CN114791492A

CN114791492A - Application of CST1 and GPX4 in combination as gastric cancer metastasis marker or prognosis marker

Info

Publication number: CN114791492A
Application number: CN202210418193.7A
Authority: CN
Inventors: 周进; 李东宝; 段开鹏; 王玉红; 陈涛; 董安琦; 任嘉裕; 董超
Original assignee: First Affiliated Hospital of Suzhou University
Current assignee: First Affiliated Hospital of Suzhou University
Priority date: 2022-04-20
Filing date: 2022-04-20
Publication date: 2022-07-26

Abstract

The invention relates to an application of CST1 and GPX4 combined as a gastric cancer metastasis marker or a prognosis marker, which is characterized in that the protein expression quantity of CST1 and GPX4 is detected, SPSS software is used for drawing an ROC curve, the sensitivity and specificity of diagnosis are obtained, the AUC value of the area under the curve is calculated, and finally, gastric cancer samples are classified according to the AUC value, the sensitivity and the specificity and are applied to the diagnosis of gastric cancer metastasis. The CST1 and the GPX4 are used as markers for carrying out combined detection on the gastric cancer, the detection specificity is strong, the sensitivity is high, false positive or false negative results which are easy to occur when the CST1 or the GPX4 are used as diagnosis markers independently can be effectively avoided, and reliable information is provided for early diagnosis of gastric cancer metastasis. The invention draws a survival curve through follow-up and survival analysis, determines that the prognosis of the patient with CST1 and GPX4 double positive is worse, and provides a biomarker for predicting the prognosis of gastric cancer. The invention provides a combined biomarker for early diagnosis and prognosis evaluation of gastric cancer metastasis with strong specificity and high sensitivity and application thereof.

Description

Application of CST1 and GPX4 in combination as gastric cancer metastasis marker or prognosis marker

Technical Field

The invention relates to the technical field of molecular biology, in particular to application of CST1 and GPX4 as a gastric cancer metastasis marker or a prognosis marker.

Background

Gastric Cancer (GC) is a malignant tumor of the digestive tract with the fifth incidence and the fourth mortality in the world, and seriously threatens human health. According to Chinese cancer epidemiological data released in 2020, in 457 ten thousand cancer cases, gastric cancer is ranked in the third place by 48 ten thousand cases, and is second only to the incidence of lung cancer and colorectal cancer. In 2020, the number of cancer deaths in China is 300 thousands, the number of gastric cancer deaths is the third order of 37 thousands, and the number of gastric cancer deaths is behind the number of lung cancer and liver cancer deaths. One of the main causes of higher mortality of gastric cancer is that most patients are in a progressive stage at the time of treatment, and even have distant metastasis, wherein peritoneal metastasis, liver metastasis and ovarian metastasis are the common distant metastasis modes of gastric cancer. Although a comprehensive treatment scheme mainly based on chemotherapy is established for metastatic gastric cancer at home and abroad, the overall curative effect is poor and the prognosis is poor. Therefore, the method has important clinical guiding significance for deeply researching and exploring the effective diagnosis and treatment target point of gastric cancer metastasis.

At present, endoscopic biopsy, serum tumor marker detection combined with imaging examinations such as CT and PETCT are the main means for evaluating whether gastric cancer has metastasis. However, serum tumor markers such as carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA 199), and carbohydrate antigen 125 (CA 125) which are commonly used in clinical practice have the disadvantages of low sensitivity, poor specificity, and the like, and cannot meet the requirement of early diagnosis of gastric cancer metastasis. In addition, the method for evaluating gastric cancer prognosis is usually used clinically to predict the long-term survival of patients according to tumor TNM stages and factors such as the primary focus of gastric cancer, infiltration depth, differentiation degree, lymph node and distant metastasis, and the like, but the heterogeneity of gastric cancer limits the application range of the method although the method is simple and easy to use.

With the vigorous development of molecular biology, key molecules in the occurrence and development of gastric cancer are searched, and relatively accurate diagnosis and prognosis evaluation indexes are established by utilizing the key molecules, so that the key molecules are hot spots and directions of the current tumor research and are also important ways for improving the survival prognosis of gastric cancer metastasis patients.

Disclosure of Invention

The technical problem to be solved is as follows: aiming at the problems that the detection marker for gastric cancer metastasis in the prior art is low in sensitivity and poor in specificity, cannot provide effective information for diagnosis and prognosis evaluation of gastric cancer metastasis and the like, the invention provides the application of the CST1 combined with the GPX4 as a gastric cancer metastasis marker or a prognosis marker, and the combined marker has the advantages of high sensitivity, good specificity and the like.

The technical scheme is as follows: the use of CST1 in combination with GPX4 as a marker for gastric cancer metastasis.

The application of CST1 combined with GPX4 as gastric cancer prognosis markers.

The application of CST1 and GPX4 in preparing a reagent or a kit for diagnosing gastric cancer metastasis.

Use of CST1 in combination with GPX4 in the preparation of a reagent or kit for the prognosis of gastric cancer.

Preferably, the detection sample of the reagent or the kit is tumor tissue, whole blood, plasma, serum, ascites or exosome, and the diagnostic reagent or the kit comprises a reagent for measuring expression of CST1 and GPX4 protein.

Preferably, the CST1 and GPX4 protein expression determination methods are as follows: representative tissue regions were selected using hematoxylin-eosin stained sections and immunohistochemical analysis was performed using the avidin-biotin complex method.

Preferably, the representative tissue region includes a gastric cancer tissue and a para-carcinoma tissue, the number of gastric cancer parenchymal cells in the gastric cancer tissue is more than 80% of the total number of cells in the gastric cancer tissue, and the para-carcinoma tissue does not contain cancer cells.

Has the beneficial effects that: (1) the invention determines two gastric cancer metastasis markers which are related to each other by high-throughput sequencing combined protein mass spectrometry for the first time: CST1 and GPX 4. The combined detection can more accurately judge the gastric cancer metastasis and prognosis and can provide a basis for the molecular pathological diagnosis of the gastric cancer. The expression of CST1 and GPX4 proteins is detected simultaneously in situ in the gastric cancer tissue cells, and the correlation between the two proteins is easy to observe and evaluate. Two related gastric cancer metastasis markers are detected on one tissue section simultaneously, so that the use amount of the specimen is saved, and the method can be applied to detection of samples with less material drawing, such as gastroscopes.

(2) The invention utilizes tissue samples of 52 gastric cancer patients collected by a first hospital affiliated to Suzhou university to prepare a tissue chip, performs immunohistochemical staining, evaluates staining results of CST1 and GPX4, utilizes SPSS software to analyze sensitivity and specificity of diagnosis, draws a ROC curve, finds that the AUC value of the area under the curve of the combined index is 0.9566, and the AUC values when CST1 or GPX4 are independently used as cancer detection markers are 0.6802 and 0.5128 respectively; in addition, the AUC of the single or combined CST1 diagnosis of the traditional tumor marker CEA is 0.6026 and 0.7189 respectively, which reflects that the CST1 and GPX4 detection provide reliable information for early diagnosis of gastric cancer metastasis.

(3) According to the invention, by analyzing the prognosis state of the gastric cancer patient, the result shows that the prognosis of the patient with positive CST1 and GPX4 proteins is the worst, and the five-year survival rate is 10.8%; single protein positive patients have intermediate prognosis, with a five-year survival rate of about 28.3%; patients who are both negative had the best prognosis, with a five-year survival rate of 57.6%. The research discovers and proves that the CST1 and the GPX4 protein can be used as biomarkers for predicting the prognosis of the gastric cancer for the first time, and the prediction effect of the biomarker is superior to that of the biomarker which is commonly used clinically at present and only utilizes clinical information to predict the prognosis.

Drawings

FIG. 1 shows immunohistochemical sections of CST1 and GPX4 proteins expressed in gastric cancer and paracancerous tissues;

FIG. 2 shows the correlation between the expression of CST1 and GPX4 proteins in gastric cancer tissues;

FIG. 3 is a ROC curve analysis graph of the combined diagnostic efficacy of CST1 and GPX4 protein markers;

FIG. 4 is a forest map of prognostic predictive efficacy of a combination of CST1 and GPX4 protein markers.

Detailed Description

The invention is further described below with reference to the figures and the specific embodiments. These specific examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

Step 1: preparing a gastric cancer tissue chip, and detecting the expression conditions of CST1 and GPX4 proteins in the gastric cancer tissue and the tissues beside the gastric cancer which are cut off by the operation of a patient by adopting an immunohistochemical method.

The selected 52 pairs of gastric cancer tissues and tissue specimens beside the gastric cancer are from the tissues (14 cases in I-II stage and 38 cases in III-IV stage) of gastric cancer patients collected in the first hospital affiliated to Suzhou university from 2010 to 2018, the diagnosis of the gastric cancer is confirmed by postoperative pathology, clinical pathological data of all patients are registered in a follow-up form after operation, the patients are followed by telephone or clinic every year for more than 24 months under the conditions of survival, recurrence and metastasis and the like, and follow-up verification is carried out again before the research experiment starts. The biological samples required for this study were obtained with patient consent prior to acquisition.

All tissue wax block specimens were from pathologist archived tissue wax blocks. Immediately fixing the tissue in 10% neutral formalin solution after the tissue is taken down in operation, taking the tissue, dehydrating, transparentizing, waxing, embedding, pathologically diagnosing the section by hematoxylin-eosin staining, and storing for a long time. Immunohistochemical KITs include Ready-to-use immunohistochemical reagent UltraSensitive SP KIT (sheep, Mooney, cat # KIT-9709), rat IgG-immunohistochemical KIT (doctor' S biosome, cat # SA 1055), endogenous biotin blocking KIT (Mooney, cat # BLK-0002), and Antibody reagent with Background Reducing Components (Dako, cat # S3022).

The antibodies used included: CST1 Primary antibody (Rabbit anti-human CST1 monoclonal antibody, Proteitech, catalog number 16025-1-AP, dilution 1: 100), GPX4 Primary antibody (Rabbit anti-human GPX4 monoclonal antibody, Abcam, catalog number ab125066, dilution 1: 2500)

Representative tissue regions were first selected using hematoxylin-eosin stained sections, and immunohistochemical analysis was further performed using the avidin-biotin complex method (i.e., SP method). The specific process of immunohistochemistry is as follows: slicing, baking at 60 deg.C for 1 hr; dewaxing and rehydration: xylene 10 min, 100% ethanol 5 min, 95% ethanol 5 min, 90% ethanol 5 min, 85% ethanol 5 min, 80% ethanol 5 min, 75% ethanol 5 min, 60% ethanol 5 min, 50% ethanol 5 min, 30% ethanol 5 min, tap water 1 min, and hydrogen peroxide 1 min; 1 part of 30% H ₂ O ₂ Adding 10 parts of distilled water, and washing with distilled water for 3 times (each time for 3 min) at room temperature for 10 min; microwave repair: immersing the slices in 0.01M citric acid buffer solution, heating to boil with the maximum fire (98-100 ℃) in microwave, cooling for about 5-10 min, and repeating for two times; naturally cooling the slices to room temperature, washing with PBS for 3 times, 5 min each time; blocking, 5% BSA, room temperature 20 min, and throwing off the redundant liquid; adding CST1/GPX4 primary antibody dropwise at 37 ℃ for 1h or at 4 ℃ overnight; PBS wash 3 times, each time 3 min; dripping biotin-labeled secondary antibody at 37 deg.C for 15-30 min; PBS wash 3 times, each time 3 min; dropwise adding SP working solution at 37 ℃ for 30 min; washing with PBS for 5 min for 3 times; dripping color developing agent into 1 mL of distilled water respectively, and mixing uniformly; after the DAB color developing agent is prepared, dropwise adding the DAB color developing agent into the slices, and detecting the reaction time (about 5 min) at room temperature under a mirror; washing with tap water, and passing through distilled water; counterstaining with hematoxylin for 2 min, and washing with tap water; and (3) dehydrating: 30% ethanol for 3 min, 50% ethanol for 3 min, 70% ethanol for 3 min, 80% ethanol for 3 min, 90% ethanol for 3 min, 95% ethanol for 3 min, 100% ethanol for 3 min, and xylene for 20 min; and sealing the film with gum and performing microscopic examination.

And 2, step: CST1 and GPX4 protein expression level fractionation

The results of step 1 were ranked.

Referring to fig. 1, each specimen was randomly examined for 5 high power field (x 400 times), and the specimens were first classified into a medium-high differentiation group and a low-low differentiation group according to the differentiation degree of the gastric cancer tissue, and further high-differentiation and low-differentiation sections were selected as representative figures. And further quantitatively scoring the staining degree by using ImageJ software to obtain specific numerical values of the staining degrees of CST1 and GPX4 in each tissue, and taking a median of the specific numerical values of CST1 and GPX4 respectively, wherein the specific numerical values which are higher than the median are defined as a CST1 or GPX4 high expression group, and the specific numerical values which are lower than the median are defined as a CST1 or a GPX4 low expression group.

Example 2

According to the 52 cases of gastric cancer tissue chip immunohistochemical scoring, the correlation between the expression of CST1 and GPX4 proteins is analyzed by Pearson correlation analysis through GraphPad Prism9 software, a linear trend and a normal distribution between two variables are generally required, the software operation selects XY and is carried out according to the guidance, and the result requires that the statistical significance is achieved, namely the resultsP<0.05. With particular reference to fig. 2, the correlation coefficient r =0.4865,P<0.0001, indicating that CST1 is in positive correlation with GPX4 protein expression in gastric cancer tissues.

Example 3

The results of the clinical pathological factor analysis of the tissue chips of 52 patients with gastric cancer by SPSS software show that the expression of CST1 and GPX4 proteins is mainly related to the differentiation degree of tumor tissues, T stage, lymph node invasion and TNM stage, but not related to the age, sex and tumor size of the patients.

Example 4

According to protein expression scores of CST1 and GPX4 of gastric cancer tissues, an SPSS software is adopted to draw an ROC curve when the CST1 and the GPX4 are used for diagnosing gastric cancer independently or jointly, and a cutoff value of diagnosis efficiency, namely a cut-off value and an AUC value of an area under the curve are determined. Specific results referring to fig. 3, the results show that the AUC value of the combined index is 0.9566, while the AUC values of the CST1 or GPX4 alone as cancer detection markers are 0.6802 and 0.5128, respectively, and the sensitivity and specificity are 84.37% and 95.02%, respectively; in addition, AUC for diagnosis of the traditional tumor marker CEA alone or in combination with CST1 is 0.6026, 0.7189, respectively.

Example 5

Collecting clinical pathological factors influencing the prognosis of gastric cancer patients, including CST1, GPX4 protein immunohistochemical expression score, TNM staging and the like, and carrying out single factor treatment by SPSS softwareAnd multi-factor Cox regression model analysis, specifically, firstly, single-factor Cox regression model analysis is carried out, the result shows that indexes such as CST1/GPX4 protein high expression, TNM staging, invasion depth and lymph node metastasis are prognostic risk factors of gastric cancer patients, the indexes are further selected for carrying out multi-factor Cox regression model analysis, and the result shows that CST1/GPX4 protein high expression and tumor invasion depth are independent risk factors for gastric cancer prognosis. The analysis result of the multifactor Cox regression model is visualized by drawing a forest map through GraphPad Prism9 software, and the result shows that the CST1/GPX4 protein is highly expressed in the risk ratio HR of the patient, as shown in FIG. 4>1，P<0.05. Survival analysis results indicate that the five-year survival rate of patients with positive CST1 and GPX4 proteins is 10.8%, and the prognosis is worst; single protein positive patients have intermediate prognosis, with a five-year survival rate of about 28.3%; patients who are both negative had the best prognosis with a five-year survival rate of 57.6%. The above results suggest that CST1 in combination with GPX4 protein is an independent risk factor for the prognosis of gastric cancer patients.

Example 6

A kit containing reagents for measuring the expression of CST1 and GPX4 proteins for diagnosing gastric cancer metastasis is prepared.

Example 7

Preparing a kit containing reagents for measuring CST1 and GPX4 protein expression, wherein the kit is used for judging gastric cancer prognosis.

Further, the reagent for quantifying the expression level of CST1 and GPX4 proteins of the present invention includes an antibody or a fragment thereof that specifically binds to CST1 and GPX4 proteins, and any antibody or fragment thereof of any structure, size, immunoglobulin class, origin, etc. may be used as long as it binds to the target protein.

Further, antibodies specific to CST1 and GPX4 proteins may be obtained by methods well known to those skilled in the art. For example, a polypeptide retaining the whole or part of the target protein or a mammalian cell expression vector incorporating a polynucleotide encoding them is prepared as an antigen, after an animal is immunized therewith, an immune cell is obtained and myeloma cells are fused to obtain a hybridoma and an antibody is collected, and finally monoclonal antibodies against the CST1 and GPX4 proteins are obtained by subjecting the obtained antibody to antigen-specific purification using the CST1 and GPX4 proteins or partial fragments thereof used as antigens.

Further, the binding of the CST1 and GPX4 protein markers to the antibody or fragment thereof may be performed by methods well known in the art. For example, washing proteins or peptides with phosphate buffer, adding fluorescent dye, mixing, and standing at room temperature for 15 min; in addition, labeling can be performed using a commercially available kit such as a biotin labeling kit.

Further, the reagents for quantifying the expression levels of CST1 and GPX4 proteins of the present invention may employ conventional methods using corresponding antibodies, such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunofluorescent staining, Western blotting (Western Blot), and the like.

Claims

Use of CST1 in combination with GPX4 as a marker for gastric cancer metastasis.
Use of CST1 in combination with GPX4 as a prognostic marker for gastric cancer.
Use of CST1 in combination with GPX4 for the preparation of a reagent or kit for the diagnosis of gastric cancer metastasis.
Use of CST1 in combination with GPX4 in the preparation of a reagent or kit for the prognosis of gastric cancer.
5. The use of claim 3 or 4, wherein the test sample of the reagent or kit is tumor tissue, whole blood, plasma, serum, ascites or exosome, and the diagnostic reagent or kit comprises reagents for determining expression of CST1 and GPX4 proteins.
6. The use of claim 5, wherein the CST1 and GPX4 protein expression determination methods are as follows: representative tissue regions were selected using hematoxylin-eosin stained sections and immunohistochemical analysis was performed using the avidin-biotin complex method.
7. The use of claim 6, wherein said representative tissue region comprises gastric carcinoma tissue and paracarcinoma tissue, wherein the number of gastric carcinoma parenchymal cells in said gastric carcinoma tissue is greater than 80% of the total number of cells in said gastric carcinoma tissue, and said paracarcinoma tissue does not contain cancer cells.