CN-121975942-A - Gastric cancer biomarker ENO3 and detection reagent thereof

Abstract

The invention discloses a stomach cancer biomarker ENO3 and a detection reagent thereof, which belong to the technical field of biological medicines, and in particular relates to a detection reagent of a stomach cancer marker, wherein the detection reagent comprises an upstream primer and a downstream primer of the stomach cancer marker and modified immunomagnetic beads, and the stomach cancer marker is enolase 3. The invention discovers and verifies that the gastric cancer marker enolase 3 promotes malignant transformation of gastric cancer by promoting glycolysis of gastric cancer cells, activating a WNT/beta-catenin signal path and up-regulating expression of matrix metalloproteinase, and simultaneously improves capture efficiency of the marker enolase 3 on trace tumor cells in peripheral blood by optimizing an immunomagnetic bead structure of the marker so as to enhance signal intensity of subsequent marker detection and provide an important tool for improving positive detection rate of gastric cancer detection based on peripheral blood.

Inventors

• NI JIAOJIAO
• GE MINGJIE
• JIANG YUCHAO
• HAN YUE
• PAN XIAOYING
• Zhao Duqin
• Yao Yinye
• Zheng Wangye

Assignees

• 浙江省肿瘤医院

Dates

Publication Date

20260505

Application Date

20260409

Claims (10)

1. 1. A detection reagent for a stomach cancer marker is characterized by comprising an upstream primer and a downstream primer of the stomach cancer marker, wherein the stomach cancer marker is enolase 3, the upstream primer has a nucleotide sequence shown as SEQ ID NO.1, and the downstream primer has a nucleotide sequence shown as SEQ ID NO.2, and the upstream primer has a nucleotide sequence shown as SEQ ID NO. 1.
2. 2. The reagent for detecting a gastric cancer marker according to claim 1, wherein the reagent for detecting gastric cancer further comprises modified immunomagnetic beads.
3. 3. The reagent for detecting gastric cancer markers according to claim 2, wherein the modified immunomagnetic beads are composite magnetic nanoparticles with oleic acid modified Fe 3 O 4 composite nanoparticles as an inner core and polymers with methacrylic acid and 2- [ [ (butylamino) carbonyl ] oxo ] ethyl acrylate as structural units as a coating layer, and the surfaces of the coating layers are fixed with enolase 3 antibodies by chemical coupling.
4. 4. The reagent for detecting gastric cancer markers according to claim 2, wherein the preparation method of the modified immunomagnetic beads comprises the steps of mixing an alkenyl derivative, divinylbenzene and sodium dodecyl sulfate, adding deionized water and oleic acid to modify Fe 3 O 4 composite nano particles, performing ultrasonic dispersion, adding acetic acid and H 2 O 2 solution under nitrogen atmosphere to react to obtain modified magnetic beads, and coupling an enolase 3 antibody to the surfaces of the modified magnetic beads by using EDC-NHS coupling technology to obtain modified immunomagnetic beads, wherein the alkenyl derivative comprises methacrylic acid and ethyl 2 [ (butylamino) carbonyl ] oxo ] acrylate.
5. 5. The reagent for detecting a gastric cancer marker according to claim 4, wherein the mass ratio of methacrylic acid to ethyl 2- [ [ (butylamino) carbonyl ] oxo ] acrylate is 1:0.5-2.
6. 6. The reagent for detecting gastric cancer markers according to claim 4, wherein the ratio of the alkenyl derivative to divinylbenzene is 1g:0.01-0.1mL.
7. 7. The reagent for detecting gastric cancer markers according to claim 4, wherein the dosage ratio of divinylbenzene to sodium dodecyl sulfate is 1mL:0.2-0.5g.
8. 8. The reagent for detecting gastric cancer markers according to claim 4, wherein the mass ratio of the sodium dodecyl sulfate to the oleic acid modified Fe 3 O 4 composite nano-particles is 1:0.5-2.
9. 9. The reagent for detecting gastric cancer markers according to claim 4, wherein the volume ratio of divinylbenzene to acetic acid is 1:0.1-0.5.
10. 10. The reagent for detecting gastric cancer markers according to claim 4, wherein the mass concentration of the H 2 O 2 solution is 20-40%, and the volume ratio of divinylbenzene to H 2 O 2 solution is 1:0.1-0.5.

Description

Gastric cancer biomarker ENO3 and detection reagent thereof Technical Field The invention relates to the technical field of biological medicines, in particular to a stomach cancer biomarker ENO3 and a detection reagent thereof. Background Gastric cancer is one of the most common malignant tumors worldwide and is also the leading cause of cancer-related death. Although significant progress has been made in terms of gastric radical surgery, assisted radiation and chemotherapy, immune combination therapy, etc., the overall prognosis for gastric cancer patients remains poor due to the problems of postoperative recurrence and distant metastasis still standing out. Therefore, the key genes and the downstream molecular mechanisms of the key genes are deeply studied, and an effective targeted intervention strategy is designed on the basis, so that the method has important significance for improving survival and prognosis of patients. An abnormality in energy metabolism is one of the fundamental characteristics of tumor cells. Even under oxygen-rich conditions, tumor cells tend to preferentially utilize aerobic glycolysis (i.e., the Warburg effect) and produce large amounts of lactic acid. The metabolic mode not only provides rapid energy supply for tumor cells, but also provides raw materials for biosynthesis of the tumor cells, thereby promoting malignant biological behaviors such as proliferation, migration, invasion and the like of the tumor cells. Enolase (Enolase, ENO) is one of the key enzymes in the glycolytic pathway responsible for catalyzing the interconversion between 2-phosphoglycerate and phosphoenolpyruvate. In mammals, enolase exists as three family members, ENO1, ENO2 and ENO3. There have been studies showing that ENO1 and ENO2 exert a carcinomatous effect in a variety of tumors. However, there are relatively few reports on the study of ENO3 in tumors, and its effect is tissue specific. For example, in liver cancer, ENO3 is considered a potential oncogene, while in colorectal cancer, ENO3 is highly expressed and is associated with a poor prognosis for patients. At present, the role of ENO3 in gastric cancer is not clear and there is a lack of systematic studies. The prior art solutions still have the limitation and challenges of, first, the lack of reliable early diagnostic biomarkers. The atypical early symptoms of gastric cancer, hidden pathological features and the limitations of imaging examinations affect the accuracy of early diagnosis. Second, the specific role of ENO3 in gastric cancer is not yet defined. Although ENO3 has been studied in other tumor types, its expression pattern, clinical significance, biological function and mechanism of action in gastric cancer are still unclear, which limits its transformation potential as a therapeutic target. Third, glycolytic targeted therapies still face significant hurdles. The glycolytic inhibitors such as 2-deoxyglucose (2-DG) which are applied at present have the problems of obvious side effect, limited curative effect and the like, and the development of high-efficiency low-toxicity metabolic intervention drugs is urgent. Therefore, the kit capable of accurately and sensitively detecting the ENO3 expression is constructed, so that the kit is helpful for revealing the role of the kit in the occurrence and development of gastric cancer, and an important tool can be provided for the establishment of early diagnosis and targeted treatment strategies. Disclosure of Invention The invention aims to provide a stomach cancer biomarker ENO3 and a detection reagent thereof, which not only provide a stomach cancer related biomarker, but also improve the capturing efficiency of the stomach cancer biomarker ENO3 on trace tumor cells in peripheral blood by optimizing the immune magnetic bead structure of the targeted stomach cancer biomarker ENO3 so as to enhance the signal intensity of the subsequent marker detection and provide an important tool for improving the positive detection rate of stomach cancer detection based on peripheral blood. The technical scheme adopted by the invention for achieving the purpose is as follows: A detection reagent for a stomach cancer marker comprises an upstream primer and a downstream primer of the stomach cancer marker, wherein the stomach cancer marker is enolase 3, the upstream primer has a sequence 5'-GAACTCCGAGATGGAGACAAAG-3', the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the downstream primer has a sequence 5'-GGACCTAGAGTCTTGTTGATGTG-3', and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2. The invention discovers and verifies that the stomach cancer marker enolase 3 (ENO 3) promotes the glycolysis of stomach cancer cells, activates a WNT/beta-catenin signal path and up-regulates the expression of matrix metalloproteinase, and promotes the malignant transformation of stomach cancer. ENO3 is not only highly expressed in gastric cancer tissues, but also has an expres