking, where purple represents AQP1, the red dotted line is a salt bridge, E represents the receptor protein EEF1A1, and A represents the ligand protein AQPQ. For example, the positively charged LYS273 of the EEF1A1 chain forms a salt bridge interaction with the ASP131 of the AQP1 protein, and the green dotted line is a hydrogen bond. For example, the AQP1 chain GLN47, ASP185, THR120, PHE35, GLN35, GLN43 and ASN42 form hydrogen bond interactions with the ASN311, SER291, ASP185, LEU77, HIS296 and GLU297 of the EE1A1 protein, and the hydrogen bond distances are 2.9, 2.9, 3.0, 3.7, 3.3, 3.1, 2.7 and 2.8 angstroms, respectively;

[0050] Co-IP verification of AQP1 and its interacting protein EEF1A1, such as Figure 4As shown, co-IP experiments revealed that AQP1 and eEF1A1 interacted with each other.

[0051] The following combined biomarker detection is used to determine the sensitivity and specificity of the combined detection of the two in the early diagnosis of nephroblastoma. The steps are as follows:

[0052] Sample collection: Collect blood, urine, or tumor tissue samples from patients with Wilms tumor for testing;

[0053] Immunohistochemistry (IHC) was used to perform double immunohistochemical staining of AQP1 and eEF1A1 on tissue sections to observe their expression patterns in nephroblastoma tissues and to evaluate their value in early diagnosis in combination with histopathological analysis.

[0054] ELISA method, using ELISA kits to detect the expression levels of AQP1 and eEF1A1 in blood or urine samples, respectively, combined with clinical patient data for statistical analysis to determine the sensitivity and specificity of combined detection of the two in the early diagnosis of nephroblastoma.

[0055] Furthermore, the present invention provides an application of a protein eEF1A1 that interacts with AQP1, a biomarker of nephroblastoma.

[0056] Including the implementation of targeted treatment strategies, the steps are as follows:

[0057] Design siRNA interference sequences. According to the gene sequence of eEF1A1, specific siRNA sequences were designed to interfere with the expression of eEF1A1 by transfecting Wilms tumor cell lines;

[0058] Cell function experiments, including cell proliferation experiments (such as CCK-8 experiments), migration experiments (such as scratch experiments) and invasion experiments (such as Transwell experiments), to evaluate the inhibitory effects of siRNA interference or small molecule inhibitors on the proliferation, migration and invasion of Wilms tumor cells;

[0059] Mouse tumor model: Wilms tumor cells transfected with eEF1A1 siRNA were injected into mice to observe tumor growth. The expression levels of AQP1 and eEF1A1 in tumor tissues were detected by immunohistochemistry and Western blot to evaluate the effect of targeted therapy.

[0060] Through early mass spectrometry detection, it was found that AQP1 and eEF1A1 had the highest degree of interaction in Wilms tumor cells. Through Co-IP experiments, the direct interaction between AQP1 and eEF1A1 in Wilms tumor cells was confirmed. The experimental results showed that AQP1 and eEF1A1 co-localized in the cells and formed a stable protein complex.

[0061] ELISA tests were performed on blood samples from 20 patients with nephroblastoma. The results showed that the sensitivity of combined detection of AQP1 and eEF1A1 was 90% and the specificity was 92%. Compared with the single detection of AQP1, the combined detection significantly improved the accuracy of early diagnosis.

[0062] The experimental method of the above ELISA test is as follows:

[0063] 1. Sample Collection

[0064] Blood samples from 20 patients with Wilms tumor were selected, and all patients were clinically diagnosed and signed informed consent;

[0065] Fasting venous blood was collected from each patient using tubes containing anticoagulants. The blood samples were immediately centrifuged to separate serum after collection and stored at -80°C for subsequent ELISA testing.

[0066] 2. ELISA Reagent Preparation

[0067] ELISA kits for AQP1 (Abcam, ab200951) and eEF1A1 (Abcam, ab214667) were used for detection;

[0068] Prepare the standard, diluent and washing solution according to the instructions of the kit.

[0069] 3.ELISA test steps

[0070] Sample processing: Dilute the serum sample appropriately according to the requirements of the kit;

[0071] Antibody coating: Seal the microplate pre-coated with AQP1 or eEF1A1 according to the kit instructions;

[0072] Add samples: Add the treated samples to the microplate and incubate for a certain period of time (usually 1 hour at 37°C);

[0073] Washing: Use washing solution to wash the plate wells thoroughly to remove unbound impurities;

[0074] Add secondary antibody: Add HRP-labeled secondary antibody and continue incubation for 30 minutes;

[0075] Washing: Wash away excess secondary antibody with washing buffer again;

[0076] Color development: Add substrate solution (usually TMB), incubate in the dark for 10-20 minutes, and the substrate changes color; Stop reaction: Add stop solution (such as 2M H2SO4) to stop the reaction;

[0077] Reading: Use a microplate reader to read the absorbance (OD value) of each well at a wavelength of 450 nm.

[0078] 4. Results Analysis

[0079] The concentrations of AQP1 and eEF1A1 in each sample were calculated based on the standard curve provided by the kit;

[0080] By comparing the concentrations of AQP1 and eEF1A1 in the negative control group, appropriate thresholds were set, and the sensitivity and specificity of AQP1 detection alone and AQP1 and eEF1A1 combined detection were calculated respectively;

[0081] In blood samples from 20 patients with Wilms tumor, the following results were obtained by ELISA testing:

[0082] For AQP1 detection alone, the sensitivity is 70% and the specificity is 80%. AQP1 has a certain diagnostic value as a control marker in the early diagnosis of Wilms tumor;

[0083] For the combined detection of AQP1 and eEF1A1, the sensitivity was increased to 90% and the specificity was increased to 92%. Compared with the detection of AQP1 alone, the combined detection can significantly improve the accuracy of early diagnosis, especially in patients with early-stage nephroblastoma, showing higher diagnostic efficacy.

[0084] like Figure 5 As shown in the figure, the sensitivity and specificity results of AQP1 detection alone and AQP1+eEF1A1 combined detection are: AQP1 detection alone has a sensitivity of 70% and a specificity of 80%; while AQP1+eEF1A1 combined detection has a sensitivity of 90% and a specificity of 92%, providing higher accuracy for early diagnosis.

[0085] By interfering with the expression of eEF1A1 by siRNA, the proliferation and migration ability of nephroblastoma cells were significantly inhibited (P<0.05). In the mouse tumor model, the tumor growth in the eEF1A1 inhibition group was significantly slowed down, and the tumor volume was reduced by more than 50% compared with the control group. The specific steps of the experiment are as follows:

[0086] 1. Cell Culture and Treatment

[0087] Cell source: Wit-49 Wilms tumor cell line;

[0088] Cell culture: cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C and humidity at 5% CO2.

[0089] siRNA transfection: Transfection was performed using specific siRNA targeting eEF1A1 (primer sequence: 5'-GGGAGGAAGGAUUGAAGGAG-3' (positive strand) 5'-CCUCCUUCAAUCCUUCAGCC-3' (reverse strand)) using Lipofectamine 3000 transfection reagent according to the manufacturer's recommendations. A control group was transfected with nonspecific siRNA.

[0090] 2. Cell Proliferation Assay

[0091] CCK-8 method: 48 hours after transfection, use Cell Counting Kit-8 (CCK-8) to detect cell proliferation. Follow the instructions to detect absorbance (OD value) and evaluate changes in cell proliferation. Perform at least three replicates for each group.

[0092] 3. Cell migration and invasion assay

[0093] Transwell migration and invasion assay: Transwell chambers were used, with the uncoated chambers used for migration assays and the coated chambers used for invasion assays. Transfected cells were added to the upper chambers and cultured for 48 hours, and the number of migrating and invading cells was quantified.

[0094] 4. Mouse Tumor Model

[0095] Establishment of mouse model: Immunodeficient mice (such as BALB / c nude mice) were used, and Wilms tumor cells (e.g. 10×10 6 cells / mouse) and were divided into two groups: control group (transfected with nonspecific siRNA) and interference group (transfected with eEF1A1 siRNA);

[0096] Tumor growth monitoring: The tumor volume of mice was measured every 3 days and the tumor volume (V = 0.5 × length × width) was calculated using a caliper. 2 );

[0097] Endpoint analysis: After mice were euthanized, tumors were removed for weight and volume measurements.

[0098] 5. Statistical Analysis

[0099] GraphPad Prism was used for data analysis, and the Student's t test was used to compare the experimental and control groups. A P value < 0.05 indicated that the difference was statistically significant.

[0100] The experimental results are as follows:

[0101] Cell proliferation: The results of CCK-8 assay showed that the cell proliferation of the interference group (eEF1A1 siRNA transfection group) was significantly reduced compared with the control group. Figure 6 As shown, inhibition of eEF1A1 significantly reduced the proliferation rate of Wilms tumor cells (P<0.00005).

[0102] Cell migration and invasion ability: Transwell experiment showed that the cell migration ability of eEF1A1 inhibition group was significantly weakened. In the migration and invasion experiments, the cell migration and invasion abilities of the interference group were significantly lower than those of the control group (P<0.005). Figure 7 The migration experiment results are shown in Figure 8 shown.

[0103] Tumor growth: In the mouse tumor model, the tumor growth of the interference group mice was significantly slowed down. Compared with the control group, the tumor weight and volume of the eEF1A1 inhibition group were significantly reduced, and the difference was statistically significant (P<0.0005 and P<0.00005). The results of the changes in mouse tumor weight are shown in Figure 2. Fig. 9 The results of the changes in mouse tumor volume are shown in Fig.10 shown.

[0104] Although the embodiments of the present invention are described in conjunction with the accompanying drawings, the patent owner may make various variations or modifications within the scope of the appended claims. As long as they do not exceed the protection scope described in the claims of the present invention, they should be within the protection scope of the present invention.

Claims

1. A method for detecting the protein eEF1A1 that interacts with the nephroblastoma biomarker AQP1, characterized in that: The following steps are involved: S1. Cell culture and preparation: The Wilms tumor cell line Wit-49 was used for in vitro culture. After the cells grew to the logarithmic growth phase, the cells were collected for subsequent experiments. S2, immunoprecipitation Co-IP experiment, total protein was extracted by cell lysate, and immunoprecipitation Co-IP was performed using anti-AQP1 antibody and anti-eEF1A1 antibody. The precipitate was analyzed by Western blot to detect the co-precipitation of AQP1 and eEF1A1 to verify the interaction between the two; S3. The direct interaction between AQP1 and eEF1A1 was confirmed experimentally, and the stability of the interaction was verified by Western blot at different time points or under drug treatment conditions. S4. Analysis of AQP1-specific interacting proteins, protein molecular docking of AQP1 and eEF1A1, and CO-IP verification of AQP1 and interacting protein EEF1A1; S5. Finally, the sensitivity and specificity of combined detection of AQP1 and eEF1A1 in the early diagnosis of nephroblastoma were determined by combining combined biomarker detection.

2. The method for detecting the protein eEF1A1 that interacts with the Wilms tumor biomarker AQP1 according to claim 1, characterized in that: The protein molecular docking of AQP1 and eEF1A1 was carried out using DISCOVERYSTUDIO software and the Zdock module to study protein-protein docking.

3. The method for detecting the protein eEF1A1 that interacts with the Wilms tumor biomarker AQP1 according to claim 1, characterized in that: The specific steps of S5 are as follows: Sample collection: Collect blood, urine, or tumor tissue samples from patients with Wilms tumor for testing; Immunohistochemistry (IHC): double-labeling of AQP1 and eEF1A1 was performed on tissue sections to observe their expression patterns in nephroblastoma tissues and to evaluate their value in early diagnosis in combination with histopathological analysis. ELISA method, using ELISA kits to detect the expression levels of AQP1 and eEF1A1 in blood or urine samples, respectively, combined with clinical patient data for statistical analysis to determine the sensitivity and specificity of combined detection of the two in the early diagnosis of nephroblastoma.

4. Use of a method for detecting a protein eEF1A1 that interacts with the nephroblastoma biomarker AQP1 according to any one of claims 1 to 3, characterized in that: Including the implementation of targeted treatment strategies, the steps are as follows: Design siRNA interference sequences. According to the gene sequence of eEF1A1, specific siRNA sequences were designed to interfere with the expression of eEF1A1 by transfecting Wilms tumor cell lines; Cell function experiments, including cell proliferation, migration and invasion experiments, were performed to evaluate the inhibitory effects of siRNA interference or small molecule inhibitors on the proliferation, migration and invasion of Wilms tumor cells; Mouse tumor model: Wilms tumor cells transfected with eEF1A1 siRNA were injected into mice to observe tumor growth. The expression levels of AQP1 and eEF1A1 in tumor tissues were detected by immunohistochemistry and Western blot to evaluate the effect of targeted therapy.

5. Use of the method for detecting the protein eEF1A1 that interacts with the biomarker AQP1 of nephroblastoma according to claim 4, characterized in that: The cell proliferation assay is a CCK-8 assay.

6. Use of the method for detecting the protein eEF1A1 that interacts with the biomarker AQP1 of nephroblastoma according to claim 4, characterized in that: The migration experiment is a scratch experiment.

7. Use of the method for detecting the protein eEF1A1 that interacts with the biomarker AQP1 of nephroblastoma according to claim 4, characterized in that: The invasion assay is a Transwell assay.

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