Application of CST1 combined with GPX4 as a metastasis marker or prognostic marker for gastric cancer

Abstract

A method of using CST1 and GPX4 as a gastric cancer metastasis marker or prognostic marker. The method detects the protein expression of CST1 and GPX4, draws an ROC curve using SPSS software, obtains the sensitivity and specificity of the diagnosis, and calculates the area under the curve AUC value. Finally, the gastric cancer samples are classified according to the AUC value, sensitivity and specificity, and applied to the diagnosis of gastric cancer metastasis. CST1 and GPX4 are used as markers for joint detection of gastric cancer, and the detection has strong specificity and high sensitivity, which can effectively avoid false positive or false negative results that are prone to occur when CST1 or GPX4 is used alone as a diagnostic marker, and provide reliable information for the early diagnosis of gastric cancer metastasis. The method draws a survival curve through follow-up and survival analysis, determines that patients with double positive results of CST1 and GPX4 have a worse prognosis, and provides a biomarker for predicting the prognosis of gastric cancer. The present invention provides a combined biomarker for early diagnosis and prognosis of gastric cancer metastasis with strong specificity and high sensitivity, and its application.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to an application of CST1 combined with GPX4 as a gastric cancer metastasis marker or prognosis marker. Background Art

[0002] Gastric cancer (GC) is the fifth most common digestive tract malignancy in the world and the fourth most common mortality rate, posing a serious threat to human health. According to the Chinese Cancer Epidemiology Data released in 2020, among the 4.57 million new cancer cases, gastric cancer ranked third with 480,000 new cases, second only to lung cancer and colorectal cancer. In 2020, there were 3 million cancer deaths in China, and gastric cancer ranked third with 370,000 deaths, behind lung cancer and liver cancer. One of the main reasons for the high mortality rate of gastric cancer is that most patients are in the advanced stage when they seek medical treatment, and even have distant metastasis, among which peritoneal metastasis, liver metastasis, and ovarian metastasis are common distant metastasis of gastric cancer. Although a comprehensive treatment plan based on chemotherapy has been developed for metastatic gastric cancer at home and abroad, the overall efficacy is poor and the prognosis is very poor. Therefore, in-depth research and exploration of effective diagnostic and treatment targets for gastric cancer metastasis has important clinical guiding significance.

[0003] At present, endoscopic biopsy, serum tumor marker detection combined with imaging examinations such as CT and PETCT are the main means to evaluate whether gastric cancer has metastasis. However, the commonly used serum tumor markers in clinical practice, such as carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA199), and carbohydrate antigen 125 (CA125), have disadvantages such as low sensitivity and poor specificity, and cannot meet the needs of early diagnosis of gastric cancer metastasis. In addition, the method commonly used in clinical practice to evaluate the prognosis of gastric cancer is based on the TNM staging of the tumor, and predicts the long-term survival of patients based on factors such as the location of the primary lesion of gastric cancer, depth of infiltration, degree of differentiation, lymph nodes and distant metastasis. Although this method is simple and easy to use, the heterogeneity of gastric cancer limits the scope of application of this method.

[0004] With the vigorous development of molecular biology, exploring the key molecules in the occurrence and development of gastric cancer and using them to establish more accurate diagnostic and prognostic indicators is the hot spot and direction of current tumor research, and it is also an important way to improve the survival prognosis of patients with gastric cancer metastasis. Summary of the invention

[0005] Technical problem to be solved: In view of the problems in the prior art that detection markers for gastric cancer metastasis have low sensitivity, poor specificity, and are unable to provide effective information for the diagnosis and prognosis of gastric cancer metastasis, the present invention provides an application of CST1 combined with GPX4 as a gastric cancer metastasis marker or prognosis marker. The combined marker has the advantages of high sensitivity and good specificity.

[0006] Technical solution: Application of CST1 combined with GPX4 as gastric cancer metastasis markers.

[0007] Application of CST1 combined with GPX4 as prognostic markers for gastric cancer.

[0008] Application of CST1 combined with GPX4 in the preparation of a reagent or kit for diagnosing gastric cancer metastasis.

[0009] Application of CST1 combined with GPX4 in the preparation of a reagent or kit for gastric cancer prognosis.

[0010] Preferably, the test sample of the reagent or kit is tumor tissue, whole blood, plasma, serum, ascites or exosomes, and the diagnostic reagent or kit comprises a reagent for determining the expression of CST1 and GPX4 proteins.

[0011] Preferably, the method for determining the expression of CST1 and GPX4 proteins is as follows: selecting representative tissue regions using hematoxylin-eosin stained sections and performing immunohistochemical analysis using avidin-biotin complex method.

[0012] Preferably, the representative tissue region includes gastric cancer tissue and adjacent cancer tissue, the number of gastric cancer parenchymal cells in the gastric cancer tissue is greater than 80% of the total number of cells in the gastric cancer tissue, and the adjacent cancer tissue does not contain cancer cells.

[0013] Beneficial effects: (1) The present invention, for the first time, uses high-throughput sequencing combined with protein spectrum analysis to identify two mutually related gastric cancer metastasis markers: CST1 and GPX4. Combined detection can more accurately determine gastric cancer metastasis and prognosis, and can provide a basis for the molecular pathological diagnosis of gastric cancer. The expression of CST1 and GPX4 proteins is simultaneously detected in situ in gastric cancer tissue cells, making it easy to observe and evaluate the correlation between the two. The simultaneous detection of two related gastric cancer metastasis markers on a tissue section helps to save specimen usage and can be applied to sample detection with less material such as gastroscopy.

[0014] (2) The present invention used tissue samples from 52 gastric cancer patients collected from the First Affiliated Hospital of Soochow University to prepare tissue chips, performed immunohistochemical staining, evaluated the staining results of CST1 and GPX4, used SPSS software to analyze the sensitivity and specificity of diagnosis, and drew the ROC curve. It was found that the area under the curve (AUC) value of the combined indicator was 0.9566, while the AUC values ​​of CST1 or GPX4 alone as cancer detection markers were 0.6802 and 0.5128, respectively; in addition, the AUC values ​​of the traditional tumor marker CEA alone or in combination with CST1 were 0.6026 and 0.7189, respectively, reflecting that the combined detection of CST1 and GPX4 provides reliable information for the early diagnosis of gastric cancer metastasis.

[0015] (3) The present invention analyzes the prognosis of gastric cancer patients, and the results show that patients with both positive CST1 and GPX4 proteins have the worst prognosis, with a five-year survival rate of 10.8%; patients with only one positive protein have an intermediate prognosis, with a five-year survival rate of about 28.3%; patients with both negative proteins have the best prognosis, with a five-year survival rate of 57.6%. This study first discovered and confirmed that CST1 combined with GPX4 protein can be used as a biomarker to predict the prognosis of gastric cancer, and its predictive efficacy is better than the current clinical method of simply using clinical information to predict prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the immunohistochemical section of the expression of CST1 and GPX4 proteins in gastric cancer and adjacent tissues;

[0017] Figure 2 The correlation between the expression of CST1 and GPX4 proteins in gastric cancer tissues;

[0018] Figure 3 This is the ROC curve analysis diagram of the combined diagnostic efficacy of CST1 and GPX4 protein markers;

[0019] Figure 4 Forest plot of the prognostic prediction effectiveness of the combination of CST1 and GPX4 protein markers. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. These specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0021] Example 1

[0022] Step 1: Prepare gastric cancer tissue chips and use immunohistochemistry to detect the expression of CST1 and GPX4 proteins in the gastric cancer tissues and adjacent tissues removed during surgery.

[0023] The 52 pairs of gastric cancer tissue and adjacent tissue specimens were selected from surgical resection tissues of gastric cancer patients admitted to the First Affiliated Hospital of Soochow University from 2010 to 2018 (14 cases of stage I-II and 38 cases of stage III-IV). The diagnosis of gastric cancer was confirmed by postoperative pathology. The clinical pathological data of all patients were registered in the form of follow-up forms after surgery, and their survival and recurrence and metastasis were followed up by telephone or outpatient clinic every year for more than 24 months. The follow-up was verified again before the start of this study. The biological samples required for this study were obtained with the consent of the patients before acquisition.

[0024] All tissue wax block specimens were from the tissue wax blocks archived by the pathology department. After the tissue was removed during surgery, it was immediately fixed in 10% neutral formalin solution. After sufficient fixation, the sample was dehydrated, transparent, waxed, and embedded. The sections were pathologically confirmed by hematoxylin-eosin staining and then stored for a long time. Immunohistochemistry kits include the ready-to-use immunohistochemistry reagent UltraSensitive SP kit (sheep, Maixin Bio, catalog number KIT-9709), rat IgG-immunohistochemistry kit (Boster Bio, catalog number SA1055), endogenous biotin blocking kit (Maixin Bio, catalog number BLK-0002), and AntibodyDiluent with Background Reducing Components (Dako, catalog number S3022).

[0025] The antibodies used include: CST1 primary antibody (rabbit anti-human CST1 monoclonal antibody, Proteintech, catalog number 16025-1-AP, dilution 1:100), GPX4 primary antibody (rabbit anti-human GPX4 monoclonal antibody, Abcam, catalog number ab125066, dilution 1:2500)

[0026] First, hematoxylin-eosin stained sections were used to select representative tissue areas, and further immunohistochemical analysis was performed using the avidin-biotin complex method (i.e., SP method). The specific process of immunohistochemistry was as follows: sections were baked at 60°C for 1 hour; dewaxing and rehydration: xylene for 10 min, 100% ethanol for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 85% ethanol for 5 min, 80% ethanol for 5 min, 75% ethanol for 5 min, 60% ethanol for 5 min, 50% ethanol for 5 min, 30% ethanol for 5 min, tap water for 1 min, hydrogen peroxide for 1 min; 1 portion of 30% H 2 O 2Add 10 portions of distilled water, incubate at room temperature for 10 min, wash with distilled water 3 times, 3 min each time; microwave repair: immerse the slices in 0.01M citrate buffer, heat to boiling at the maximum power (98℃-100℃) in the microwave, cool (about 5-10 min), repeat twice; let the slices cool naturally to room temperature, wash with PBS 3 times, 5 min each time; block with 5% BSA, room temperature for 20 min, and shake off excess liquid; add CST1 / GPX4 primary antibody, 37℃, 1h, or 4℃ overnight; wash with PBS 3 times, 3 min each time; add biotin-labeled secondary antibody, 37℃, 15-30 min; wash with PBS 3 times, 3 min each time; add SP working solution, 37℃, 30 min; wash with PBS 3 times, 5 min each time; add color developer in 1 mL of distilled water and mix well; after DAB color developer is prepared, add it to the slices, room temperature, and detect the reaction time under the microscope (about 5 min); rinse with tap water and rinse with distilled water; counterstain with hematoxylin for 2 min and rinse with tap water; dehydrate: 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, xylene for 20 min; seal with gum and examine under a microscope.

[0027] Step 2: CST1 and GPX4 protein expression level grading

[0028] The results of step 1 are graded.

[0029] See also Figure 1 Five high-power microscopic fields (×400 times) were randomly detected for each specimen. First, the gastric cancer tissues were divided into moderately and highly differentiated groups and low and undifferentiated groups according to the degree of differentiation. Highly differentiated and lowly differentiated sections were further selected as representative images. ImageJ software was further used to quantitatively score the staining degree to obtain the specific values ​​of CST1 and GPX4 staining degrees in each tissue, and the medians of the specific values ​​of CST1 and GPX4 were taken respectively. The values ​​above the median were defined as the CST1 or GPX4 high expression group, and the values ​​below the median were defined as the CST1 or GPX4 low expression group.

[0030] Example 2

[0031] Based on the immunohistochemical scores of the above 52 gastric cancer tissue chips, the correlation between CST1 and GPX4 protein expression was analyzed by Pearson correlation analysis using GraphPad Prism9 software. Usually, a linear trend and normal distribution are required between the two variables. The software operation selects "XY" and proceeds according to the guidance. The results are required to be statistically significant. P <0.05. For specific results, see Figure 2 , correlation coefficient r=0.4865,P <0.0001, indicating that the expression of CST1 and GPX4 proteins in gastric cancer tissues was positively correlated.

[0032] Example 3

[0033] SPSS software was used to analyze clinical pathological factors of tissue microarrays of 52 gastric cancer patients. The specific results are shown in the table below. The results showed that the expression of CST1 and GPX4 proteins was mainly related to the degree of tumor tissue differentiation, T stage, lymph node invasion, and TNM stage, but not to the patient's age, gender, or tumor size.

[0034]

[0035] Example 4

[0036] According to the expression scores of CST1 and GPX4 proteins in gastric cancer tissues, SPSS software was used to draw the ROC curves of CST1 and GPX4 alone and in combination for the diagnosis of gastric cancer, and the cut-off value of diagnostic efficacy and the area under the curve (AUC) value were determined. Figure 3 The results showed that the AUC value of the combined index was 0.9566, while the AUC values ​​of CST1 or GPX4 alone as cancer detection markers were 0.6802 and 0.5128, respectively, with sensitivity and specificity of 84.37% and 95.02%, respectively; in addition, the AUC of the traditional tumor marker CEA alone or combined with CST1 diagnosis were 0.6026 and 0.7189, respectively.

[0037] Example 5

[0038] Clinical pathological factors affecting the prognosis of gastric cancer patients were collected, including CST1 and GPX4 protein immunohistochemical expression scores, TNM stage, etc., and univariate and multivariate Cox regression model analysis was performed using SPSS software. Specifically, univariate Cox regression model analysis was first performed, and the results showed that indicators such as high expression of CST1 / GPX4 protein, TNM stage, invasion depth, and lymph node metastasis were risk factors for the prognosis of gastric cancer patients. These indicators were further selected for multivariate Cox regression model analysis, and the results showed that high expression of CST1 / GPX4 protein and tumor invasion depth were independent risk factors for the prognosis of gastric cancer. Subsequently, the results of the multivariate Cox regression model analysis were visualized by drawing a forest map using GraphPad Prism9 software, see Figure 4 The results showed that the risk ratio of patients with high expression of CST1 / GPX4 protein was HR>1. P<0.05. The results of survival analysis showed that the five-year survival rate of patients with positive CST1 and GPX4 proteins was 10.8%, the worst prognosis; patients with single positive protein had an intermediate prognosis, with a five-year survival rate of about 28.3%; patients with both negative proteins had the best prognosis, with a five-year survival rate of 57.6%. The above results suggest that CST1 combined with GPX4 protein is an independent risk factor for the prognosis of gastric cancer patients.

[0039] Example 6

[0040] A kit is prepared, which contains reagents for determining the expression of CST1 and GPX4 proteins, and is used for diagnosing gastric cancer metastasis.

[0041] Example 7

[0042] A kit is prepared, wherein the kit contains reagents for determining the expression of CST1 and GPX4 proteins, and the kit is used for judging the prognosis of gastric cancer.

[0043] Furthermore, the reagents for quantifying the expression levels of CST1 and GPX4 proteins of the present invention include antibodies or fragments thereof that specifically bind to CST1 and GPX4 proteins. Antibodies or fragments thereof of any structure, size, immunoglobulin class, origin, etc. can be used as long as they bind to the target protein.

[0044] Furthermore, antibodies specific to CST1 and GPX4 proteins can be obtained by methods known to those skilled in the art, such as preparing a polypeptide retaining the whole or part of the target protein or a mammalian cell expression vector incorporating a polynucleotide encoding them as an antigen, using the polypeptide to immunize an animal, obtaining immune cells and fusing them with myeloma cells to obtain hybridomas and collect antibodies, and finally obtaining monoclonal antibodies against CST1 and GPX4 proteins by subjecting the obtained antibodies to antigen-specific purification using CST1 and GPX4 proteins or partial fragments thereof used as antigens.

[0045] Furthermore, the binding of CST1 and GPX4 protein markers to antibodies or fragments thereof can be carried out by methods known in the art, such as washing the protein or peptide with phosphate buffer, adding a fluorescent dye, mixing and leaving at room temperature for 15 minutes; in addition, commercial kits such as a biotin labeling kit can also be used for labeling.

[0046] Furthermore, the reagents for quantifying the expression levels of CST1 and GPX4 proteins of the present invention can be prepared by using corresponding antibodies in a common manner, such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunofluorescence staining, Western Blot, and the like.

Claims

1. Use of a reagent for detecting the expression of CST1 combined with GPX4 proteins in the preparation of a reagent or kit for diagnosing gastric cancer metastasis or prognosis, It is characterized in that The diagnostic reagent or kit comprises a reagent for determining the expression of CST1 and GPX4 proteins. The method for determining the expression of CST1 and GPX4 proteins is as follows: selecting representative tissue areas using hematoxylin-eosin stained sections and performing immunohistochemical analysis using an avidin-biotin complex method. The representative tissue areas include gastric cancer tissue and adjacent cancer tissue. The number of gastric cancer parenchymal cells in the gastric cancer tissue is greater than 80% of the total number of cells in the gastric cancer tissue, and the adjacent cancer tissue does not contain cancer cells.

2. The use according to claim 1, It is characterized in that The detection sample of the reagent or kit is tumor tissue, whole blood, plasma, serum, ascites or exosomes.

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