Application of marker LAP3 in gastric cancer detection

Abstract

The invention provides a gastric cancer detection marker LAP3, the LAP3 can be applied to gastric cancer detection, and meanwhile, the invention provides a kit for detecting the expression level of the gastric cancer marker LAP3. The invention provides a novel high-sensitivity and high-specificity marker for detection of gastric cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of disease detection, in particular to a marker for gastric cancer detection. BACKGROUND

[0002] The formation of gastric cancer undergoes multiple stages of pathological changes, including the occurrence of mutations and transformed cells under the action of stimulating factors, which causes the transformation of gastric gland epithelium to intestinal epithelium, leading to changes in the environment inside the stomach cavity, and an increased possibility of carcinogen generation. Factors that cause gastric cancer include both genetic factors and environmental factors, such as family history, diet, smoking, alcohol consumption, Helicobacter pylori infection, and Epstein-Barr virus infection.

[0003] Due to the lack of typical clinical signs in the early stage, and the high invasion and metastasis ability of gastric cancer, many patients are found to have advanced gastric cancer at the time of first diagnosis, and the prognosis is poor. In addition, the preliminary diagnosis of gastric cancer mainly relies on gastroscopy plus biopsy, which is an invasive operation that brings great pain to the patients. It also hinders the popularization and application of gastric cancer examination, and delays the detection time of gastric cancer. For gastric cancer without metastasis, early gastric cancer generally adopts endoscopic treatment (EMR or ESD) or surgical resection, and the five-year survival rate can reach more than 60%. For advanced metastatic gastric cancer, chemotherapy is generally used as the treatment method, and the five-year survival rate is not more than 30%. However, the emergence of chemotherapy resistance often leads to treatment failure.

[0004] With the development of immunodetection technology and molecular detection technology, the detection of proteins or genetic materials related to cancer can effectively assist in the diagnosis of cancer. However, at present, there is a lack of specific and highly sensitive biomarkers in the field of gastric cancer detection, resulting in a lack of non-invasive screening methods for cancer, which seriously affects the detection rate of early gastric cancer. For example, the current biomarkers that can be used for early diagnosis of gastric cancer include carcinoembryonic antigen (CEA), carbonhydrate antigen 19-9 (CA19-9), carbonhydrate antigen 724 (CA724), and carbonhydrate antigen 125 (CA125), etc. However, the sensitivity and specificity of these indicators in gastric cancer are relatively low, and the positive rate of CEA in gastric cancer is only 4.3%, the positive rates of CA19-9, CA724 and CA125 are 1.5%, 4.8% and 1.9% respectively, and the positive rate of combined detection of the above indicators is only 10.4%. Therefore, it is urgent to explore biomarkers with high sensitivity and specificity to improve the detection rate of early gastric cancer. SUMMARY

[0005] The application aims to provide an application of a marker LAP3 in detection of gastric cancer to solve the above problems.

[0006] To achieve the above object, the application adopts the following technical scheme: The application of a marker LAP3 in detection of gastric cancer.

[0007] The application of a marker LAP3 in detection of gastric cancer reagents.

[0008] In an alternative embodiment, the detection of gastric cancer includes gastric cancer screening, early diagnosis of gastric cancer, evaluation of gastric cancer treatment effect and prognosis of gastric cancer.

[0009] A reagent for detecting gastric cancer, wherein the reagent can detect a gastric cancer marker LAP3.

[0010] In an alternative embodiment, the reagent for detecting the gastric cancer marker LAP3 is an immunodiagnostic reagent or a molecular diagnostic reagent.

[0011] In an alternative embodiment, the detection method of the immunodiagnostic reagent is enzyme-linked immunoassay, double-antibody sandwich method, competition method or capture method.

[0012] In an alternative embodiment, the detection method of the molecular diagnostic reagent is PCR technology, molecular hybridization, gene sequencing, nucleic acid mass spectrometry or biochip.

[0013] In an alternative embodiment, the reagent for detecting the gastric cancer marker LAP3 is an enzyme-linked immunoassay reagent, a colloidal gold reagent, a chemiluminescence reagent, a time-resolved fluorescence immunoassay reagent, a flow cytometry fluorescence detection reagent, an immunoturbidimetry detection reagent or a mass spectrometry detection reagent.

[0014] In an alternative embodiment, the reagent is used for detection of a sample, and the sample includes whole blood, plasma, serum, urine, cerebrospinal fluid and saliva.

[0015] In an alternative embodiment, the reagent for detecting the gastric cancer marker LAP3 can specifically detect LAP3 protein or LAP3 protein expression, such as detecting LAP3 protein or LAP3 protein expression related nucleic acid, protein or compound.

[0016] In an alternative embodiment, the molecule for specifically detecting LAP3 protein is an antibody or a nucleic acid probe; preferably a monoclonal antibody.

[0017] In an alternative embodiment, the molecule for specifically detecting LAP3 protein carries a detectable label.

[0018] The application also provides a method for assisting in detecting gastric cancer, comprising the following steps: a) detecting LAP3 in a sample from a subject, and determining the LAP3 concentration in the sample; and b) comparing the LAP3 concentration in the sample with a reference cutoff value.

[0019] In an alternative embodiment, the reference cutoff value of the LAP3 concentration for distinguishing gastric cancer is 205.1 nmol / L. When the LAP3 concentration in the sample is higher than the reference cutoff value, the subject has a high risk of gastric cancer.

[0020] In an alternative embodiment, the LAP3 concentration range for detecting a high risk of gastric cancer is 256.46 nmol / L-260.34 nmol / L.

[0021] In an alternative embodiment, the LAP3 concentration range for detecting a low risk of gastric cancer is 194.39 nmol / L-201.33 nmol / L.

[0022] Compared with the prior art, the application has the following advantages: The serum protein marker LAP3 for detecting gastric cancer in the application has the advantages of high sensitivity and strong specificity in diagnosing gastric cancer, and can be used for gastric cancer screening, early diagnosis of gastric cancer, evaluation of gastric cancer treatment, and prognosis of gastric cancer. It has a wide application prospect in the immunodiagnosis of gastric cancer, molecular diagnosis of gastric cancer, and targeted therapy of gastric cancer. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the application, and therefore should not be regarded as a limitation on the scope of the application.

[0024] Figure 1 For laser capture microdissection (LCM) purification of gastric tissue, A, B, and C represent the NGM before cutting, after cutting, and after purification, respectively; D, E, and F represent the AH before cutting, after cutting, and after purification, respectively; G, H, and I represent the GPDAC before cutting, after cutting, and after purification, respectively; J, K, and L represent the LMGAC before cutting, after cutting, and after purification, respectively; Figure 2 For non-redundant protein and peptide identification of ProteinPilot; Figure 3 For MS / MS spectrum of identified protein matching peptide, the left side of the table is the m / z theoretical value of the peptide amino acid sequence, the gray area is the actual detection value consistent with the theoretical value, and the right side of the table is the peptide fingerprint of the peptide; Figure 4The differential expression of LAP3 in gastric adenocarcinoma and normal tissues; Figure 5 For immunohistochemical staining to detect the expression of LAP3 protein in gastric cancer tissues, A1, A2, A3 are paracancerous tissues; B1, B2, B3 are well-differentiated adenocarcinoma tissues; C1, C2, C3 are moderately differentiated adenocarcinoma tissues; D1, D2, D3 are poorly differentiated adenocarcinoma tissues; E1, E2, E3 are lymph node metastasis cancer tissues, A1-E1 is 40 times, A2-E2 is 100 times, A3-E3 is 400 times; Figure 6 For ELISA method to detect the expression of LAP3 protein in serum of different groups; Figure 7 ROC curve for LAP3 protein alone detection. DETAILED DESCRIPTION

[0025] As used herein: Leucine aminopeptidase 3 (LAP3) gene is located on human chromosome 4p15, composed of 13 exons, encoding LAP3 protein molecules. Leucine aminopeptidase 3 (LAP3) belongs to the M1 aminopeptidase family, which is an aminopeptidase that catalyzes the hydrolysis of leucine residues and can be secreted in human serum. It is expressed in a variety of cells and mainly localized in the cytoplasm.

[0026] "Individual", "subject", or "patient" refers to a vertebrate. In certain embodiments, the vertebrate refers to a mammal. Mammals include, but are not limited to, livestock animals such as cows, sport animals, pets such as cats, dogs, and horses, primates, mice, and rats. In certain embodiments, the mammal refers to a human.

[0027] As used herein, the term "sample" or "test sample" refers to a composition obtained or derived from a subject of interest that contains cells and / or other molecular entities to be characterized and / or identified, e.g., based on physical, biochemical, chemical, and / or physiological characteristics. In one embodiment, the definition encompasses blood and other liquid samples of biological origin and tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom. The source of a tissue sample can be solid tissue, like from a fresh, frozen and / or preserved organ or tissue sample or biopsy or punch; blood or any blood component; a body fluid; and cells or plasma from a subject at any time during gestation or development.

[0028] Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture fluid, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[0029] Gastric cancers as described herein include tubular adenocarcinoma, papillary adenocarcinoma, mucous adenocarcinoma, signet ring cell carcinoma, mixed carcinoma, adenosquamous carcinoma, squamous cell carcinoma, undifferentiated carcinoma.

[0030] In one embodiment, the sample is a clinical sample. In another embodiment, the sample is used in a diagnostic assay. In some embodiments, the sample is obtained from a primary or metastatic tumor. A representative tumor tissue mass / biopsy is often obtained using a tissue biopsy. Alternatively, tumor cells can be indirectly obtained in the form of tissue / fluid known or believed to contain tumor cells of interest. For example, a sample of a lung cancer lesion can be obtained by resection, bronchoscopy, fine needle aspiration, bronchial brushing, or from sputum / saliva, pleural fluid or blood.

[0031] As used herein, a "reference sample" refers to any sample, standard, or level used for comparison purposes, and a "reference cutoff" can be adapted based on the reference sample situation. In one embodiment, the reference sample is obtained from a healthy and / or non-diseased portion (e.g., tissue or cells) of the same subject or patient's body. In another embodiment, the reference sample is obtained from untreated tissue and / or cells of the same subject or patient's body. In yet another embodiment, the reference sample is obtained from a healthy and / or non-diseased portion (e.g., tissue or cells) of the body of an individual that is not the subject or patient. In still another embodiment, the reference sample is obtained from an untreated tissue and / or cell portion of the body of an individual that is not the subject or patient.

[0032] In certain embodiments, the reference sample is a single sample or a combination of multiple samples from the same subject or patient at one or more time points different from the time at which the test sample is obtained. For example, the reference sample is obtained from the same subject or patient at an earlier time point than the time at which the test sample is obtained. Such a reference sample can be useful if the reference sample is obtained during the initial diagnosis of the cancer and the test sample is obtained later, when the cancer has metastasized.

[0033] In certain embodiments, the reference sample comprises all types of biological samples as defined above under the term "sample" obtained from one or more individuals that are not the subject or patient. In certain embodiments, the reference sample is obtained from one or more individuals that have a angiogenic disorder (e.g., cancer) that is not the subject or patient.

[0034] In certain embodiments, the reference sample is a combined plurality of samples from one or more healthy individuals that are not the subject or patient. In certain embodiments, the reference sample is a combined plurality of samples from one or more individuals that have a disease or disorder (e.g., an angiogenic disorder such as, for example, cancer) that is not the subject or patient. In certain embodiments, the reference sample is a pooled RNA sample from normal tissue or a pooled plasma or serum sample from one or more individuals that are not the subject or patient. In certain embodiments, the reference sample is a pooled RNA sample from tumor tissue or a pooled plasma or serum sample from one or more individuals that have a disease or disorder (e.g., an angiogenic disorder such as, for example, cancer) that is not the subject or patient.

[0035] In certain embodiments, the term "elevated" refers to a total increase of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater in the level of a protein or nucleic acid as detected by standard methods known in the art, such as those described herein, as compared to a reference sample. In certain embodiments, the term elevated refers to an increase in the level of expression / amount of a gene or biomarker in a sample, wherein the increase is at least about 1.5X, 1.75X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 25X, 50X, 75X, or 100X the level of expression / amount of the corresponding gene or biomarker in a reference sample.

[0036] In certain embodiments, the term "decreased" refers herein to a total decrease of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater in the level of a protein or nucleic acid as detected by standard methods known in the art, such as those described herein, as compared to a reference sample. In certain embodiments, the term decreased refers to a decrease in the level of expression / amount of a gene or biomarker in a sample, wherein the decrease is at least about 0.9X, 0.8X, 0.7X, 0.6X, 0.5X, 0.4X, 0.3X, 0.2X, 0.1X, 0.05X, or 0.01X the level of expression / amount of the corresponding gene or biomarker in a reference sample.

[0037] "Detection" includes any means of detection, including direct and indirect detection. The content of detection includes gastric cancer screening, gastric cancer early diagnosis, gastric cancer efficacy evaluation and gastric cancer prognosis judgment, etc. and gastric cancer related detection.

[0038] The term "label" as used herein refers to a compound or composition that directly or indirectly provides for the detection of the agent to which it is coupled or fused. The label can be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can catalyze chemical changes to a detectable substrate compound or composition.

[0039] The antibodies described above in the present application include, but are not limited to, natural antibodies, polyclonal antibodies, monoclonal antibodies, monovalent antibodies, bispecific antibodies, heteroconjugate antibodies, multispecific antibodies, human antibodies, humanized antibodies, de-immunized antibodies, or chimeric antibodies, single-chain antibodies, Fv fragments, Fab fragments, F(ab') fragments, fragments generated from Fab expression libraries, anti-idiotypic (anti-Id) antibodies, and epitope binding fragments of any of the above. The antibodies can be functional fragments thereof, such as Fab, Fab', F(ab')2, Fv, and single binding domain fragments. The antibodies can also be single-chain antibodies, such as scFv.

[0040] The term "marker" as used herein refers generally to a molecule, including a gene, protein, carbohydrate structure, or glycolipid, whose expression in / on a mammalian tissue or cell can be detected by standard methods (or methods disclosed herein) and is predictive, diagnostic, and / or prognostic of the sensitivity of the mammalian tissue or cell to a treatment regimen based on cancer development. In certain embodiments, the expression of such biomarker is determined to be higher or lower than that observed in a reference sample. The expression of such biomarkers can be determined using high-throughput multiplexed immunoassays, such as those available from Rules Based Medicine, Inc. or Meso Scale Discovery. The expression of biomarkers can also be determined using, for example, PCR or FACS assays, immunohistochemical assays, or gene chip-based assays.

[0041] The term "array" or "microarray" as used herein refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes (e.g., oligonucleotides), on a substrate. The substrate can be a solid substrate (such as a glass slide) or a semi-solid substrate (such as a nitrocellulose membrane). The nucleotide sequences can be DNA, RNA, or any permutation thereof.

[0042] As used herein, a "gene," "target gene," "target biomarker," "target sequence," "target nucleic acid," or "target protein" is a polynucleotide or protein of interest that one desires to detect. Generally, as used herein, a "template" is a polynucleotide that contains a target nucleotide sequence. In some instances, the terms "target sequence," "template DNA," "template polynucleotide," "target nucleic acid," "target polynucleotide," and variations thereof are used interchangeably.

[0043] "Amplification" as used herein generally refers to the process of generating multiple copies of a desired sequence. "Multiple copies" refers to at least two copies. A "copy" does not necessarily refer to perfect sequence complementarity or identity to the template sequence. For example, a copy can include nucleotide analogs such as deoxyinosine, intentional sequence alterations such as those introduced by primers that hybridize to, but are not complementary to, the template, and / or sequence errors that occur during amplification.

[0044] A "native sequence" polypeptide includes a polypeptide having the same amino acid sequence as a polypeptide derived from nature. As such, a native sequence polypeptide can have the amino acid sequence of a naturally occurring polypeptide from any mammal. Such native sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence" polypeptide expressly encompasses naturally occurring truncations or secreted forms of the polypeptide (e.g., extracellular domain sequences), naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants.

[0045] The embodiments of the present application will be described in detail with specific examples below, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions are not specified in the examples, and the conventional conditions or the conditions recommended by the manufacturer are followed. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market.

[0046] Example 1 Preparation of tissue samples and sequencing

[0047] 1.1 Preparation of frozen sections

[0048] Different tissue samples (all confirmed by pathology) collected were taken out of liquid nitrogen, and the tissue blocks were placed on the disc carrier of the freezing section machine, O.C.T embedding agent was added dropwise, and frozen until the tissue blocks were completely embedded. The frozen tissues were serially sectioned, and each tissue sample was observed by H.E staining before, during and after freezing sectioning to confirm whether it contained the target tissue. After every 10-15 sections were cut, routine H.E staining was performed again to observe and confirm the presence or absence of the target tissue.

[0049] 1.2 Hematoxylin-eosin (H.E) staining

[0050] The frozen section was placed in 75% ethanol pre-cooled at 4 ℃ for 1 min, washed with 4 ℃ pre-cooled ultrapure water for 2 times, 5 S each time, dyed in eosin staining solution for 10 S, dyed in hematoxylin staining solution for 10 min, differentiated in 1% hydrochloric acid alcohol for 3 S, returned to blue in tap water, dehydrated in alcohol, transparent in xylene, and sealed in gum before being placed under a microscope for observation of the tissue.

[0051] 1.3 Methyl green staining of frozen sections

[0052] The frozen section was placed in 75% ethanol pre-cooled at 4 ℃ for 1 min, washed with 4 ℃ pre-cooled ultrapure water for 2 times, 5 S each time, dyed in 0.5% methyl green staining solution (containing protease inhibitor) pre-cooled at 4 ℃ for 30 S, washed with 4 ℃ pre-cooled ultrapure water for 2 times, 5 S each time, decolorized in 95% ethanol pre-cooled at 4 ℃ for 1 time, 5 S each time, air-dried at room temperature for 3-5 min, and then purified by laser capture microdissection (LCM) to obtain the target tissue.

[0053] 1.4 Purification of different tissues by LCM

[0054] The methyl green-stained frozen tissue section was placed on the stage of a laser capture microdissection device, a collection tube was installed, and the outline of the target tissue to be selected was outlined on the display screen using a mouse. The mouse was synchronized with the laser to automatically cut the target tissue in the section. According to the field of view, magnification, and distribution of the target tissue, the laser aperture, speed, and intensity were adjusted. After the target tissue was cut, it fell into the collection tube for collection. The cutting time of each tissue should not be too long (to prevent degradation of the protein in the tissue). Before cutting, 2-3 μL of protease inhibitor was added to the cover of the collection tube. After cutting, the purified tissue was collected by centrifugation at 4 ℃ and stored in a -80 ℃ refrigerator.

[0055] The LCM technique as described above was used to separate and purify the glands in NGM, AH, GPDAC, and LMGAC tissues from normal gastric mucosa (NGM), atypical hyperplasia gastric mucosa (AH), gastric poorly differentiated adenocarcinoma (GPDAC), and lymph node metastatic carcinoma (LMGAC), respectively, to reduce the interference of non-target tissues such as fibrous tissue, inflammatory cells, and blood vessels in the interstitium. The above tissues were cut after being routinely stained with H.E. After the histological type was determined to meet the experimental requirements, frozen sections were prepared. After the prepared frozen sections were stained with methyl green, the corresponding tissues were purified by LCM (e.g. Figure 1 ). The field of view was adjusted according to the position and size of the different tissues in the frozen section. The purity of the target tissue obtained after cutting was ≥95%.

[0056] 1.5 Extraction and concentration determination of total protein of the tissue

[0057] After the LCM purification of different gastric tissues, protein lysate (10 mM PMSF, 65 mM DTT, 7 M Urea, 2 M Thiourea) was added, mixed, lysed on ice for 1 h, and the tissue proteins were fully lysed by ultrasonic. The mixture was centrifuged at 12000 rpm at 4 ℃ for 45 min, and the supernatant was collected as the total protein extracted from the purified tissue. The protein solution was adjusted to pH 8.0-9.0 with 50 mM NaOH. A small part of the protein was taken to determine the concentration, and the rest was stored at -80 ℃ for later use.

[0058] The protein concentration was determined by using a micro BCA protein assay kit. According to the principle that protein and Cu2+ are combined in an alkaline environment, Cu2+ is reduced to Cu1+-BCA, which is combined with Cu1+ to form a stable blue-violet compound, there is a high absorption value at 562 nm wavelength, and the protein concentration is proportional to the principle. According to the kit instructions, first draw the standard curve, calculate the linear regression equation, refer to the linear regression equation, according to the absorbance value, calculate the protein concentration of different samples.

[0059] Because of the genetic individual differences in the occurrence of gastric cancer, the purified tissues were from different tissues of 20 gastric cancer patients, and the purified tissues were classified and mixed for iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) labeling and two-dimensional liquid chromatography-mass spectrometry identification.

[0060] 1.6 Isotope iTRAQ labeling of proteins

[0061] After acetone precipitation of the sample protein, dissolve with lysis solution. Take about 100 μL (about 100 μg) of sample protein and place it in a 1.5 mL EP tube, add 300 μL of precipitant 1, shake well, and place on ice for 15 min; add 300 μL of precipitant 2, shake well, centrifuge at 12000 rpm at 4 ℃ for 5 min, discard the supernatant; add 40 μL of washing agent 1, centrifuge at 12000 rpm at 4 ℃ for 5 min, discard the supernatant; add 25 μL of graded water, shake well; add 1 ml of washing agent 2 (acetone, pre-cooled at -20 ℃ for 1 h) and 5 μL of washing additive 2, shake well, incubate at -20 ℃ for 30 min (the time can be appropriately extended), shake every 10 min for 30 s during the incubation; centrifuge at 12000 rpm at 4 ℃ for 5 min, discard the supernatant; after drying, add 1 μL (calculated based on 100 μg of sample) of reducing reagent, mix well, and incubate at 60 ℃ for 1 h; add 1 μL of cysteine blocking reagent, mix well, and place at room temperature for 10 min; centrifuge at 12000 rpm at 4 ℃ for 15 min, discard the supernatant; add acetone pre-cooled at 4 ℃ (acetone: sample volume ratio = 5:1), precipitate at -20 ℃ for 1 h, centrifuge at 12000 rpm at 4 ℃ for 15 min, discard the supernatant; add 60 μL of iTRAQ decomposition buffer; add 10 μL (calculated based on 100 μg of sample), 1 mg / mL trypsin, and incubate at 37 ℃ overnight (12-16 h).

[0062] iTRAQ™ reagents 113, iTRAQ™ reagents 115, iTRAQ™ reagents 117 and iTRAQ™ reagents 119 were labeled by adding 70 μL of ethanol to each reagent and mixing. Each tissue protein sample was added with the corresponding iTRAQ reagent (normal gastric mucosa: iTRAQ™ reagents 113; gastric low differentiated adenocarcinoma: iTRAQ™ reagents 115; atypical hyperplasia: iTRAQ™ reagents 117; lymph node metastasis cancer: iTRAQ™ reagent 119) and mixed. Incubation was performed at room temperature for 1 h. iTRAQ labeling reaction was terminated by adding 100 μL of ultrapure water and placing at room temperature for 30 min. All protein samples were mixed, homogenized, and lyophilized. The samples were dissolved in 500 μL of 0.1 % FA deionized water, centrifuged at 10000 rpm for 1 min at room temperature. The C18 desalting column (Sep-pak C18 Cartridges, Vac 1 cc, waters, USA) was washed twice with 500 μL of pure acetonitrile (dusting) and twice with 500 μL of 0.1 % FA water (water balance). The iTRAQ reagent labeled sample was slowly eluted in the C18 desalting column, washed twice with 500 μL of 0.1 % FA water, eluted once with 400 μL of 0.1 % FA 50 % acetonitrile, and collected the eluent (containing the sample) and lyophilized.

[0063] 1.7 LC-MS / MS peptide analysis

[0064] 1.7.1 First dimension strong cation column (SCX) separation

[0065] Dissolve the sample in 1 mL SCX buffer (25 % v / v acetonitrile, 10 mM KH2PO4, pH 2.6), mix well, then load the sample solution into a Polysulfoethyl column (2.1 mm x 100 mm, 5 pm, 200 A, The Nest Group, Inc. MA) and separate using a 20 AD HPLC chromatography system, A phase: 10 mM KH2PO4, 25 % CAN (acetonitrile, Fisher scientific, Fair lawn, New Jersey), PH 2.6, B phase: 10 mM KH2PO4, 350 mM KCL, 25 % CAN, PH 2.6; UV detection wavelength: 214 nm / 280 nm, flow rate: 200 pL / min; time: 60 min, salt gradient: from 5 min 5 % to 40 min rising to 25 % (adjusted with A liquid), the specific salt gradient is as follows: 1) 5 mM, 5 % CAN; 2) 30 mM, 5 % CAN; 3) 60 mM, 5 % CAN, 4) 100 mM, 5 % CAN, 5) 250 mM, 5 % CAN; 6) 500 mM, 50 % CAN, vacuum centrifugal concentration, then dissolved in 50 pL RPLC A phase (5 % ACN, 0.1 % formic acid) for second dimension analysis.

[0066] 1.7.2 Second dimension reversed-phase liquid chromatography-mass spectrometry (RPLC-MS)

[0067] An Up column (Zorbax 300SB-C18 column, 0.1 x 15 mm, 5 μm, 300 Å, microm, USA) was used for chromatographic separation with a Shimadzu 20 AD HPLC system, chromatographic separation time: 90 min, gradient: from 5 min 5 % to 70 min rising to 35 %, A: 5 % ACN, 0.1 % formic acid, B: 95 % ACN, 0.1 % formic acid, flow rate: 300 nL / min, tandem mass spectrometry (MS / MS) analysis was performed with a Q-STAR XL (Applied Biosystem, USA) system, information dependent acquisition mode (IDA) was used for mass spectrometry analysis, first full scan once in the range of 400-1800 m / z, then select the top 4 ion peaks with the highest intensity for secondary ion scanning. Ion spectrum 100-2000 m / z range accumulated 2 S, set to increase all ion mode, using ±0.015 % (150 ppm) tolerance range dynamic exclusion, set 30 S for exclusion time. Mass spectrometry conditions: ion source spray voltage is 1.7 kV, full scan MS uses Orbitrap for positive ion mode scanning, scanning range is 300-1800 m / z, resolution is set to 60,000 (m / z 400), when performing tandem mass spectrometry (MS / MS) analysis, Orbitrap AGC is set to 1 x 106 ions, maximum introduction time is 150 ms, Orbitrap performs 4 MS / MS scans on the top 4 ions with intensity greater than 500, using higher energy C-trap dissociation (HCD) for peptide ion fragmentation.

[0068] Protein identification and analysis of Example 2

[0069] 2.1 ProteinPilot software identifies proteins

[0070] The ProteinPilot™ software (version 4.2, revision 1340) of SCIEX Company was used as a protein analysis tool to analyze the protein mass spectrometry data in the tissue of Example 1.

[0071] The list of detected non-redundant proteins is as follows Figure 2The Protein Detected column shows the proteins detected, ranked by unusedProtScore from high to low. The columns include the coverage of all matching peptides (% Cov), the coverage of peptides with confidence > 95% [% Cov (95%)], the Accession # of the protein in the IPI database, the name of the protein, the species of origin, the number of peptides with confidence > 95% [Peptides (95%)], the quantitation information, and the corresponding P value. The Peptide Quantitation column shows the mass spectrometry identification information of all matching peptides (including confidence, peptide sequence, modification, mass error of experimental value and theoretical value, mass-to-charge ratio, charge number of precursor ion, etc.) and quantitation information. The Peptide Quantitation Information column is the mass spectrometry quantitation information of the selected peptide (such as LGEHNIEVLEGNEQFINAAK) and Precursor MS Region. The identified protein matching peptide coverage is color-coded, with the identified protein matching peptide being color-coded, green indicating a peptide with confidence > 95%, red indicating a peptide with confidence > 75%, yellow indicating a peptide with confidence > 50%, and gray indicating an unmatched peptide. The MS / MS spectrum of the peptide is shown in Figure 1, with the m / z theoretical value of the peptide amino acid sequence on the left and the peptide fingerprint of the peptide on the right. Figure 3

[0072] The peptide fingerprint of the peptide identified by mass spectrometry was searched in the database, and bioinformatics analysis was performed, and a total of 243 differentially expressed proteins were obtained. Table 1 shows some of the differentially expressed proteins and their changes, listing the IPI number of the protein in the identified protein database, the name of the protein, and the protein quantification values in the comparison of different tissues. The identified proteins with a quantification ratio greater than 1.5 were up-regulated, and those with a quantification ratio less than 0.667 were down-regulated. Among them, 153 were up-regulated in the gastric cancer tissue, and 90 were down-regulated. In addition, some of the differentially expressed proteins were progressively up-regulated in the process of gastric mucosal epithelial carcinogenesis, and some of the differentially expressed proteins were progressively down-regulated in the process of gastric mucosal epithelial carcinogenesis.

[0073] Table 1 Quantitative proteomics identification of differentially expressed proteins in each stage of gastric mucosal epithelial carcinogenesis

[0074] ​Note: No: protein number; Accession#: IPI number of protein in database; Protein Name: protein name; AH vs. NGM, GPDAC vs. NGM, LMGAC vs. NGM, GPDAC vs. AH, LMGAC vs. AH, LMGAC vs. GPDAC represent the ratio of protein expression in two stages; NGM: normal gastric mucosa, AH: atypical hyperplasia; GPDAC: gastric poorly differentiated adenocarcinoma; LMGAC: lymph node metastasis cancer. IPI number is the number in IPI database (IPI human database).

[0075] 2.2 Analysis of markers in TCGA database

[0076] The expression of LAP3 in gastric cancer tissues was analyzed by TCGA (The Cancer Genome Atlas) database. The inventors found that LAP3 protein was highly expressed in gastric cancer tissues by quantitative proteomics technology in the early stage, and further analyzed the expression of LAP3 in gastric adenocarcinoma by using TCGA database. The results are shown in Figure 4 As shown, compared with normal tissues, the expression of LAP3 in gastric adenocarcinoma was significantly increased (P<0.05).

[0077] 2.3 Clinical verification markers

[0078] The expression of LAP3 protein in gastric cancer tissue chip (containing 206 cases of gastric adenocarcinoma tissues, 206 cases of paracancerous tissues and 65 cases of lymph node metastasis cancer tissues) was verified by immunohistochemical technique. The experimental results are shown in Figure 5 As shown, LAP3 protein is mainly located in the cytoplasm. The positive rates of LAP3 protein in paracancerous gastric mucosa, high-moderately differentiated adenocarcinoma, low differentiated adenocarcinoma and lymph node metastasis cancer tissues were 55.83% (115 / 206), 77.27% (102 / 132), 93.24% (69 / 74) and 76.92% (50 / 65), respectively. As shown in Table 2, the expression of LAP3 protein in gastric adenocarcinoma tissues was significantly increased (P<0.001).

[0079] Table 2: Expression statistics of LAP3 protein in gastric cancer tissues

[0080] Note: 1. Compared with paracancerous gastric mucosa tissues, **** P <0.0001; *** P <0.001; ** P <0.01.

[0081] 2, The difference between well-differentiated adenocarcinoma and moderately differentiated adenocarcinoma is small, but both are significantly different from poorly differentiated adenocarcinoma, so high-moderate is combined as a group.

[0082] 3, The positive staining intensity and the percentage of positive cells were observed, the average value was calculated, and the formula was used to calculate the immune response score (IRS = staining intensity x percentage of positive cells). Staining intensity score: 0 points: no color; 1 point: light yellow; 2 points: brown yellow; 3 points: brown. Positive cell percentage score: 1 point: ≤10; 2 points: 11%-50%; 3 points: 51%-80%; 4 points: >80%. Final score = staining intensity score x positive cell percentage score. According to the final score, different positive levels are divided: 0 points for negative (-), 1-4 points for weakly positive (+), 5-8 points for moderate positive (++), and 9-12 points for strong positive (+++).

[0083] The expression of LAP3 protein in the serum of 60 patients with gastric adenocarcinoma and 32 normal healthy people was detected by ELISA. The patient serum was the preoperative serum of gastric cancer patients treated and operated in the First Affiliated Hospital of Nanhua University in 2021, and the serum specimen of healthy people who came to the hospital for physical examination was used as the control group. Among the 60 patients with gastric cancer, there were 35 male patients and 25 female patients. The age was between 31-82 years old, and the average age was 61 years old. Thirteen males and 19 females were selected as the control group, and the age was between 44-85 years old, and the average age was 62 years old. There was no significant difference in general data such as gender (□2=2.359, P=0.125) and age (t=0.634, P=0.528) between the two groups (P>0.05), which were comparable. The experimental results are as follows Figure 6 As shown in Table 3, the concentration of LAP3 in the serum specimens of 60 patients with gastric cancer was 258.40±1.94 nmol / L, and the concentration of LAP3 in the serum specimens of 32 normal healthy people was 197.86±3.47 nmol / L. The expression of LAP3 in the serum of patients with gastric cancer was higher than that in normal healthy people (P<0.0001).

[0084] Table 3 Expression analysis of LAP3 protein in the serum of patients with gastric cancer

[0085] **** Compared with the health examination group, P <0.0001.

[0086] The clinicopathological data of the above 60 gastric cancer patients were analyzed, and the results are shown in Table 4. The expression of LAP3 in the serum of the patients was not related to the patients' gender (P=0.258), age (P=0.781), tumor size (P=0.463), degree of differentiation (P=0.464), TNM stage (P=0.224) and lymph node metastasis (P=0.212).

[0087] Table 4. Relationship analysis between serum LAP3 protein expression and clinicopathological parameters

[0088] 2.4 Diagnostic evaluation of serum LAP3 protein detection for gastric cancer

[0089] The performance of LAP3 protein in gastric cancer diagnosis was assessed by plotting ROC curves, and the results are as follows: Figure 7 As shown, the area under the curve for serum LAP3 protein was 0.713, suggesting that LAP3 protein has clinical value in the screening, diagnosis, treatment monitoring, and prognostic assessment of gastric cancer. When the Youden index (sensitivity + specificity - 1) reaches its maximum value, the optimal cutoff point for LAP3 is 205.1 nmol / L, at which point its diagnostic sensitivity is 100% and its specificity is 50%.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

[0091] Furthermore, those skilled in the art will understand that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are intended to be within the scope of this application and form different embodiments. For example, in the foregoing claims, any of the claimed embodiments can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of this application and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

Claims

1. Application of the biomarker LAP3 in the detection of gastric cancer.

2. Application of the biomarker LAP3 in reagents for detecting gastric cancer.

3. The application of the biomarker LAP3 according to claim 2 in a gastric cancer detection reagent, wherein the gastric cancer detection includes gastric cancer screening, early diagnosis of gastric cancer, evaluation of gastric cancer treatment efficacy, and prognosis assessment of gastric cancer.

4. A reagent for detecting gastric cancer, wherein the reagent can detect the gastric cancer marker LAP3; Preferably, the reagent is an immunodiagnostic reagent or a molecular diagnostic reagent; Preferably, the reagent is an enzyme-linked immunosorbent assay (ELISA) reagent, a colloidal gold reagent, a chemiluminescent reagent, a time-resolved fluorescence immunoassay reagent, a flow cytometry reagent, an immunoturbidimetric assay reagent, or a mass spectrometry reagent.

5. The reagent for detecting gastric cancer according to claim 4, wherein the reagent is used for the detection of a sample, the sample comprising whole blood, plasma, serum, urine, cerebrospinal fluid, and saliva.

6. The reagent for detecting gastric cancer according to claim 4, wherein the reagent for detecting the gastric cancer marker LAP3 is capable of specifically detecting LAP3 protein or LAP3 protein expression.

7. The reagent for detecting gastric cancer according to claim 4, wherein the molecule specifically detecting LAP3 protein is an antibody or a nucleic acid probe.

8. The reagent for detecting gastric cancer according to claim 7, wherein the molecule specifically detecting LAP3 protein carries a detectable marker.

9. A method for assisting in the detection of gastric cancer, comprising the following steps: a) Detect LAP3 in samples from subjects to determine the LAP3 concentration in the samples; b) Compare the LAP3 concentration in the samples with a reference threshold.

10. The method for assisting in the detection of gastric cancer according to claim 9, wherein the reference threshold value for LAP3 is 205.1 nmol / L.

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