CN108872600B

CN108872600B - Application of PDLIM4 as gastric cancer marker

Info

Publication number: CN108872600B
Application number: CN201810749863.7A
Authority: CN
Inventors: 吕文堂; 陈慕熙; 尤姝
Original assignee: Guangdong Zhongke Qingzi Medical Technology Co ltd
Current assignee: Guangdong Zhongke Qingzi Medical Technology Co.,Ltd.
Priority date: 2018-07-09
Filing date: 2018-07-09
Publication date: 2021-06-15
Anticipated expiration: 2038-07-09
Also published as: CN113189345B; CN113189344A; CN113189344B; CN113189345A; CN108872600A

Abstract

The invention provides application of PDLIM4 as a gastric cancer diagnosis marker. The application of PDLIM4 provided by the invention is that PDLIM4 is used as a protein molecular marker for detecting gastric cancer. PDLIM4 has obvious differential expression in gastric cancer cell and cancer paracell, so that it can be used to detect whether the patient has gastric cancer based on its expression in gastric cell.

Description

Application of PDLIM4 as gastric cancer marker

Technical Field

The invention relates to the technical field of screening/detecting of gastric cancer, in particular to application of PDLIM4 as a gastric cancer marker.

Background

The gastric cancer is a malignant tumor originated from gastric mucosa epithelium, the incidence rate of the gastric cancer is the first in various malignant tumors in China, the incidence rate of the gastric cancer is obviously different regionally, and the incidence rate of the gastric cancer is obviously higher in northwest and east coastal areas of China than in south areas. The good hair age is more than 50 years old, and the ratio of the incidence rates of men and women is 2: 1. Gastric cancer tends to be younger due to changes in dietary structure, increased working pressure, infection with helicobacter pylori, and the like. Gastric cancer can occur in any part of the stomach, more than half of which occur in antrum, and the greater curvature, lesser curvature, anterior and posterior walls of the stomach can be affected. Most of gastric cancers belong to adenocarcinoma, have no obvious symptoms in the early stage, or have nonspecific symptoms such as epigastric discomfort, eructation and the like, are often similar to the symptoms of chronic stomach diseases such as gastritis, gastric ulcer and the like, and are easy to ignore, so the early diagnosis rate of the gastric cancers in China is still low at present. The prognosis of gastric cancer is related to the pathological stage, location, tissue type, biological behavior, and therapeutic measures of gastric cancer.

So far, the abnormal expression of less than 10 genes has been determined to be related to the occurrence and development of gastric cancer, but the abnormal expression rate of the determined gastric cancer related genes in gastric cancer is not high, the pathogenesis of gastric cancer is not clarified so far, and the early diagnosis rate of gastric cancer still needs to be improved. In addition, the survival rate of gastric cancer patients is not obviously improved in the traditional gastric cancer operation and chemotherapy in China and a plurality of gene therapy methods matched with the traditional gastric cancer operation and chemotherapy in recent years, so that the search for a new gastric cancer related gene, especially a gastric cancer high expression gene, has important significance for discussing the pathogenesis and early diagnosis of gastric cancer.

PDLIM4(HGNC accession number 16501; Entrez Gene accession number 8572; Ensembl accession number ENSG 00000131435; OMIM accession number 603422; UniProtKB accession number P50479) belongs to the FOZ/LIM protein family and plays an important role in the development process.

Disclosure of Invention

By screening proteins differentially expressed in human gastric cancer tissues and corresponding paraneoplastic tissues, the inventors of the present application found a protein differentially expressed in gastric cancer tissues and corresponding paraneoplastic tissues (up-regulated expression in cancer tissues), and identified it as PDLIM4 by mass spectrometry. Immunoblotting experiments confirmed that PDLIM4 was indeed differentially expressed in gastric cancer tissues and corresponding paracancerous tissues (up-regulated expression in cancer tissues). Immunohistochemical experiments on 55 human gastric cancer tissues and paracarcinoma tissues further prove that PDLIM4 is differentially expressed (up-regulated) in the human gastric cancer tissues and the paracarcinoma tissues, and immunological experiments on serum also show that PDLIM4 is highly expressed in human gastric cancer patients.

Based on the correlation between PDLIM4 and gastric cancer, PDLIM4 can be used as a protein molecular marker to detect the expression level of the PDLIM4 and can be used for detecting the gastric cancer.

It is therefore a primary object of the present invention to provide PDLIM4 for use in detecting gastric cancer.

The PDLIM4 provided by the invention is applied as a protein molecular marker for detecting gastric cancer.

The application of PDLIM4 in detecting the protein molecular marker of gastric cancer provided by the invention is a kit for preparing a quantitative proteome technology.

It is another object of the present invention to provide the use of an antibody to PDLIM 4.

The PDLIM4 antibody provided by the invention is applied to preparation for detecting gastric cancer and/or a kit for detecting gastric cancer.

According to the present invention, antibodies against PDLIM4 include monoclonal antibodies and polyclonal antibodies.

It is yet another object of the present invention to provide a method for detecting in vitro whether the expression of PDLIM4 is abnormal in gastric tissue.

The method provided by the invention comprises the following steps: the amount of PDLIM4 in the stomach tissue/serum to be tested was detected and compared with the amount of PDLIM4 in normal stomach tissue/serum.

In view of the fact that no association of PDLIM4 with gastric cancer has been reported so far. Therefore, the discovery of the invention provides a brand-new approach for the diagnosis and/or treatment of gastric cancer.

Furthermore, the present invention also relates to the following embodiments:

1. a method of diagnosing gastric cancer, the method comprising:

a) obtaining a suspected cancer tissue of the individual,

b) determining the expression level of PDLIM4 therein,

c) obtaining the same tissue of a normal individual or a normal tissue of the same individual, and

d) the expression level of PDLIM4 in the sample was determined as a control expression level,

e) comparing the expression level of PDLIM4 obtained in b) with the control expression level of PDLIM4 obtained in d), determining that the individual has gastric cancer when the expression level of PDLIM4 obtained in b) is increased compared to the control expression level of PDLIM4 obtained in d).

2. The method of embodiment 1, wherein the tissue is stomach tissue.

3. The method according to

embodiment

1 or 2, wherein said expression level is detected by any one method selected from the group consisting of:

(a) detecting mRNA encoding PDLIM4, and

(b) detecting the protein of PDLIM 4.

4. The method according to any one of embodiments 1-3, wherein the amount of PDLIM4 is determined by using an antibody against PDLIM 4.

5. The method according to any one of embodiments 1-4, wherein said antibody to PDLIM4 is a monoclonal or polyclonal antibody.

6. A method of diagnosing gastric cancer, the method comprising:

a) obtaining the serum of the individual and obtaining the serum of the individual,

b) determining the expression level of PDLIM4 therein,

c) obtaining the serum of a normal individual,

d) determining the expression level of PDLIM4 therein as a control expression level, and

7. The method of embodiment 6, wherein the expression level of PDLIM4 is determined by using an antibody against PDLIM 4.

8. The method according to embodiment 6 or 7, wherein the antibody against PDLIM4 is a monoclonal or polyclonal antibody.

9. Use of an antibody against PDLIM4 in the preparation of a kit for diagnosing gastric cancer in an individual.

10. A kit for diagnosing gastric cancer in a subject, the kit comprising

a) Reagents for determining the expression level of PDLIM4, and

b) instructions for use.

Other aspects and advantages of the invention will become apparent by reference to the following description of specific embodiments thereof, taken in conjunction with the accompanying drawings.

Drawings

FIG. 1 is a relative distribution diagram of protein expression levels obtained by analyzing data of an immunoblot of PDLIM 4.

FIG. 2 is a graph showing the relative distribution of protein expression levels obtained by analyzing the data of the results of the PDLIM4 immunohistochemistry experiment.

FIG. 3 is a data analysis relative distribution diagram of the experimental results of protein content in PDLIM4 serum.

Detailed Description

In a preferred embodiment, fragments of the proteins are advantageously used. The fragment should be identical to the sequence of the protein in the sample or a fragment thereof obtained by enzymatic digestion (e.g., trypsin digestion). For ease of manipulation, the fragments are preferably 5 to 50 amino acids in length, preferably 6 to 30 amino acids, more preferably 6 to 25 amino acids in length. Furthermore, the optimal transitions of the fragments should have a good signal-to-noise ratio. The sample is preferably labeled with a heavy isotope (e.g., 13C, 14N) to distinguish it from an unlabeled sample.

Table 1 PDLIM4 peptide fragment sequence.

The step of early detection of a patient with gastric cancer comprises adding an amount of heavy isotope labeled fragments to an enzyme (e.g., trypsin) digested patient serum sample and detecting the levels of the fragments in the patient serum using quantitative proteomics techniques to determine the level of PDLIM4 in the serum; the measured level is compared with a critical value determined from the level of a normal person, and if the measured level is higher than the critical value, it indicates that the gastric cancer has a high probability.

The critical value can be determined by one skilled in the art according to conventional means. For example, one skilled in the art can plot a receiver operating characteristic curve (ROC curve) based on measured serum protein content data of normal persons (group), and then determine a cut-off value (cut-off).

In a more preferred embodiment, the quantitative proteomics techniques can be combined with synthetic peptide fragment based absolute quantification techniques (AQUA) so that direct absolute measurement of PDLIM4 in multiple samples can be performed. Comparing the measured level with the level of normal control serum, and if the measured level is higher than the level of normal control serum, it indicates that the subject has gastric cancer.

In another preferred embodiment, the substance that specifically binds to human PDLIM4 protein is an antibody. The term "antibody" is used herein in its broadest sense and specifically includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies). The term "antibody" also includes fragments of intact molecules, such as Fab and F (ab')2, which are capable of binding in response to an antigen. Fab and F (ab')2 fragments lack the Fe fragment of intact antibodies, are cleared more rapidly from the circulatory system, and have lower nonspecific tissue binding than intact antibodies. Fab and F (ab')2 and other antibody fragments useful in the present invention can also be used to detect and quantify human PDLIM4, provided they exhibit the desired activity for specific binding. These fragments can generally be generated by proteolytic cleavage with papain (to generate Fab fragments) or pepsin (to generate F (ab')2 fragments).

The polyclonal antibody and the monoclonal antibody used in the present invention can be produced by a conventional method. Generally, a suitable animal, preferably a mouse, rat, rabbit or goat, is first immunized with the protein. Rabbit and goat are preferred for the preparation of polyclonal antisera, since the available serum volume is large and labelled anti-rabbit and anti-goat antibodies can be obtained. Immunization is generally carried out by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and then injecting the mixture or emulsion parenterally, usually subcutaneously or intramuscularly. After 2-6 weeks, the immunization is boosted by one or more injections with protein in saline (preferably incomplete zolpidem U). The blood of the immunized animal is drawn into a glass or plastic container, the blood is incubated at 25 ℃ for I hours, and then at 4 ℃ for 2-18 hours to obtain the polyclonal antiserum. Monoclonal antibodies can be made by the standard method of Kohler and Milstein [ Nature (1975)256:495-96] or modifications thereof. Typically, mice or rats are immunized as described above. However, rather than bleeding the animal and then extracting the serum, the spleen (and optionally several large lymph nodes) is removed and dispersed into single cells. If desired, spleen cells can be screened by adding a cell suspension (after removal of non-specifically adhered cells) to the protein antigen-coated plate or well. Antigen-specific membrane-bound immunoglobulin-expressing B cells bind to the plate and are not washed away as are other substances in the suspension. The resulting B cells or all dissociated splenocytes are then induced to fuse with myeloma cells to form hybridomas, which are cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, "HAT"). The resulting hybridomas were inoculated by limiting dilution and assayed for production of antibodies that specifically bind to the immunizing antigen (and not to unrelated antigens). The selected monoclonal antibody-secreting hybridomas are then cultured in vitro (e.g., in tissue culture flasks or hollow fiber reactors) or in vivo (e.g., in mouse ascites).

The determination of antigens in serum using antibodies can be accomplished using immunofluorescence techniques using fluorescently labeled antibodies in conjunction with optical microscopy, flow cytometry, or fluorometric detection. The assay methods include, for example, incubating a biological sample (e.g., a biological fluid such as serum) in the presence of an antibody capable of identifying a detectable label, and then detecting the antibody by any of a number of methods well known in the art.

The biological sample may be treated with a solid support or carrier, such as nitrocellulose, or other solid support or carrier capable of immobilizing cells, cell particles, or soluble proteins. The support or carrier is then washed with a suitable buffer and treated with detectably labeled antibody according to the invention as described above. The solid support or carrier is then washed a second time with buffer to remove unbound antibody. The amount of label bound to the solid support or carrier can then be detected by conventional means. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylase, natural and modified celluloses, polyacrylamides, gabbros and magnetite. The structure of the support or carrier may be spherical (e.g., within a bead), cylindrical (e.g., the inner surface of a test tube or the outer surface of a rod). Additionally, the surface may be flat, such as a plate, test strip, or the like. Preferred supports or carriers include polystyrene beads. Those skilled in the art will know of many other suitable carriers for binding antibodies or antigens, or will be able to determine such carriers by routine experimentation.

In the present invention, the antibody may be linked to an enzyme and then used in an enzyme immunoassay. Then, when the enzyme is contacted with a suitable substrate, the enzyme reacts with the substrate to produce a chemical that can be detected (e.g., by spectrophotometric analysis, fluorometric analysis, or visual inspection). Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, S-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Detection can be accomplished using colorimetric methods using a chromophore substrate for the enzyme. Detection may also be accomplished by visually comparing the extent of enzyme reaction with a similarly prepared standard.

Detection can be accomplished by a variety of other immunoassays. For example, by radiolabelling the antibody, it can be detected by using Radioimmunoassay (RIA). The radioisotope can be detected by scintillation counting or the like or by autoradiography.

The antibodies of the invention may also be labeled with fluorescent compounds. When a fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected by fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The antibody may also be detectably labeled with a fluorescent metal such as 152E or other lanthanide metals. These metals can be attached to the antibody through chelating groups of these metals, such as diethylenetriaminepentaacetic acid (ETPA). Antibodies can also be detectably labeled by coupling them to chemiluminescent compounds. The presence of luminescence is then detected during the chemical reaction to detect the presence of the chemiluminescent-labeled antibody. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, thermal acridine ester, imidazole, acridine salt and oxalate ester. Likewise, the antibodies of the invention may also be labeled with a bioluminescent compound. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of bioluminescent proteins can be determined by detecting luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase and aequorin.

The antibodies of the invention may also be used in immunoassay assays, such as sandwich assays. In a typical immunoassay, an amount of unlabeled antibody (or antibody fragment) is bound to a solid support or carrier, and an amount of detectably labeled soluble antibody is added, so that the ternary complex formed between the solid-phase antibody, antigen, and labeled antibody can be detected and/or quantified. Typical and preferred immunoassay tests include "forward" tests in which an antibody bound to a solid phase is first contacted with a sample to be tested, and antigen is extracted from the sample by forming a binary solid phase antibody-antigen complex. After an appropriate incubation time, the solid support or carrier is washed to remove liquid sample residues (including unreacted antigen, if any) and then contacted with a solution containing an unknown amount of labeled antibody. After a second incubation period to allow the labeled antibody to complex with the antigen bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labeled antibody.

In a third aspect, the present invention provides a kit for detecting whether a subject has gastric cancer, the kit comprising an amino acid sequence shown in table 1.

In a preferred embodiment, the amino acid sequence may also carry a label. The label is preferably an isotopic label. The kit may further comprise instructions that provide instructions to a user to use the above-described polypeptide sequences to detect whether a subject has gastric cancer, as described above.

Examples

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

The experimental procedures for which specific conditions are not indicated in the following examples are generally carried out according to conventional conditions such as those described in Sambrook et al, molecular cloning, A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.

In the following examples of the present invention, urea, 3- [ (3-cholamidopropyl) _ diethylammonium ] -1-propanesulfonic acid (CHAPS), Sodium Dodecylsulfonate (SDS), Dithiothreitol (DTT), Tris (hydroxymethyl) aminoethane (Tris), Iodoacetamide (IAA) were purchased from Bio-Rad; chemical reagents such as beta-mercaptoethanol were purchased from Sigma; trypsin (Trypsin, sequencing grade) was purchased from Promega; itraq (anaerobic tags for relative and absolute quantification) reagent purchased from AB SCIEX; SCX and SAX columns and pH buffer kits were purchased from waters.

In the following examples of the invention, the formulations of the solutions used are as follows:

lysis solution 8mol/L urea, 4% CHAPS, 40mmol/L Tris and 65mmol/L DTT.

TBST: tris 2.42g/L, sodium chloride 8g/L, Tween-201 ml/L, pH adjusted to 7.6 with HCl.

Example 1 preparation of protein samples of cancer tissue and tissue adjacent to cancer of gastric cancer. Protein samples of cancer tissues and tissues adjacent to the cancer, which are prepared by an Enzymatic Sample Preparation (ESP) method, are quantified by itraq (immunological tags for relative and absolute quantification) differential proteomics. As a result, PDLIM4 was found to be highly expressed in the gastric cancer tissue. Immunoblotting experiments and immunohistochemical experiments further confirmed that PDLIM4 was indeed differentially expressed in cancer tissues of gastric cancer and in paracancerous tissues. The present invention has been completed based on this finding.

Therefore, PDLIM4 can be used as a protein molecular marker to detect the expression level of the gastric cancer, namely PDLIM4 can be used as a protein molecule for detecting the gastric cancer.

Cancer tissues and tissues adjacent to the cancer were confirmed by 2 pathologists in 4 cases of patients with gastric cancer, and the pathological data of the 4 cases are shown in Table 2.

Table 2: pathological data of 4 patients with gastric cancer.

The preparation method of the in-solution enzymatic hydrolysis sample is used for preparing the protein samples of the cancer tissues and the tissues beside the cancer of the gastric cancer of the 4 patients (the cancer tissues and the tissues beside the cancer are all paired samples taken from the same gastric cancer patient), and the specific process is as follows:

the surgically excised fresh tissue mass was quickly placed on ice and quickly cut into several small macroscopic, non-necrotic areas. After washing the tissue pieces several times with a precooled PBS solution, rapidly grinding the tissue pieces into cell pellets in liquid nitrogen, dissolving the cell pellets in a lysis solution respectively, then intermittently sonicating the cell pellets for 2min using a sonicator (Soniprep 150, UK, MSE Co.) under ice bath conditions, centrifuging the cell pellets at 15000g/min and 4 ℃ for 1h, taking the supernatant, and quantifying the total protein by a modified Bradford method (see Bio-Rad Co., Ltd.).

Example 2: screening of differentially expressed proteins in cancer tissue and tissue adjacent to cancer

Screening for differentially expressed proteins was performed using the iTRAQ technique (see Pichler P et al anal chem.2010aug 1; 82(15):6549-58.) in combination with the in-solution enzymatic hydrolysis technique (see William m.old et al mol Cell proteomics.2005; 4: 1487-:

200ug of each of the 4 pairs of gastric cancer tissue and paracancerous tissue protein samples obtained in example 1 were digested into peptides by in-solution digestion, and then replaced with iTRAQ reaction reagents. Each 100ug was mixed with iTRAQ reagent and allowed to stand at room temperature for 1 hr. 8uL of hydroxylamine was added, and the mixture was allowed to stand at room temperature for 15min to terminate the reaction. Combine 8 samples, shake to mix well, remove impurities such as salt in the sample.

The peptide fragment mixture was first analyzed on an SCX/SAX column, then on a fully automated multi-dimensional liquid chromatography tandem mass spectrometer LTQ-active (available from Thermo Fisher), the obtained raw data was subjected to database search using Maxquant software (german mapple institute), and the identified proteins were quantitatively analyzed with the quantitative results shown in table 2.

Table 3: quantification of PDLIM4 in gastric cancer tissues and paracarcinoma tissues:

according to the results in table 3, the expression level of PDLIM4 in the gastric cancer tissue was about 2 times higher than that in the paracarcinoma tissue, and the expression in the gastric cancer tissue was significantly up-regulated.

Example 3: verification of PDLIM4

The pathological data of 6 pairs of gastric cancer tissues and corresponding protein samples of tissues adjacent to the cancer are shown in Table 4.

Table 4: pathological data of 6 cases of gastric cancer specimens

Western blot analysis of protein samples of the above 6 pairs of gastric cancer tissues and corresponding paracarcinoma tissues using purchased anti-PDLIM 4 antibody was performed as follows:

20ug of protein samples were separated by 12% SDS-PAGE and transferred to PVDF membrane (available from GE Healthcare);

primary antibody Rabbit anti-human PDLIM4 polyclonal antibody (purchased from Abcam company, diluted 1: 1000) was used, incubated overnight at 4 ℃ and washed three times with TBST for 5 minutes each;

the secondary antibody was an anti-rabbit antibody (purchased from Santa Cruz, I:10000 dilution), incubated at room temperature for I hours, and washed three times with TBST for 10 minutes each;

ECL plus reagent (available from GE Healthcare) was added to the reaction mixture and reacted for 5 minutes, followed by detection by exposure to X-ray film.

The immunoblot was subjected to data analysis using Gel-Pro Analyzer Gel quantitative analysis software (Media Cybernetic Co., Ltd.) to obtain a relative distribution graph of the protein expression level, and the results are shown in FIG. 1.

The results in FIG. 1 show that the expression level in the stomach cancer tissue is significantly higher than that in the corresponding paracarcinoma tissue, the average ratio is 2.56, and the P value (paired t test) is 0.002.

According to the results of fig. 1, PDLIM4 was highly expressed in gastric cancer tissues, which is consistent with mass spectrometry results.

Example 4. To further confirm the expression difference between the gastric cancer tissue and the para-carcinoma tissue of PDLIM4, 55 pairs of gastric cancer tissue and corresponding para-carcinoma tissue samples were randomly selected and immunohistochemical studies were performed using a gastric cancer tissue chip, wherein the specific data of the selected samples are shown in table 5.

Table 5: gastric cancer tissue chip data.

The experimental procedure for immunohistochemical studies was as follows:

baking the tissue slices in a constant temperature box at 60 ℃ for about 3 hours, taking out, and sequentially carrying out dewaxing hydration treatment by using dimethylbenzene, dimethylbenzene/ethanol (I: I), 100% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 50% ethanol and water;

PBS wash 3 times, each for 5 minutes; 0.3% H₂O₂(methanol dilution) soaking for half an hour, washing for 3 times with PBS, 5 minutes each time;

repairing antigen under high pressure, washing with double-distilled water for 2 times, 5 minutes each time; PBS wash 2 times, 5 minutes each time; 10% serum was blocked for 20 min at room temperature;

adding rabbit anti-human PDLIM4 polyclonal antibody (purchased from Abcam company, I:50 dilution), standing overnight at 4 deg.C, washing with PBST for 3 times, each for 5 min;

biotin-labeled goat anti-rabbit antibodies (from ABC kit, purchased from VECTOR corporation) were added, incubated at room temperature for 30 minutes, and washed 3 times with PBST, 5 minutes each; PBS wash 2 times, 5 minutes each time;

ABC solution (from ABC kit, purchased from VECTOR corporation) was added, incubated at room temperature for 30 minutes, washed 3 times with PBS, each for 5 minutes;

DAB solution (purchased from Shanghai Biotechnology engineering service, Ltd.) is developed; hematoxylin (purchased from VECTOR, H3404) staining for 20 seconds;

tissue sections were dehydrated and cleared with 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol, xylene/ethanol (I: I), xylene in that order.

Then, the gel was mounted on a neutral resin and observed under a microscope. The tissue chip was evaluated and the results showed: in the tissue chip, 55 pairs of effective samples are scored according to the staining intensity and the positive rate, wherein the scoring standard of the staining intensity is that the negative is O score, the weak positive is I score, the medium positive is 2 score and the strong positive is 3 score; the positive rate scoring standard is that less than 5 percent is O score, 5 to 30 percent is I score, 31 to 60 percent is 2 score, and more than 60 percent is 3 score. The score of the staining intensity and the score of the positive rate were added to obtain a total score of the tissue sections, and the score was shown in Table 6.

Table 6: the gastric cancer tissue chip is used for comprehensive scoring.

In FIG. 2, the average total score of the cancer cells of stomach cancer tissue was 3.27 and the average total score of the paracarcinoma tissue was 2.25, which were very significantly different from each other, and the P value was 0.00002, as calculated from the results in Table 6. It was statistically found that PDLIM4 was highly expressed in gastric cancer cell lines in more than 73% (40) of the sample pairs. The results were consistent with the previous mass spectrometry results and immunoblotting results.

Example 5. In order to further find the expression difference between the gastric cancer serum and the normal human serum of PDLIM4, 30 samples of the gastric cancer serum and 30 samples of the normal human serum were randomly selected and subjected to enzyme-linked immunosorbent assay, wherein the specific data of the selected samples are respectively shown in Table 7.

Table 7: gastric cancer serum sample information.

Table 7: information on normal serum samples.

FIG. 3 shows that PDLIM4 is highly expressed in serum of gastric cancer patients according to the results of ELISA. The results were consistent with previous mass spectrometry results, immunoblotting results, and immunohistochemistry results.

In conclusion, PDLIM4 has obvious differential expression in cancer tissues of gastric cancer and tissues beside the cancer, and the expression is increased in serum of gastric cancer patients, which is obviously closely related to the occurrence and development of gastric cancer, so the expression level can be used for detecting the gastric cancer. Accordingly, it will be apparent to those skilled in the art that antibodies specific to PDLIM4, including various monoclonal and polyclonal antibodies to PDLIM4, can be used to detect gastric cancer, or to prepare a preparation or kit for detecting gastric cancer, since they can be used to detect the expression level of PDLIM 4.

Although the biological functions and tumor-associated mechanisms of PDLIM4 dynamics remain to be further studied, it is certain to use it as a marker for detecting gastric cancer. PDLIM4 can be used as a potential marker of gastric cancer, and the intracellular biological function of PDLIM4 suggests that PDLIM4 can be used as a prognostic molecular marker of gastric cancer and a target molecule for clinical treatment.

Claims

1. Use of an antibody against PDLIM4 in the preparation of a kit for diagnosing gastric cancer in an individual.

2. The use of claim 1, wherein the antibody to PDLIM4 is a monoclonal or polyclonal antibody.