Biomarker for prognosis of gastric cancer patient

Abstract

The invention belongs to the technical field of biology, and particularly relates to a prognosis biomarker for gastric cancer patients. According to the present invention, the gastric cancer prognosis evaluation method based on the ZNF154 or the combination of the ZNF154 and the EGR1 and the protein expression level has advantages of simple operation and low cost, can provide the more accurate prognosis evaluation for the clinic, and provides the important basis for the personalized treatment of the patient.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a biomarker for the prognosis of gastric cancer patients. Background Art

[0002] Gastric cancer is a malignant tumor originating from the gastric mucosal epithelium, with a high incidence and mortality rate worldwide. According to the World Health Organization, the incidence and mortality rate of gastric cancer rank fourth and sixth among all malignant tumors, respectively (Wang Lu (review), Ning Wenfeng (review). New progress in the study of the molecular mechanism of gastric cancer [J]. Chinese Journal of Modern Medicine, 2016, 18 (10): 106-108.)

[0003] The occurrence of gastric cancer is related to a variety of factors, including Helicobacter pylori infection, poor eating habits, genetic factors, etc. (Luo Jinsong, He Miao, Yang Guoquan. Clinical diagnosis, formation mechanism and clinical treatment of gastric cancer [J]. Genomics and Applied Biology, 2021, 40(02): 936-940.). Early gastric cancer usually has no obvious symptoms and is easily ignored, leading to delayed treatment (Wang Yuanfang. Analysis of clinical pathological characteristics of 97 cases of early gastric cancer and their relationship with prognosis [J]. Shanxi Medical Journal, 2020, 49(2): 180-182.). Therefore, early diagnosis and treatment are crucial to improving the survival rate of gastric cancer patients. Studies have shown that the 5-year survival rate of early gastric cancer can reach more than 90% (Wang Li, Zang Cuicui, Zhang Yan. Application value of Jin's pulse characteristics in the diagnosis of gastric cancer [J]. Research on Integrated Traditional Chinese and Western Medicine, 2023, 15(4): 275-277, 288.410), while the 5-year survival rate of advanced gastric cancer is significantly reduced. The treatment of gastric cancer is mainly surgical radical resection. Preoperative and postoperative chemotherapy, molecular targeted drug therapy and immunotherapy can prolong the patient's survival to a certain extent. In recent years, more and more studies have shown that the expression level of specific proteins is closely related to the occurrence, development and prognosis of tumors. Therefore, finding new biomarkers to achieve early prognosis assessment is a hot topic in current research.

[0004] As transcription factors, ZNF154 and EGR1 are involved in a variety of biological processes, including cell proliferation, differentiation, and apoptosis. The methylation status of the ZNF154 gene has been shown to be closely related to the development, diagnosis, and prognosis of cancer in a variety of cancers. These findings not only reveal the potential role of ZNF154 in the occurrence and development of cancer, but also provide new targets for the early diagnosis and treatment of cancer.

[0005] Previous studies have found that in malignant tumors such as lung cancer, breast cancer, and head and neck squamous cell carcinoma, high expression of EGR1 indicates that patients have better long-term benefits. As a tumor suppressor gene, EGR1 affects the malignant progression of tumors. EGR1 regulates the expression of genes such as PTEN, CCND1, and Fibronectin, activates signaling pathways such as NF-κB, MAPK, and AMPK, thereby affecting the malignant behavior of tumor cells (SUN M, NIE FQ, ZANG C, et al. The Pseudogene DUXAP8 Promotes Non-small-cell Lung Cancer Cell Proliferation and Invasion by Epigenetically Silencing EGR1 and RHOB[J]. Mol Ther, 2017, 25(3): 739-751.; SHAJAHAN-HAQ AN, BOCA SM, JIN L, et al. EGR1regulates cellular metabolism and survival in endocrine resistant breast cancer[J]. Oncotarget, 2017, 8(57): 96865-96884.).

[0006] At present, the prognosis evaluation of gastric cancer mainly relies on clinical pathological characteristics and traditional biomarkers (such as CA19-9 and CEA). Although these markers can reflect the patient's prognosis to a certain extent, their sensitivity and specificity are limited, and they cannot accurately reflect the molecular characteristics of the tumor. In recent years, some studies have attempted to use gene expression profiles or other molecular markers for prognosis evaluation, but these methods are often complex, costly, and require specialized equipment and technical support. Therefore, establishing a prognosis evaluation method for gastric cancer is of great significance for the formulation of personalized chemotherapy regimens for gastric cancer patients and improving the long-term survival of patients. Summary of the invention

[0007] The present invention finds that the expression levels of ZNF154 and EGR1 in gastric cancer are significantly correlated with the patient's prognosis, and proposes a method for evaluating the prognosis of gastric cancer based on the score of ZNF154 and EGR1 expression levels. Based on this, the present invention is completed.

[0008] In a first aspect, the present invention provides a biomarker for the prognosis of gastric cancer patients, wherein the biomarker is ZNF154. When the ZNF154 protein expression level score in the biological sample of the subject is ≥2, the prognosis of the subject is good.

[0009] Furthermore, the biological sample of the subject is a tissue section.

[0010] In a second aspect, the present invention provides an application of a biomarker in the preparation of a reagent for predicting the prognosis of gastric cancer patients, wherein the reagent is a reagent that can detect the expression level of ZNF154 protein; when the ZNF154 protein expression level score in the subject's biological sample is ≥2, the subject has a good prognosis.

[0011] Furthermore, the biological sample of the subject is a tissue section.

[0012] In a third aspect, the present invention provides a kit for predicting the prognosis of gastric cancer patients, the kit comprising a reagent for detecting the expression level of ZNF154 protein; when the ZNF154 protein expression level score in the biological sample of the subject is ≥2, the prognosis of the subject is good.

[0013] Furthermore, the biological sample of the subject is a tissue section.

[0014] Furthermore, the kit can be prepared as an ELISA detection kit and / or a colloidal gold detection kit.

[0015] Furthermore, the diagnostic method of the kit includes a direct method, an indirect method, a double antibody sandwich method and / or a competitive method.

[0016] In a fourth aspect, the present invention provides a biomarker for the prognosis of gastric cancer patients, wherein the biomarker is a combination of ZNF154 and EGR1, and when the ZNF154 protein expression level score in the subject's biological sample is ≥2, and the EGR1 protein expression level score in the subject's biological sample is ≥2, the subject has a good prognosis.

[0017] Furthermore, the biological sample of the subject is a tissue section.

[0018] In a fifth aspect, the present invention provides an application of a biomarker in the preparation of a reagent for predicting the prognosis of gastric cancer patients, wherein the biomarker is a combination of ZNF154 and EGR1, and the reagent is a reagent that can detect the expression levels of ZNF154 and EGR1 proteins; when the ZNF154 protein expression level score in the subject's biological sample is ≥2, and the EGR1 protein expression level score in the subject's biological sample is ≥2, the subject has a good prognosis.

[0019] Furthermore, the biological sample of the subject is a tissue section.

[0020] In a sixth aspect, the present invention provides a kit for predicting the prognosis of gastric cancer patients, the kit containing reagents for detecting the expression levels of ZNF154 and EGR1 proteins; when the ZNF154 protein expression level score in the subject's biological sample is ≥2, and the EGR1 protein expression level score in the subject's biological sample is ≥2, the subject has a good prognosis.

[0021] Furthermore, the biological sample of the subject is a tissue section.

[0022] Furthermore, the kit can be prepared as an ELISA detection kit and / or a colloidal gold detection kit.

[0023] Furthermore, the diagnostic method of the kit includes a direct method, an indirect method, a double antibody sandwich method and / or a competitive method.

[0024] Beneficial Effects

[0025] The method for evaluating the prognosis of gastric cancer based on the protein expression level of ZNF154 or the combination of ZNF154 and EGR1 proposed in the present invention has the advantages of simple operation and low cost, can provide more accurate prognosis evaluation for clinicians, and provide an important basis for personalized treatment of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 ZNF154 is lowly expressed in gastric cancer tissues and gastric cancer cells.

[0027] Figure 2 ZNF154 inhibits the proliferation and migration of gastric cancer cells.

[0028] Figure 3 ZNF154 significantly inhibited the growth of subcutaneous tumors in nude mice.

[0029] Figure 4 ZNF154 interacts with EGR1.

[0030] Figure 5 ZNF154 regulates the activity of NF-κB pathway in gastric cancer cells.

[0031] Figure 6 Low expression of ZNF154 is associated with poor prognosis.

[0032] Figure 7 Low EGR1 expression is associated with poor prognosis.

[0033] Figure 8 ZNF154 and EGR1 jointly predict patient prognosis. Fig. 9 Survival curves of different factors were used to evaluate the prognosis of patients. DETAILED DESCRIPTION

[0034] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0036] Example 1 Subject inclusion criteria

[0037] A. Inclusion Criteria

[0038] Age requirement: Patients must be between 18 and 75 years old.

[0039] Diagnosed as gastric cancer: diagnosed as primary gastric cancer (including gastric adenocarcinoma, gastric signet ring cell carcinoma, gastric squamous cell carcinoma, etc.) through pathological or histological examination.

[0040] Untreated: The patient did not receive radiotherapy, chemotherapy, targeted therapy, or immunotherapy before surgical specimen collection.

[0041] Functional status: The patient's ECOG score (Eastern Cooperative Oncology Group) is 0-2, indicating that he can carry out daily activities and has basic self-care ability.

[0042] Informed consent: Patients voluntarily participated in the study and signed the informed consent form.

[0043] B. Exclusion criteria

[0044] History of other malignancies: The patient has a history of other malignancies.

[0045] Serious comorbidities: Patients have serious comorbidities that affect surgery or sample collection, such as uncontrolled cardiovascular disease, liver and kidney failure, severe respiratory disease, etc.

[0046] Previous treatment history: Patients who have received preoperative chemotherapy, radiotherapy, or targeted therapy will be excluded.

[0047] Infection: The presence of active systemic infection (such as HIV, active HBV or HCV infection that is not controlled), or other serious infection symptoms may affect surgery or specimen collection.

[0048] Pregnancy or lactation: Pregnant or lactating women are not included to avoid the potential effects of surgery and treatment on mother and fetus.

[0049] Mental illness or cognitive impairment: The patient has severe mental illness or cognitive impairment and is unable to cooperate with the surgical procedure or understand and sign the informed consent form.

[0050] Example 2 Sample Collection

[0051] A. Sample Type

[0052] Tumor tissue samples were collected from patients with gastric cancer.

[0053] B. Collection Methods

[0054] Samples were obtained during surgery or by endoscopic biopsy, deblooded with saline, fixed in formalin, and subsequently embedded in tissue.

[0055] C. Tissue Section Preparation

[0056] The fixed tissue samples were dehydrated, cleared, and waxed, and finally cut into 4-5 μm thick sections. The sections were placed on glass slides and dried to ensure that the sections were firmly attached.

[0057] Example 3 Expression level of ZNF154 in gastric cancer tissues and gastric cancer cells

[0058] The primer sequences used are shown in Table 1.

[0059] A. Detection of mRNA expression levels

[0060] (1) The RNA of gastric cancer tissue was lysed and extracted using TRIzol reagent;

[0061] (2) reverse transcribe RNA into cDNA using a first-strand cDNA synthesis kit;

[0062] (3) The expression level of mRNA was analyzed using a qPCR kit on an ABI 7500qPCR machine (the primers used for PCR are shown in Table 1 );

[0063] (4) Adopt 2 -ΔΔCt The method normalizes the expression level of each gene relative to the same cDNA and compares these levels to its control.

[0064] B. Detection of protein expression levels

[0065] (1) Grind the tissue with a grinding rod, lyse the cells or tissue with a protein lysis buffer, and centrifuge;

[0066] (2) Add buffer according to the ratio of protein supernatant to 5x loading buffer = 4:1, mix well, and heat;

[0067] (3) Load equal amounts of protein and molecular weight markers onto the wells of SDS-PAGE gel for electrophoresis. After the protein is transferred from the gel to the membrane, add 5% skim milk powder for blocking, and incubate the membrane with appropriately diluted primary antibodies in blocking buffer;

[0068] (4) Incubate with ZNF154 primary antibody overnight, wash the membrane with TBST, incubate with corresponding secondary antibody, wash the membrane with TBST, develop color with super luminescent solution, and observe protein expression.

[0069] C. Test Results

[0070] like Figure 1 As shown in the results, it was found that the mRNA and protein levels of ZNF154 in gastric cancer tissues were significantly lower than those in normal gastric tissues (p<0.05), and were significantly correlated with lymph node metastasis, distant metastasis, poor differentiation and diffuse subtypes (P<0.01). The mRNA and protein levels of ZNF154 in gastric cancer cells (AGS, HGC27, MGC803, NCI-N87) were significantly lower than those in normal gastric cells GES-1 cells.

[0071] Table 1. Primer sequences

[0072]

[0073]

[0074] Example 4 Immunohistochemical staining

[0075] A. Antibody Selection

[0076] Select primary antibodies specific for ZNF154 and EGR1 and ensure they are validated and highly specific.

[0077] B. Staining Method

[0078] (1) Wax removal and dehydration: After removing the wax with xylene, the mixture is dehydrated with a gradient of ethanol;

[0079] (2) Antigen retrieval: Antigen retrieval is performed in a microwave oven or pressure cooker to enhance the specificity of antibody binding;

[0080] (3) Blocking nonspecific binding: Block the sections with a blocking solution containing 5% bovine serum albumin (BSA) at room temperature;

[0081] (4) Primary antibody incubation: The primary antibodies of ZNF154 and EGR1 were added to the sections and incubated overnight;

[0082] (5) Secondary antibody incubation: Incubate with appropriate HRP- or fluorescently labeled secondary antibodies at room temperature for 1 h;

[0083] (6) Color development reaction: Use DAB (3,3'-diaminobenzidine) to carry out color development reaction and observe the results.

[0084] C. Morphological Analysis

[0085] Assess protein expression through qualitative and quantitative analysis of cell morphology, distribution, and intensity.

[0086] D. Quantitative analysis

[0087] The sections were observed under an optical microscope, and the expression of ZNF154 and EGR1 was scored. The score method was usually used to evaluate the expression level (staining intensity: negative 0 point, light yellow 1 point, brown 2 points, yellowish brown 3 points; percentage of gastric cancer cell staining area: <25% for 1 point, 25-50 for 2 points, 50-75 for 3 points, >75% for 4 points; the final score was the product of staining intensity and area score) (as shown in Table 2).

[0088] Table 2. Correlation between ZNF154 expression and clinical pathological parameters of gastric cancer

[0089]

[0090] Example 5 ZNF154 inhibits gastric cancer cell proliferation and migration

[0091] AGS and HGC27 cells were used to study the cell biological function of ZNF154.

[0092] A. MTT test

[0093] The cells were seeded into well plates, and the survival rate was determined every day using a 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay kit;

[0094] The absorbance at 490 nm was detected using an enzyme reader.

[0095] B. Colony formation assay

[0096] The cells were seeded into tissue culture plates and cultured;

[0097] Ten days later, cells were fixed with 4% paraformaldehyde and then stained with 0.2% crystal violet;

[0098] Take photos, observe, and record data.

[0099] C. Transwell experiment

[0100] Insert the 8-μm pore membrane into the Transwell instrument;

[0101] Inoculate 2 × 10 4 Cells, incubation;

[0102] Cells were fixed with 4% paraformaldehyde and then stained with 0.2% crystal violet;

[0103] Take photos, observe, and record data.

[0104] D. Test results

[0105] like Figure 2As shown, overexpression of ZNF154 inhibited the proliferation and migration ability of gastric cancer cells, while silencing ZNF154 increased the proliferation and migration ability of gastric cancer cells.

[0106] Example 6 ZNF154 significantly inhibits the growth of subcutaneous tumors in nude mice

[0107] HGC27 cells (2×10 6 ) were injected subcutaneously into both flanks of 4-week-old BALB / C female athymic mice. During the experiment, the diameter and weight of the animals were measured and recorded regularly (every 2-3 days) until day 24, when the nude mice bearing xenograft tumors were euthanized. Xenograft tumors were carefully isolated and accurately weighed. Tumor volume was calculated by measuring the longest (a) and shortest (b) dimensions of the tumor using the formula ab2 / 2, where a is the longest dimension and b is the shortest dimension.

[0108] like Figure 3 As shown, the subcutaneous tumors in nude mice in the ZNF154 overexpression group were significantly smaller than those in the control group, and the tumorigenic ability of ZNF154-OE cells was weakened.

[0109] Example 7 ZNF154 interacts with EGR1

[0110] ZNF154 overexpression gastric cancer cell lines were collected (3 samples per group, approximately 5*10 5 cells), transcriptome sequencing was used to screen downstream signaling molecules and further experimentally verify them.

[0111] like Figure 4 As shown, the protein expression of EGR1 was also increased after ZNF154 overexpression, and the mRNA expression of ZNF154 and EGR1 in gastric cancer tissues was significantly positively correlated. Immunoprecipitation showed that EGR1 protein was detected in the protein complex pulled down by ZNF154 antibody.

[0112] Example 8 ZNF154 regulates NF-κB pathway activity in gastric cancer cells

[0113] Through the transcriptome data enrichment analysis in Example 7, it was found that ZNF154 may affect the activity of the NF-κB pathway in gastric cancer cells. Western blotting experiments confirmed that ZNF154 regulates the nuclear import and export of P65, thereby regulating the activity of the NF-κB pathway. Chromatin immunoprecipitation using ZNF154 antibodies revealed that ZNF154 binds to the IκB-α promoter (e.g. Figure 5 shown).

[0114] Example 9 Prognostic Assessment

[0115] A. Statistical Analysis

[0116] The expression scores of ZNF154 and EGR1 and the clinical information of the patients (such as tumor stage, pathological parameters, and follow-up survival) were collected and divided into high expression group and low expression group (score <=1 for Low; score >=2 for High) according to their respective immunohistochemical scores (such as Figure 6 ), and survival analysis was performed.

[0117] B. Prognosis prediction

[0118] The Kaplan-Meier survival curve was used to evaluate the prognosis of the patients.

[0119] like Figure 7-9 As shown, low expression of ZNF154 and low expression of EGR1 both indicate poor prognosis of gastric cancer patients; CEA, Ki67, and Her-2 have no significant correlation with the prognosis of gastric cancer patients, and the predictive effect of prognosis is poor. When ZNF154 and EGR1 are used together, the prognosis is best when both are highly expressed, with a median survival time of 104 months, and the prognosis is worst when both are low, with a median survival time of 21 months, and when one is high and the other is low, it is between the two, with a median survival time of 43 months.

Claims

1. A biomarker for the prognosis of gastric cancer patients, wherein the biomarker is ZNF154. When the ZNF154 protein expression level score in the subject's biological sample is ≥2, the subject has a good prognosis.

2. Use of a biomarker in the preparation of a reagent for predicting the prognosis of gastric cancer patients, wherein the reagent is a reagent that can detect the expression level of ZNF154 protein; when the ZNF154 protein expression level score in the subject's biological sample is ≥2, the subject has a good prognosis.

3. A kit for predicting the prognosis of gastric cancer patients, the kit comprising a reagent for detecting the expression level of ZNF154 protein; when the ZNF154 protein expression level score in the subject's biological sample is ≥2, the subject has a good prognosis.

4. A biomarker for the prognosis of gastric cancer patients, wherein the biomarker is a combination of ZNF154 and EGR1. When the ZNF154 protein expression level score in the subject's biological sample is ≥2, and the EGR1 protein expression level score in the subject's biological sample is ≥2, the subject has a good prognosis.

5. Use of a biomarker in the preparation of a reagent for predicting the prognosis of gastric cancer patients, wherein the biomarker is a combination of ZNF154 and EGR1, and the reagent is a reagent that can detect the expression levels of ZNF154 and EGR1 proteins; when the ZNF154 protein expression level score in the subject's biological sample is ≥2, and the EGR1 protein expression level score in the subject's biological sample is ≥2, the subject has a good prognosis.

6. A kit for predicting the prognosis of gastric cancer patients, the kit comprising reagents for detecting the expression levels of ZNF154 and EGR1 proteins; when the ZNF154 protein expression level score in the subject's biological sample is ≥2, and the EGR1 protein expression level score in the subject's biological sample is ≥2, the subject has a good prognosis.

7. The method according to any one of claims 1 to 6, wherein the biological sample of the subject is a tissue section.

8. The kit for predicting the prognosis of gastric cancer patients according to claim 3 or 6, wherein the kit can be prepared as an ELISA detection kit and / or a colloidal gold detection kit.

9. The kit for predicting the prognosis of gastric cancer patients according to claim 3 or 6, wherein the diagnostic method of the kit comprises a direct method, an indirect method, a double antibody sandwich method and / or a competitive method.

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