Application of exosome marker for stomach cancer diagnosis in preparation of stomach cancer diagnosis product
By screening and detecting the exosome LncSENP6 gene, a non-invasive gastric cancer diagnosis method was developed, which solved the invasive problem of early diagnosis of gastric cancer and improved the early diagnosis rate of gastric cancer.
Patent Information
- Application Number
- CN202510408480.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-07-04
AI Technical Summary
Existing gastric cancer diagnosis methods such as gastroscopy are invasive and lack of effective blood-based markers for early diagnosis, resulting in a low early diagnosis of gastric cancer.
High-throughput sequencing technology was used to screen out the exosome LncSENP6 genes related to gastric cancer, and their specific high expression in the plasma of gastric cancer patients was detected by real-time fluorescence quantitative PCR technology. A kit containing LncSENP6 primers was developed for gastric cancer diagnosis.
The early diagnosis of gastric cancer is achieved by non-invasively detecting blood, which improves the early diagnosis rate and diagnostic accuracy of gastric cancer.
Smart Images
Figure CN120249489A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the technical field of disease diagnosis and treatment, and specifically relates to the application of an exosome marker for gastric cancer diagnosis in the preparation of products for diagnosing gastric cancer. Background Art
[0002] Gastric cancer is a tumor originating from the gastric mucosa epithelium, characterized by a high incidence and high mortality rate. Currently, gastroscopy is used as a tool for diagnosing gastric cancer, but gastroscopy examination is somewhat invasive, and the acceptance level of the public towards gastroscopy is not very high. Therefore, the early diagnosis of gastric cancer patients has become one of the hotspots in clinical research. There is an urgent need to find a tumor marker to solve the current international problem, improve the early diagnosis rate of gastric cancer, and further improve the treatment effect of gastric cancer. Exosomes, as one of the main active substances, non-coding RNA, are the biological basis for the function of tumor exosomes and a part of the intercellular communication system, and they have attracted extensive attention as biomarkers. In addition, exosomes are very stable in the blood and can be isolated for clinical evaluation even in the early stages of the disease.
[0003] Therefore, there is an urgent need to develop gastric cancer screening, early diagnosis, and treatment monitoring technologies based on gastric cancer markers to provide comprehensive guidance for its precision medicine. Summary of the Invention
[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide the application of an exosome marker for gastric cancer diagnosis in the preparation of products for diagnosing gastric cancer. Specifically, the following technical solutions are adopted: In the first aspect, the present invention provides the application of an exosome marker for gastric cancer diagnosis in the preparation of products for diagnosing gastric cancer, wherein the exosome marker is LncSENP6; the nucleotide sequence of LncSENP6 is as shown in SEQ ID NO.5.
[0005] SEQ ID NO.5:
[0006] In the above gene sequence, capital letters represent the coding region, which can be transcribed into mRNA and further translated into proteins, while lowercase letters represent the non-coding region.
[0007] The present invention screens out the exosomal LncSENP6 gene closely related to gastric cancer expression through high-throughput sequencing technology, and real-time fluorescence quantitative PCR technology detects that it shows specific high expression in the plasma of gastric cancer patients, which can be used as a biomarker for early gastric cancer.
[0008] As a further preferred embodiment, the product includes a chip or a reagent or a kit.
[0009] In a second aspect, the present invention provides a product for diagnosing gastric cancer, and the product contains a reagent for quantitatively detecting the above-mentioned exosomal marker.
[0010] In a third aspect, the present invention provides a primer pair for amplifying the above-mentioned exosomal marker, and the primer pair includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.1; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2.
[0011] SEQ ID NO.1 (LncSENP6-F): 5’-GGTGCCTATGTCTCAGCCTCTT-3’.
[0012] SEQ ID NO.2 (LncSENP6-R): 5’-GCCATAGAACTGATGAGAGGGAG-3’.
[0013] In a fourth aspect, the present invention provides the application of the above-mentioned primer pair in the preparation of a kit or a reagent for diagnosing gastric cancer.
[0014] In a fifth aspect, the present invention provides a kit for diagnosing gastric cancer, and the kit includes the above-mentioned primer pair.
[0015] As a further preferred embodiment, the kit further includes a primer pair for amplifying the internal reference gene β-actin.
[0016] As a further preferred embodiment, the primer pair for amplifying the internal reference gene β-actin includes an upstream primer for the internal reference gene β-actin and a downstream primer for the internal reference gene β-actin; the nucleotide sequence of the upstream primer for the internal reference gene β-actin is as shown in SEQ ID NO.3; the nucleotide sequence of the downstream primer for the internal reference gene β-actin is as shown in SEQ ID NO.4.
[0017] SEQ ID NO.3 (β-actin-F): 5’-TCGCCATTGCATTGGGAT-3’.
[0018] SEQ ID NO.4 (β-actin-R): 5’-ATATCGGCCGACACCACAT-3’.
[0019] The kit provided above in the present invention includes a primer pair for detecting the LncSENP6 gene in exosomes and a primer pair for detecting the internal reference gene β-actin. By extracting plasma exosome mRNA, reverse transcribing the mRNA into cDNA, and specifically detecting the gene expression level of exosomal LncSENP6 in plasma using real-time fluorescence quantitative PCR with the cDNA as a template, it has high diagnostic value and can be used for the screening of pre-cancerous lesions of gastric cancer, with broad application prospects.
[0020] As a further preferred embodiment, the kit further includes exosome-specific capture antibodies for CD9, CD81, or TSG101.
[0021] In a sixth aspect, the present invention provides the application of an exosome marker for gastric cancer diagnosis in constructing a prognostic prediction model for gastric cancer, where the exosome marker is LncSENP6; the nucleotide sequence of LncSENP6 is as shown in SEQ ID NO.5.
[0022] The beneficial effects of the present invention are as follows: The present invention detected and verified gastric cancer plasma samples and healthy human plasma samples through high-throughput sequencing technology and fluorescence quantitative PCR technology, and found that exosomal LncSENP6 is highly specifically expressed in gastric cancer plasma exosomes, verifying that exosomal LncSENP6 is closely related to the occurrence of gastric cancer. Therefore, exosomal LncSENP6 can be used as a plasma exosome marker for diagnosing gastric cancer and can provide an important reference for the early diagnosis of gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.
[0024] Figure 1 Shown is the electron microscopy identification of exosomes.
[0025] Figure 2 Shown is the Western blot identification of exosome-specific protein markers CD9, CD81, and TSG101.
[0026] Figure 3 Shown are the results of whole transcriptome sequencing of plasma exosomes.
[0027] Figure 4 Shown is the differential expression of the LncSENP6 gene.
[0028] Figure 5 Shown is the ROC curve analysis. Specific embodiments
[0029] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present application.
[0030] Example 1 Screening of exosomal LncSENP6 (1) Extraction of plasma exosomes: Collect 10 plasma samples from gastric cancer patients and 10 plasma samples from healthy individuals for the extraction of exosomes. The kit for extracting plasma exosomes is purchased from Shanghai Yumeibo Biotechnology Co., Ltd. Observe the extracted exosomes under an electron microscope. The results are as Figure 1 shown. It can be seen from the results that the exosomes have a complete morphological structure and are evenly distributed.
[0031] Then, the exosomes are purified by differential centrifugation (centrifuged at 10,000×g for 30 min to remove cell debris, and ultracentrifuged at 100,000×g for 90 min to collect the exosome precipitate), specifically captured by an affinity chromatography column coated with CD9, CD81, and TSG101 antibodies, and finally washed three times with PBS buffer (pH 7.4) to obtain high-purity exosomes; the purified exosomes are lysed with RIPA lysis buffer (containing 1% protease inhibitor) for 30 min, and the supernatant is taken after centrifugation at 12,000×g for 15 min to obtain exosome-specific protein markers (CD9, CD81, TSG101), and Western blot identification is performed. The results are as Figure 2 shown. The exosome-specific protein markers CD9, CD81, and TSG101 are positive in the exosome samples, indicating that the exosomes have been successfully prepared and enriched.
[0032] Finally, the samples of plasma exosomes from 10 gastric cancer patients and 10 healthy individuals extracted are subjected to whole transcriptome sequencing to further observe the changes in the LncRNAs expression profiles in plasma exosomes of gastric cancer patients and healthy individuals. The results are as Figure 3As shown, the results show that 6 LncRNA molecules with the largest differential expression multiples related to gastric cancer are screened out, and among them, the candidate molecule LncSENP6 with the largest differential expression multiple in the plasma exosomes of gastric cancer patients is selected.
[0033] (2)Extraction of total mRNA from plasma exosomes: The EXOsome RNA isolation Kit was used to extract the total mRNA of exosomes.
[0034] (3)Reverse transcription of mRNA into cDNA using a reverse transcription kit: The configuration of the reverse transcription reaction system is shown in Table 1, and the reverse transcription reaction conditions set on the PCR instrument are shown in Table 2. The reverse-transcribed cDNA was aliquoted into two 200 μL PCR tubes and stored at -20 °C for later use.
[0035] Table 1 Reverse transcription reaction system Table 2 Reverse transcription reaction conditions (4)Taking the prepared 10 cDNA samples of healthy human plasma as the control group and 10 cDNA samples of gastric cancer patient plasma as the observation group, the expression levels of the LncSENP6 gene in the plasma of 10 healthy people and 10 gastric cancer patients were detected by real-time fluorescence quantitative PCR technology to further verify the candidate molecule LncSENP6 related to gastric cancer screened out by the high-throughput sequencing results.
[0036] Example 2 This example provides a kit for diagnosing gastric cancer, which includes a primer pair for the LncSENP6 gene in exosomes and a primer pair for detecting the internal reference gene β-actin; Primers were designed according to the LncSENP6 gene. The upstream primer sequence of the primer pair for the LncSENP6 gene is shown as SEQ ID NO.1, and the downstream primer sequence is shown as SEQ ID NO.2. Using β-actin as the internal reference gene to normalize the target gene LncSENP6, the upstream primer sequence of the primer pair for amplifying the internal reference gene β-actin is shown as SEQ ID NO.3, and the downstream primer sequence is shown as SEQ ID NO.4.
[0037] SEQ ID NO.1: 5’-GGTGCCTATGTCTCAGCCTCTT-3’ (LncSENP6-F) SEQ ID NO.2: 5’-GCCATAGAACTGATGAGAGGGAG-3’ (LncSENP6-R) SEQ ID NO.3: 5'-TCGCCATTGCATTGGGAT-3' (β-actin-F) SEQ ID NO.4: 5'-ATATCGGCCGACACCACAT-3' (β-actin-R) Example 3 Effect evaluation of the kit for diagnosing gastric cancer (1) Take 2 mL of the patient's plasma sample. After removing cell debris by centrifugation at 2000×g for 10 min, the differential centrifugation method is used (centrifugation at 10000×g for 30 min to remove large particle impurities, and ultracentrifugation at 100000×g for 70 min to precipitate exosomes) to obtain the crude exosome extract. Further, exosomes are specifically captured by an affinity chromatography column coated with CD9 / CD81 / TSG101 antibodies. After washing with PBS buffer, it is concentrated to 50 μL through a 100 kDa ultrafiltration tube, and the protein concentration measured by the BCA method should be >0.5 μg / μL.
[0038] (2) Use TRIzol LS reagent to lyse exosomes. After chloroform stratification and centrifugation at 12000×g, the miRNeasy kit is used to extract total RNA, and the A260 / A280 ratio (1.8 - 2.0) is detected by NanoDrop and the RNA integrity (RIN > 7.0) is evaluated by Agilent 2100. Subsequently, reverse transcription is carried out with PrimeScript RT Master Mix, and the reaction system contains specific primers for LncSENP6 and β-actin, and is incubated at 37°C for 15 min to synthesize cDNA.
[0039] (3) Using the SYB Green method and primers for the LncSENP6 gene and the β-actin internal reference gene, qPCR reaction is carried out on a fluorescence quantitative PCR instrument to amplify the cDNA sequence corresponding to exosomal LncSENP6 in plasma. The fluorescence quantitative PCR reaction system is shown in Table 3.
[0040] Table 3 Fluorescence quantitative PCR reaction system The relative expression levels of exosomal LncSENP6 in plasma in the healthy control group and gastric cancer patients are achieved by plotting a scatter plot using Origin drawing software.
[0041] The results are as Figure 4 shown. The LncSENP6 gene is lowly expressed in the healthy control group and highly expressed in gastric cancer patients. Gastric cancer patients and the healthy control group can be clearly distinguished by the expression of the LncSENP6 gene in plasma.
[0042] The relative expression levels of exosomal LncSENP6 gene in plasma of gastric cancer patients and healthy controls were obtained to evaluate the predictive ability of exosomal LncSENP6, and statistical analysis was performed using the ROC curve.
[0043] The results of the ROC curve analysis were as Figure 5 shown. The AUC values of exosomal LncSENP6 in diagnosing gastric cancer were 0.953, indicating that exosomal LncSENP6 could specifically distinguish gastric cancer patients from healthy controls. By detecting the level of exosomal LncSENP6 in plasma, the purpose of screening for gastric cancer could be achieved.
[0044] The embodiments of the present application have been described above in conjunction with the accompanying drawings. Specific examples have been used herein to elaborate on the principles and implementation manners of the present application. The description of the above embodiments is only for helping to understand the core idea of the present application. However, the present application is not limited to the above specific implementation manners. The above specific implementation manners are merely illustrative and not restrictive. Under the inspiration of the present application, those of ordinary skill in the art can also make many forms without departing from the purpose of the present application and the scope protected by the claims, and all of them belong to the protection scope of the present application.
Claims
1. Use of an exosome marker for gastric cancer diagnosis in the preparation of a product for diagnosing gastric cancer, characterized in that, The exosome marker is LncSENP6; the nucleotide sequence of LncSENP6 is as shown in SEQ ID NO.
5.
2. The application according to claim 1, wherein The product includes a chip, a reagent or a kit.
3. A product for diagnosing gastric cancer, characterized in that, The product contains a reagent for quantitatively detecting the exosome marker described in claim 1.
4. A primer pair for amplifying the exosome marker described in claim 1, characterized in that, The primer pair includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.1; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.
2.
5. Use of the primer pair according to claim 4 in the preparation of a kit or reagent for diagnosing gastric cancer.
6. A kit for diagnosing gastric cancer, characterized in that, The kit includes the primer pair according to claim 4.
7. The kit according to claim 4, wherein The kit further includes a primer pair for amplifying the internal reference gene β-actin.
8. The kit according to claim 7, wherein, The primer pair for amplifying the internal reference gene β-actin includes an upstream primer for the internal reference gene β-actin and a downstream primer for the internal reference gene β-actin; the nucleotide sequence of the upstream primer for the internal reference gene β-actin is as shown in SEQ ID NO.3; the nucleotide sequence of the downstream primer for the internal reference gene β-actin is as shown in SEQ ID NO.
4.
9. The kit according to claim 8, wherein It also contains one or more of the CD9 exosome capture antibody, the CD81 exosome capture antibody, and the TSG101 exosome capture antibody.
10. Use of an exosome marker for gastric cancer diagnosis in constructing a prognostic prediction model for gastric cancer, characterized in that, The exosome marker is LncSENP6; the nucleotide sequence of LncSENP6 is as shown in SEQ ID NO.5.