Application of FIGN protein in cytoplasm as a marker in the preparation of diagnostic products for lung adenocarcinoma and / or breast cancer

Abstract

The present invention relates to the use of FIGN protein in the cytoplasm as a marker in the preparation of a diagnostic product for lung adenocarcinoma and / or breast cancer, and is related to the biomedical field. The use of FIGN protein in the cytoplasm as a marker in the preparation of a diagnostic product for lung adenocarcinoma and / or breast cancer comprises a product for detecting the expression level of FIGN protein in the cytoplasm of a sample collected from the lung and / or breast of a subject to be diagnosed. The diagnostic predictive biomarker and diagnostic kit of the present invention have sensitivities of 88.89% and 87.87% in breast cancer and lung adenocarcinoma tumor tissues, respectively, and sensitivities of 92.22% and 90.00% in normal breast and lung tissues, respectively. These provide new tools and methods for the early diagnosis of breast cancer, lung adenocarcinoma, and other conditions, and have important clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an application of FIGN protein in cytoplasm as a marker in the preparation of a lung adenocarcinoma and / or breast cancer diagnosis product. Background Art

[0002] Early diagnosis of tumors is of great significance for treatment and prognosis. Breast cancer and lung adenocarcinoma are common types of malignant tumors, and their diagnosis mainly relies on histopathological examination and molecular marker detection. Currently, commonly used markers for early diagnosis of breast cancer and lung adenocarcinoma include CK7 and CK19. However, these existing markers have certain limitations in clinical application, such as insufficient sensitivity or specificity, which makes it difficult to diagnose early in some cases. Therefore, there is an urgent need for a new auxiliary marker to improve the diagnostic efficacy of existing markers and solve the problems in clinical pathological diagnosis. Summary of the Invention

[0003] The main purpose of the present invention is to provide an application of a cytoplasmic FIGN protein marker in the preparation of lung adenocarcinoma and breast cancer diagnostic products, in order to at least partially solve the above technical problems.

[0004] To achieve the above objectives, the present invention proposes an application of FIGN protein in the cytoplasm as a marker in the preparation of a lung adenocarcinoma and / or breast cancer diagnostic product, wherein the lung adenocarcinoma and / or breast cancer diagnostic product includes a product for detecting the expression level of FIGN protein in the cytoplasm of a sample, wherein the sample is collected from the lung and / or breast to be diagnosed of the subject.

[0005] Based on the above technical solutions, it can be seen that the application of the cytoplasmic FIGN protein marker of the present invention in the preparation of lung adenocarcinoma and breast cancer diagnostic products has at least one of the following beneficial effects compared to the prior art:

[0006] This study establishes an effective diagnostic tool and indicator for tumor and normal tissue by detecting changes in the nuclear and cytoplasmic localization of FIGN in tumor tissues such as breast cancer and lung adenocarcinoma, as well as in normal tissue. Experimental results demonstrate that this method has sensitivities of 88.89% and 81.67% in breast cancer and lung adenocarcinoma tumor tissues, respectively, and 92.22% and 90.00% in the corresponding normal breast and lung tissues, respectively. This provides a new tool for early diagnosis of tumors, facilitates targeted and appropriate treatment, improves patient prognosis, and ultimately enhances patient quality of life and reduces their financial burden. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments.

[0008] Figure 1 The present invention is a flow chart of using the markers for diagnosing lung adenocarcinoma or breast cancer for immunohistochemical staining detection. DETAILED DESCRIPTION

[0009] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0010] The terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. The singular forms "a", "said" and "the" used in the present invention are also intended to include plural forms, unless the context clearly indicates other meanings.

[0011] Existing studies have shown that Fidgetin (FIGN) is a microtubule-cleaving enzyme (protein) belonging to the AAA+ protein family. Its functions are involved in various cellular activities, including spindle formation, cell division, neural regeneration, and ciliogenesis. Fidgetin cleaves microtubules in an ATP-dependent manner, mainly acting on the labile part of microtubules, especially the minus end of microtubules, thereby regulating the dynamic reorganization and disassembly of microtubules. Under normal circumstances, the FIGN protein is localized to the cell nucleus and plays an important role in maintaining the dynamic balance of the cytoskeleton. However, the localization changes of FIGN in tumor tissue and its application in tumor diagnosis have not been fully studied. After novel search, it was found that FIGN is currently considered to be a poor prognosis gene (protein) for hepatocellular carcinoma, but the early diagnostic significance of FIGN in breast cancer and lung adenocarcinoma is still unclear.

[0012] This study conducted in-depth research on this situation. Through comparative experiments on tissues such as breast cancer and lung adenocarcinoma and normal tissues, it was found that FIGN protein showed significant changes in nuclear and cytoplasmic localization in tumor tissues and normal tissues: FIGN protein was mainly localized in the nucleus in normal breast tissue and lung tissue, while it was mainly localized in the cytoplasm in tumor tissues such as breast cancer and lung adenocarcinoma, which provides new possibilities for tumor diagnosis.

[0013] Based on this unique discovery, the present invention proposes a predictive biomarker for diagnosing lung adenocarcinoma or breast cancer, namely the FIGN protein. This predictive biomarker, unlike existing prognostic markers, can distinguish tumor tissue from normal tissue with high specificity, enabling more accurate early diagnosis.

[0014] Among them, the predictive biomarker for diagnosing lung adenocarcinoma or breast cancer is diagnosed by the subcellular distribution of FIGN protein in the cytoplasm of samples collected from subjects for the corresponding diagnosis of lung adenocarcinoma or breast cancer. More preferably, the diagnosis is achieved by the subcellular distribution of FIGN protein in the nucleus and cytoplasm of the collected samples, which can more accurately determine whether it is a tumor cell or a normal cell.

[0015] The present invention also provides a lung adenocarcinoma and / or breast cancer diagnosis kit, comprising:

[0016] One or more reagents for detecting the expression level of FIGN protein in the cytoplasm of a lung adenocarcinoma or breast cancer tissue sample obtained from a subject.

[0017] The lung adenocarcinoma and / or breast cancer diagnostic kit may further include a nuclear-cytoplasmic separation kit for separating the nucleus and cytoplasm of a sample collected from a subject. The nuclear-cytoplasmic separation kit may be an existing commercially available product, such as the PARISTI Kit or BioVision's nuclear / cytoplasmic separation kit, or a specially designed nuclear-cytoplasmic separation kit.

[0018] The lung adenocarcinoma and / or breast cancer diagnostic kit may further comprise a microplate coated with a first antibody, wherein the first antibody is a polyclonal or monoclonal antibody against FIGN protein, capable of binding to FIGN protein and immobilizing it on a solid phase carrier.

[0019] The lung adenocarcinoma and / or breast cancer diagnostic kit may also include an enzyme-labeled secondary antibody for detecting the primary antibody bound to the FIGN protein. This secondary antibody is typically labeled with HRP (horseradish peroxidase) to produce a detectable signal in the subsequent color development step; and a substrate solution for the color development reaction, typically including TMB (3,3',5,5'-tetramethylbenzidine) or other similar colorants. When HRP comes into contact with the substrate solution, a color change occurs, the intensity of which is proportional to the concentration of the FIGN protein in the sample.

[0020] The lung adenocarcinoma and / or breast cancer diagnostic kit can also be designed to present results using chemiluminescence or digital PCR methods for markers; preferably, for example, amino acid recognition sites encoding FIGN protein in the nucleus and cytoplasm can be designed and diagnosed using chemiluminescence. Thus, a chemiluminescence detection kit can be obtained.

[0021] The present invention also provides a nucleic acid detection library comprising at least one of the above-mentioned predictive biomarkers for diagnosing lung adenocarcinoma and / or breast cancer.

[0022] The present invention also provides a lung adenocarcinoma and / or breast cancer diagnosis system, comprising:

[0023] A protein detection module, used to perform quantitative detection of FIGN protein in the cytoplasmic components of the sample; wherein the sample is collected from the lung and / or breast of the subject to be diagnosed, and the protein detection module uses immunohistochemical staining technology for detection; and

[0024] The lung adenocarcinoma and / or breast cancer diagnostic kit as described above.

[0025] The present invention also proposes an application of FIGN protein in the cytoplasm as a marker in the preparation of lung adenocarcinoma and / or breast cancer diagnosis products.

[0026] Among them, the lung adenocarcinoma and / or breast cancer diagnostic product includes a product for detecting the expression level of FIGN protein in the cytoplasm of a sample, and the sample is collected from the lung and / or breast of the subject to be diagnosed.

[0027] Wherein, the product for detecting the expression level of FIGN protein in the cytoplasm of a sample includes a substance capable of binding to FIGN protein; and / or

[0028] The product for detecting the expression level of FIGN protein in the cytoplasm of a sample is, for example, selected from reagents, kits, test strips, gene chips, high-throughput sequencing platforms, antibody chips, and instrument platforms.

[0029] Among them, when the product for detecting the expression level of FIGN protein in the cytoplasm of the sample is an instrument platform capable of detecting the expression level of FIGN protein, for example, the instrument platform comprises a protein detection module for quantitative detection of FIGN protein in the cytoplasmic components of the sample, wherein the sample is collected from the lung and / or breast to be diagnosed of the subject.

[0030] The present invention will be further described below through specific embodiments. It should be noted that the following embodiments are merely illustrative and are not intended to limit the present invention. Based on the embodiments of the present invention shown below, all other embodiments obtained by ordinary technicians in the relevant technical field without making creative efforts are within the scope of protection of the embodiments of the present invention.

[0031] Example 1

[0032] 1. Instruments and Equipment

[0033] Table 1 shows the instruments and equipment used in the implementation of the present invention.

[0034]

[0035] 2. Experimental Materials

[0036] Table 2 shows the experimental materials used in the implementation of the present invention.

[0037]

[0038] 3. Antibodies

[0039] Rabbit anti-human FIGN polyclonal antibody (ab232756) Abcam, USA

[0040] 4. Sequence

[0041] MQWTPEHAQWPEQHFDITSTTRSPAHKVEAYRGHLQRTYQYAWANDDISALTASNLLKKYAEKYSGILEGPVDRPVLSNYSDTPSGLVNGRKNESEPWQPSLNSEAVYPMNCVPDVITASKAGVSSALPPADVSASIGSSPGVASNLTEPSYSSSTCGSHTVPSLHAGLPSQEYAPGYNGSYLHSTY SSQPAPALPSPHPSPLHSSGLLQPPPPPPPPPALVPGYNGTSNLSSYSYPSASYPPQTAVGSGYSPGGAPPPPSAYLPSGIPAPTPLPPTTVPGYTYQGHGLTPIAPSALTNSSASSLKRKAFYMAGQGDMDSSYGNYSYGQQRSTQSPMYRMPDNSISNTNRGGFDRSAETSSLAFKPTKQLMSS EQQRKFSSQSSRALTPPSYSTAKNSLGSRSSESFGKYTSPVMSEHGDEHRQLLSHPMQGPGLRAATSSNHSVDEQLKNTDTHLIDLVTNEIITQGPPVDWNDIAGLDLVKAVIKEEVLWPVLRSDAFSGLTALPRSILLFGPRGTGKTLLGRCIASQLGATFFKIAGSGLVAKWLGEAEKIIHASFL VARCRQPSVIFVSDIDMLLSSQVNEEHSPVSRMRTEFLMQLDTVLTSAEDQIVVICATSKPEEIDESLRRYFMKRLLIPLPDSTARHQIIVQLLSQHNYCLNDKEFALLVQRTEGFSGLDVAHLCQEAVVGPLHAMPATDLSAIMPSQLRPVTYQDFENAFCKIQPSISQKELDMYVEWNKMFGCSQ

[0042] 5. Clinical Sample Collection

[0043] The experimental samples of the experimental set came from breast cancer and lung adenocarcinoma (tumor tissue) and corresponding adjacent normal tissues (normal tissue) archived from 2015 to 2018 after surgery in the First Affiliated Hospital of Anhui Medical University, including 90 cases of breast cancer and 60 cases of lung adenocarcinoma.

[0044] The validation set used different experimental samples: 90 breast cancer cases (tumor tissue) and corresponding adjacent normal tissues (normal tissue) from the archived collection of postoperative breast cancer at the First Affiliated Hospital of Anhui Medical University between 2018 and 2020; and 30 lung adenocarcinoma cases (tumor tissue) and corresponding adjacent normal tissues (normal tissue) from the archived collection of postoperative lung adenocarcinoma at the First Affiliated Hospital of Anhui Medical University between 2015 and 2018. All patients were informed and provided written consent.

[0045] The pathological sections corresponding to each tissue specimen were confirmed by two senior pathologists, and the clinical relevant data corresponding to each tissue specimen were collected.

[0046] 6. Immunohistochemistry detection method (main steps such as Figure 1 shown)

[0047] (1) Cut the paraffin sample into 3-4 μm white slices, place them on a slide holder, and bake them in a 65°C oven for 2 h.

[0048] (2) Dewaxing and hydration: quickly take out the slides from the oven and place them in xylene No. 1 tank for 10 minutes, xylene No. 2 tank for 10 minutes, anhydrous ethanol tank for 3 minutes, 95% ethanol tank for 3 minutes, 85% ethanol tank for 3 minutes, and 75% ethanol tank for 3 minutes;

[0049] (3) Rinse under tap water for 3 minutes to remove the alcohol (avoid directing water flow on the slide);

[0050] (4) Prepare citrate repair solution (6.46 g citrate powder + 2 L ultrapure water), pour it into a small aluminum cylinder (completely covering the tissue on the white slide), place the small aluminum cylinder in a pressure cooker, add appropriate amount of tap water to the pressure cooker, and boil;

[0051] (5) Place the slide holder in a small aluminum cylinder, cover it, and when the pressure cooker lid starts to rotate and emit steam, count for 90 seconds (high pressure repair to fully expose the antigen);

[0052] (6) Place the pressure cooker under running water to cool down. After 10-15 minutes, remove the pressure cooker cover and continue to cool down to room temperature under running water.

[0053] (7) Circle drawing: Take out the repaired slices and place them in a wet box. Use a special oil-based pen for immunohistochemistry to draw a circle 3-4 mm away from the edge of the tissue.

[0054] (8) Wash twice with PBS (phosphate buffered saline) and once with PBST (phosphate buffered saline containing Tween 20), 3 min each time;

[0055] (9) Blocking: Shake off the PBST on the slices, add 1 drop (about 50 μL-80 μL, enough to fully cover the tissue) of endogenous peroxidase to each slice, and incubate at room temperature in the dark for 30 minutes;

[0056] (10) Washing: Wash twice with PBS and once with PBST, 3 min each time;

[0057] (11) Adding primary antibody: Shake off the PBST on the slice, dilute the primary antibody with PBS in proportion, and add 80 μL of primary antibody dilution solution;

[0058] (12) Place the light-proof humidified box in a 4°C refrigerator and incubate overnight;

[0059] (13) Remove the tube from the light-proof humidified box and rewarm it at room temperature for 30 minutes. Wash it twice with PBS and once with PBST, 3 minutes each time.

[0060] (14) Shake off the PBST on the slices, add 80 μL of secondary antibody solution, and incubate at room temperature for 30 min;

[0061] (15) Wash with PBS 3 times, 3 min each time;

[0062] (16) Shake dry the slide, add 80 μL of DAB (3,3'-diaminobenzidine) colorimetric solution, observe the tissue under a microscope and observe if it shows positive coloration, then immediately place it in tap water to stop the reaction;

[0063] (17) Rinse the slide holder under running water for 3 minutes:

[0064] (18) Counterstain with hematoxylin for 10 seconds (the specific time depends on the freshness of the hematoxylin, etc., and the stain should be rinsed with water until it turns blue);

[0065] (19) Rinse the slide holder under running water for 3 minutes until the water in the aluminum cylinder is clear;

[0066] (20) Dehydration and transparency: Place the sections in 75% alcohol, 85% alcohol, 95% alcohol and anhydrous ethanol for 1 min each, and immerse them in xylene No. 1 and xylene No. 2 tanks for 5 min each;

[0067] (21) Sealing: Add an appropriate amount of neutral resin fixing glue, cover with a coverslip, and press gently to evenly cover the tissue;

[0068] (22) Microscopic examination: The specimens can be observed immediately or left to air at room temperature overnight (to allow the xylene to evaporate completely), and photographed for record.

[0069] (23) Result judgment: Only areas with intact tissue structure and uniform staining were evaluated, and fields with necrosis, hemorrhage or folding were excluded. Observe under a high-power microscope (40x objective lens) and randomly select the 5 most representative tumor cell-dense areas for counting. Count 100 tumor cells with clear morphology in each observation area, excluding interstitial cells and inflammatory cells. The positive judgment standard is the appearance of specific brown granular staining in the cytoplasm / nucleus, which must be clearly distinguished from the negative control. The proportion of positive cells is divided into five levels: 0 point: positive cells ≤5%; 1 point: positive cells 6-25%; 2 points: positive cells 26-50%; 3 points: positive cells 51-75%; 4 points: positive cells >75%. Staining intensity score: 0 point: no staining (consistent with the negative control); 1 point: weak positive (light brown); 2 points: moderate positive (brown); 3 points: strong positive (dark brown). The positive cell percentage score is multiplied by the staining intensity score to obtain a comprehensive score. A comprehensive score ≥4 points indicates positive expression, and a comprehensive score <4 points indicates negative expression.

[0070] 7. Results

[0071] Table 3 Statistics of test results of paraffin specimens of breast cancer and lung adenocarcinoma tumor tissues and normal tissues in the experimental set

[0072]

[0073] Table 4. Detection results statistics of paraffin specimens of breast cancer and lung adenocarcinoma tumor tissues and normal tissues in the validation set

[0074]

[0075] As shown in the test results and statistics in Tables 3 to 8:

[0076] (1) Among 90 breast cancer paraffin specimens, 80 were localized in the cytoplasm, which means that 80 breast cancer patients were diagnosed among 90 suspected breast cancer patients, with a sensitivity of 80 / 90×100%=88.89%; among 90 normal breast paraffin specimens, 83 were localized in the nucleus, which means that 83 non-breast cancer patients were diagnosed among 90 suspected breast cancer patients, with a sensitivity of 83 / 90×100%=92.22%. Among 60 lung adenocarcinoma paraffin specimens, 49 were localized in the cytoplasm, which means that 49 lung adenocarcinoma patients were diagnosed among 60 suspected lung adenocarcinoma patients, with a sensitivity of 49 / 60×100%=81.67%; among 60 normal lung paraffin specimens, 58 were localized in the nucleus, which means that 58 non-lung adenocarcinoma patients were diagnosed among 60 suspected lung adenocarcinoma patients, with a sensitivity of 58 / 60×100%=96.67%.

[0077] (2) Validation set: 79 of 90 breast cancer paraffin specimens were located in the cytoplasm, with a sensitivity of 79 / 90×100%=87.78%; 81 of 90 normal breast paraffin specimens were located in the nucleus, with a sensitivity of 81 / 90×100%=90.00%. 30 of 30 lung adenocarcinoma paraffin specimens were located in the cytoplasm, meaning that 30 of 30 suspected lung adenocarcinoma patients were diagnosed as lung adenocarcinoma patients, with a sensitivity of 30 / 30×100%=100.00%; 23 of 30 normal lung paraffin specimens were located in the nucleus, meaning that 23 of 30 suspected lung adenocarcinoma patients were diagnosed as non-lung adenocarcinoma patients, with a sensitivity of 23 / 30×100%=76.67%.

[0078] This indicates that the nuclear and cytoplasmic subcellular localization detection of the FIGN protein of the present invention can become an effective predictive biomarker for diagnosing breast cancer and lung adenocarcinoma.

[0079] Table 5 Detection results of paraffin specimens of breast cancer and normal tissues in the experimental set

[0080]

[0081]

[0082]

[0083] Table 6 Detection results of paraffin specimens of breast cancer and normal tissues in the validation set

[0084]

[0085]

[0086]

[0087] Table 7 Detection results of paraffin specimens of lung adenocarcinoma and normal tissues in the experimental set

[0088]

[0089]

[0090]

[0091] Table 8 Detection results of paraffin specimens of lung adenocarcinoma and normal tissues in the validation set

[0092]

[0093]

[0094] Example 2 Nuclear protein and cytoplasmic protein extraction kit + Western Blot detection

[0095] The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0028) uses cytoplasmic protein extraction reagents A and B to fully swell cells under hypotonic conditions, disrupting the cell membrane and releasing cytoplasmic proteins. The nuclei are then pelleted by centrifugation. Nuclear proteins are extracted using a high-salt nuclear protein extraction reagent. Separate nuclear and cytoplasmic protein samples are quantified and adjusted to the same concentration.

[0096] Then, the expression levels of cytoplasmic FIGN protein and nuclear FIGN protein are detected by Western Blot. The specific steps include, for example:

[0097] Protein extraction: Use a total protein extraction kit or a nucleoplasmic fractionation kit to extract FIGN protein from the nucleus and cytoplasm.

[0098] Protein quantification: The FIGN protein concentration was determined using the BCA assay or Coomassie Brilliant Blue method.

[0099] Electrophoresis and membrane transfer: The FIGN protein sample was subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane.

[0100] Antibody detection: Use specific antibodies (such as anti-NF-κB, anti-IκBα, etc.) for primary and secondary antibody incubation, and finally detect the target protein by chemiluminescence or imaging technology.

[0101] Example 3 Subcellular separation + ELISA detection system

[0102] Use a specialized nuclear / cytoplasmic fractionation kit (such as the PARISTI Kit or BioVision's Nuclear / Cytoplasmic Isolation Kit) to separate the nucleus and cytoplasm. Follow the kit instructions to obtain plasma proteins and nuclear proteins, respectively. Quantify the nuclear and plasma protein samples separately and adjust them to the same concentration.

[0103] Then, the expression levels of cytoplasmic FIGN protein and nuclear FIGN protein were detected according to the standard double-antibody sandwich ELISA procedure.

[0104] The above examples all achieved relatively good experimental results. It can be seen that the marker, kit and detection system of the present invention can well detect malignant tumors such as breast cancer and lung adenocarcinoma, with a simple detection method and high accuracy.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a diagnostic product for lung adenocarcinoma and / or breast cancer using FIGN protein in the cytoplasm as a marker, characterized in that: The lung adenocarcinoma and / or breast cancer diagnostic product includes a product for detecting the expression level of FIGN protein in the cytoplasm of a sample, wherein the sample is collected from the lung and / or breast of the subject to be diagnosed.

2. The use according to claim 1, characterized in that The product for detecting the expression level of FIGN protein in the cytoplasm of a sample is selected from the group consisting of reagents, kits, test papers, gene chips and antibody chips.

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