A plasma exosomal circRNA as a gastric cancer biomarker, its kit and application

Abstract

The present invention provides a plasma exosomal circRNA that can be used as a gastric cancer diagnostic marker, as well as a kit and application thereof, belonging to the technical field of biological and molecular diagnosis. The present invention provides the application of the plasma exosomal circRNA marker in the preparation of a gastric cancer diagnostic kit and / or a gastric cancer diagnostic agent. The present invention first discovers that hsa_circ_0000513 exists in plasma exosomes, and through experiments, it is confirmed that: in the plasma of gastric cancer patients, the expression level of the plasma exosomal hsa_circ_0000513 of the present invention is significantly increased, and it is a novel biomarker with potential value for the early diagnosis and prognosis judgment of gastric cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological and molecular diagnostics, and particularly relates to a gastric cancer plasma exosome circRNA biomarker, a kit thereof, and an application thereof. Background Art

[0002] Gastric cancer is the fifth most common cancer and the third leading cause of cancer death globally. Due to the fact that the diagnosis of gastric cancer often occurs at an advanced stage, its mortality rate is very high. Currently, the diagnosis of gastric cancer mainly relies on tissue pathological biopsy, which has limitations such as invasiveness and tumor heterogeneity. Detection of blood tumor markers such as CEA and CA199 has low sensitivity and specificity and cannot meet the needs of gastric cancer screening and early diagnosis. Therefore, there is an urgent need to establish a rapid, sensitive, specific, and convenient detection method for the diagnosis of gastric cancer.

[0003] Exosomes are nanoscale vesicles secreted by cells, containing some bioactive molecules such as lipids, proteins, and nucleic acids from the source cells, and can mediate specific intercellular communication. It has been found that an important way of signal transduction between tumor cells is through RNA in exosomes, especially non-coding RNA, such as circular RNA. Moreover, the lipid bilayer structure of exosomes enables it to protect the contents from degradation by external enzymes and the like. Plasma samples are commonly used body fluid specimens in clinics, which are simple and quick to collect and are not affected by platelet aggregation and release of exosomes. Therefore, plasma exosomes can better reflect the human body state. Thus, plasma exosomes of tumor patients have great application prospects in the early diagnosis and treatment of tumors.

[0004] Circular RNA (circRNA) is a non-coding RNA formed by the selective back-splicing of its precursor mRNA. CircRNA has cell and tissue specificity, as well as diversity in quantity and type. Its circular structure makes it stable and conserved, and it is widely distributed in vivo and participates in the progression of various diseases, especially the invasion, migration, proliferation, and drug resistance of tumors. Some studies have found that circRNA is highly enriched in exosomes and is correlated with tumor progression. Therefore, liquid biopsy of exosomal circRNA may be developed into a novel non-invasive biomarker and a potential target molecule for cancer.

[0005] Thus, it can be seen that developing a new plasma exosome circRNA biomarker for the diagnosis of gastric cancer will have great promoting value for the early diagnosis, treatment, and prognosis judgment of gastric cancer. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a plasma exosome circRNA biomarker hsa_circ_0000513, which can be used as a biomarker for diagnosing gastric cancer. By detecting and analyzing hsa_circ_0000513 in plasma exosomes, it helps to study the pathogenesis of gastric cancer and can be used for early diagnosis and prognosis judgment of gastric cancer.

[0007] To achieve the above object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides an application of a plasma exosome circRNA biomarker in the preparation of a kit for diagnosing gastric cancer and / or a reagent for diagnosing gastric cancer, wherein the plasma exosome circRNA biomarker is hsa_circ_0000513.

[0009] Preferably, the nucleotide sequence of the biomarker hsa_circ_0000513 is as shown in SEQ ID NO:1.

[0010] The present invention also provides a primer pair for detecting the above plasma exosome circRNA biomarker, and the primer pair is an upstream and downstream primer for detecting the content of hsa_circ_0000513.

[0011] Preferably, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.3.

[0012] The present invention also provides an application of the above primer pair in the preparation of a kit for diagnosing gastric cancer and / or a reagent for diagnosing gastric cancer.

[0013] The present invention also provides a kit for diagnosing gastric cancer, and the kit includes the above primer pair.

[0014] Preferably, the kit further includes a reverse transcription reagent, a fluorescence quantitative PCR reagent, and a primer pair for detecting the internal reference β-actin.

[0015] Preferably, the reverse transcription reagent is HiScript III 1st Strand cDNA Synthesis Kit(+gDNA wiper)R312, and the fluorescence quantitative PCR reagent is AceQ qPCR SYBR GREEN Master Mix.

[0016] Preferably, the primer pair for detecting the internal reference β-actin is an upstream and downstream primer for detecting the content of β-actin, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is as shown in SEQ IDNO.5.

[0017] Preferably, the contents of hsa_circ_0000513 and β-actin in plasma exosomes are measured by the kit, and the relative content of hsa_circ_0000513 is represented by △CT = CThsa_circ_0000513 - CTβ-actin.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides the application of plasma exosome circRNA markers in the preparation of gastric cancer diagnostic kits and / or gastric cancer diagnostic agents. The present invention discovers for the first time that hsa_circ_0000513 exists in plasma exosomes. By detecting circRNA in plasma exosomes of patients, hsa_circ_0000513 is screened out. It is found that its expression level is significantly increased in plasma exosomes of gastric cancer patients, and it can be used as a new biomarker for gastric cancer diagnosis, providing a new basis for the early diagnosis of gastric cancer, contributing to the research on the pathogenesis of gastric cancer, and having great promotion value for the early diagnosis, treatment, and prognosis judgment of gastric cancer. Description of the Drawings

[0020] Figure 1 : Structural simulation diagram of hsa_circ_0000513;

[0021] Figure 2 : Amplification curves of hsa_circ_0000513 and β-actin detected by real-time fluorescence quantitative PCR provided by the examples of the present invention;

[0022] Figure 3 : Melting curves of hsa_circ_0000513 and β-actin detected by real-time fluorescence quantitative PCR provided by the examples of the present invention;

[0023] Figure 4 : Sequencing results of the hsa_circ_0000513 PCR product after TA cloning provided by the examples of the present invention;

[0024] Figure 5 : Agarose gel analysis after QPCR reactions of cDNA and gDNA with the hsa_circ_0000513 primer provided by the examples of the present invention;

[0025] Figure 6 : Relative change amounts of circRNA before and after RNase R enzyme treatment of RNA detected by QRT-PCR provided by the examples of the present invention;

[0026] Figure 7: Expression level of plasma exosomal hsa_circ_0000513 provided by the embodiments of the present invention;

[0027] Figure 8 : Schematic diagram of specificity and sensitivity of ROC curve analysis of plasma exosomal hsa_circ_0000513 for gastric cancer diagnosis provided by the embodiments of the present invention. Detailed implementation manners

[0028] The present invention provides an application of a plasma exosomal circRNA marker in the preparation of a kit for diagnosing gastric cancer and / or a reagent for diagnosing gastric cancer, and the plasma exosomal circRNA marker is hsa_circ_0000513 (the structural simulation diagram is as Figure 1 shown). The nucleotide sequence of the marker hsa_circ_0000513 in the present invention is as shown in SEQ ID NO:1.

[0029] The present invention also provides a method for extracting a plasma exosomal circRNA marker. Preferably, the exosomal circRNA marker in the present invention is extracted from plasma. Preferably, the plasma exosomal circRNA marker is extracted using a plasma exosomal circRNA extraction kit, and the extraction kit includes ExoQuick Plasma prep and Exosome precipitation kit. The reagents used for extracting the plasma exosomal circRNA are miRNeasy Serum / Plasma Kit, absolute ethanol, RNase-free ddH2O, and chloroform. In the present invention, the ExoQuick Plasma prep and Exosome precipitation kit is purchased from SBI, product number EXOQ5TM-1, and the miRNeasy Serum / Plasma Kit is purchased from QIAGNE, product number 217184. The present invention uses a plasma exosomal circRNA extraction kit to extract plasma exosomal circRNA, which can simply and accurately extract exosomal circRNA from plasma, facilitating the detection, analysis, and application of plasma exosomal circRNA in laboratories and clinics.

[0030] The present invention provides a primer pair for detecting the above-mentioned plasma exosomal circRNA marker. The primer pair is the upstream and downstream primers for detecting the content of hsa_circ_0000513. The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.3.

[0031] The present invention provides the application of the above primer pair in the preparation of a kit for diagnosing gastric cancer and / or a reagent for diagnosing gastric cancer.

[0032] The present invention provides a kit for diagnosing gastric cancer, and the kit comprises the above primer pair. Preferably, the kit further comprises a reverse transcription reagent, a fluorescence quantitative PCR reagent, and a primer pair for detecting the internal reference β-actin. In the present invention, the β-actin is an internal reference gene required for relative quantification by fluorescence quantitative PCR and is used for the quantitative detection of hsa_circ_0000513 in plasma exosomes. In the present invention, the reverse transcription reagent is HiScript III 1st Strand cDNA Synthesis Kit(+gDNA wiper)R312, purchased from Vazyme Co., Ltd., with the product number R312-01; the fluorescence quantitative PCR reagent is AceQ qPCR SYBR GREEN Master Mix, purchased from Vazyme Co., Ltd. with the product number Q111-02. In the present invention, the primer pair for detecting the internal reference β-actin is the upstream and downstream primers for detecting the content of β-actin. The nucleotide sequence of the upstream primer is shown as SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.5.

[0033] The present invention measures the contents of hsa_circ_0000513 and β-actin in plasma exosomes through the kit, and represents the relative content of hsa_circ_0000513 with △CT = CT hsa_circ_0000513 - CT β-actin.

[0034] In the present invention, all primers are synthesized by Sangon Biotech (Shanghai).

[0035] In the present invention, unless otherwise specified, the chemical reagents used are all conventional commercially available reagents, and the technical means used are all conventional technical means well-known to those skilled in the art.

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Example 1

[0038] Extraction of circRNA from plasma exosomes

[0039] (1) Collection and preparation of plasma samples:

[0040] 1) Use a purple tube anticoagulated with EDTA to collect 10 ml of elbow venous blood from the detection object. After blood collection, gently invert and mix 5 times immediately to fully mix.

[0041] 2) Use a horizontal centrifuge. At 4°C, centrifuge the collected blood at a speed of 3000 rpm / min for 10 min to preliminarily separate the upper-layer plasma.

[0042] 3) Use a pipette to aspirate the upper-layer plasma and transfer it to a 1.5 mL EP tube. Again, at 4°C, centrifuge it at 3000 g / min for 15 min with a horizontal centrifuge to remove residual cells.

[0043] 4) Use a pipette to transfer and dispense the centrifuged upper-layer plasma into 1.5 mL EP tubes (260 μL / tube), and store it at -80°C for later use.

[0044] (2) Extraction of plasma exosomes:

[0045] 1) Take out the plasma sample stored at -80°C and place it at 4°C to melt. Use a pipette to aspirate 250 μL of the plasma sample into a new RNase-free EP tube.

[0046] 2) Add 2 μL of thrombin, gently pipette to mix well, and let it stand and incubate at room temperature for 5 min.

[0047] 3) At 10000 rpm / min at room temperature, centrifuge for 5 min (there are visible fibrin pellets at the bottom of the test tube). Use a pipette to transfer the supernatant to a new 1.5 mL EP tube.

[0048] 4) Add 63 μL of ExoQuick Plasma prep and Exosome precipitation kit, pipette to mix well, and let it stand at 4°C for 30 min.

[0049] 5) At 4°C, centrifuge at 1500 g for 30 min, and discard the supernatant.

[0050] 6) Again at 4°C, centrifuge at 1500 g for 5 min.

[0051] 7) Use a 10 μL pipette to carefully aspirate the supernatant to completely remove the residual liquid. Add 200 μL of ready-made PBS, gently pipette to mix well, and store it at -80°C or keep it at 4°C for later use.

[0052] (3) Extraction of exosomal circRNA:

[0053] 1) Add 5 volumes of QIAzol Lysis Reagent to the exosome solution kept at 4°C above, vortex for 2 min to fully lyse it, and let it stand at room temperature for 5 min.

[0054] 2) Add an equal volume of chloroform to the exosome solution, vortex for 15 seconds, and let stand at room temperature for 3 minutes;

[0055] 3) Centrifuge at 12,000 g for 15 min at 4°C and carefully transfer the upper clear aqueous phase to a new EP tube.

[0056] 4) Add 1.5 times the volume of pre-cooled anhydrous ethanol and mix thoroughly by inverting 10 times;

[0057] 5) Pipette 700 μL of the above mixture into the centrifuge column (already placed in the collection tube), centrifuge at room temperature, centrifuge at 12000 g for 15 seconds, discard the filtrate, add the remaining mixture to the centrifuge column, and repeat the above steps;

[0058] 6) Add 700 μL of RWT buffer to the centrifuge column, centrifuge at room temperature, 12000 g, 15 s, and discard the filtrate;

[0059] 7) Add 500 μL of RPE buffer to the centrifuge column, centrifuge at room temperature, 12,000 g for 15 seconds, and discard the filtrate;

[0060] 8) Add 500 μL of 80% ethanol to the centrifuge column and centrifuge at room temperature, 12,000 g, for 2 min. Discard the filtrate and collection tube.

[0061] 9) Place the spin column in a new collection tube and centrifuge at 12,000 g for 5 minutes at room temperature to dry the membrane in the spin column.

[0062] 10) Discard the collection tube and place the spin column in a new 1.5 mL EP tube (provided with the kit). Add 15 μL of RNase-free water to the center of the spin column membrane and let it stand for 5 minutes.

[0063] 11) Centrifuge at 12,000 g for 1 min at room temperature. Collect the filtrate in the EP tube as the RNA solution and store at -80°C or reverse transcribe immediately.

[0064] Example 2

[0065] Detection and identification of plasma exosomes has_circ_0000513

[0066] (1) cDNA preparation:

[0067] The RNA obtained in Example 1 was used as a template for reverse transcription to prepare cDNA. The reaction system for reverse transcription is shown in Table 1, and the reaction conditions for reverse transcription are shown in Table 2.

[0068] Table 1 Reverse transcription reaction system

[0069]

[0070] Table 2 Reverse transcription reaction conditions

[0071]

[0072] The product obtained by reverse transcription can be temporarily stored at 4°C and immediately subjected to Q-PCR reaction, or stored at -20°C for use within half a year, or stored at -80°C for long-term storage.

[0073] (2) Real-time fluorescence quantitative PCR reaction

[0074] Take the reaction product obtained by reverse transcription in step (1) for qPCR, and the reaction system is shown in Table 3.

[0075] Table 3 qPCR reaction system

[0076]

[0077] In QuantStudio TM 3 Real-Time PCR System, perform the reaction, and the amplification conditions are shown in Table 4.

[0078] Table 4 qPCR amplification conditions

[0079]

[0080] (3) Analysis of real-time fluorescence quantitative PCR reaction results:

[0081] In step (2), the amplification curves and melting curves of hsa_circ_0000513 and β-actin are detected by real-time fluorescence quantitative PCR reaction, as shown in Figure 2 、 3 . It can be seen that the amplification curves of hsa_circ_0000513 and β-actin detected by real-time fluorescence quantitative PCR provided in the embodiments of the present invention are in an "S" shape, and the melting curves are single peaks, indicating that the specificity of the detection of hsa_circ_0000513 and β-actin in the present invention is good.

[0082] Recover the PCR product for TA cloning plasmid construction and sequencing, as shown in Figure 4 . After TA cloning and sequencing of the hsa_circ_0000513 PCR product provided in the embodiments of the present invention, it is found that it contains the reverse connection point of hsa_circ_0000513, proving that the primer specifically amplifies hsa_circ_0000513;

[0083] (4) Circularization identification of hsa_circ_0000513:

[0084] 1) Perform QPCR reactions on cDNA and gDNA from the same type of cells (the reaction system and program are the same as above), collect the PCR products for agarose gel electrophoresis analysis, as Figure 5 shown, after performing QPCR reactions on cDNA and gDNA with the hsa_circ_0000513 primers provided in the examples of the present invention and then conducting agarose gel analysis, it was found that it could not be amplified in gDNA, but amplified well in cDNA, which conforms to the characteristics of circular RNA primers;

[0085] 2) Treat the total cellular RNA with RNase R enzyme, and use QRT-PCR to detect the relative change in circRNA before and after treatment. The results are as Figure 6 shown. The relative change in circRNA before and after treating RNA with RNase R enzyme detected by QRT-PCR in the examples of the present invention shows that the mRNA β-actin decreased relatively due to being digested by RNase R enzyme, and the circRNA hsa_circ_0000513 showed relative enrichment, which conforms to the characteristics of circular RNA primers.

[0086] Example 3

[0087] Value of gastric cancer plasma exosome has_circ_0000513 as a diagnostic marker:

[0088] (1) The present invention detected 53 gastric cancer patients who visited the Department of Gastroenterology of Zhenjiang First People's Hospital from January to December 2021, and simultaneously detected the plasma samples of 62 healthy controls with similar ages and the same genders. The two groups of samples were collected, sampled, aliquoted, and stored under the same conditions, and the content of plasma exosome hsa_circ_0000513 was detected and analyzed. Detect has_circ_0000513 according to the collection of plasma, extraction of exosome circRNA, reverse transcription, and real-time fluorescence quantitative PCR in Examples 1 and 2.

[0089] (2) Data processing: This experiment adopted a relative quantitative analysis method, using β-actin as the internal reference gene, and representing the relative content of has_circ_0000513 in the sample with △CT = CThsa_circ_0000513 - CTβ-actin, and used Graphpad Prism8 for plotting and analysis.

[0090] (3) Result analysis:

[0091] The present invention compared the relative expression levels of plasma exosome has_circ_0000513 in gastric cancer patients and healthy control groups. The results are as Figure 7As shown, it can be seen that the expression level of hsa_circ_0000513 in plasma exosomes of gastric cancer patients is significantly increased compared with that of the healthy control group.

[0092] The present invention analyzes the diagnostic performance of the expression level of plasma exosomal hsa_circ_0000513 in gastric cancer patients and healthy subjects. The results are as Figure 8 shown. The area under the curve (AUC) of the ROC curve obtained by the present invention for analyzing the diagnostic performance of plasma exosomal hsa_circ_0000513 in gastric cancer is 0.702, the sensitivity is 74.4%, and the specificity is 61.5%. This indicates that the plasma exosomal hsa_circ_0000513 provided in the embodiments of the present invention has good specificity and sensitivity for the diagnosis of gastric cancer.

[0093] It can be seen that plasma exosomal hsa_circ_0000513 can be used as a non-invasive marker for the diagnosis of gastric cancer, providing a basis for the diagnosis of gastric cancer.

[0094] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structure or equivalent process transformation made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of the present invention.

Claims

1. Use of a reagent for detecting plasma exosomal circRNA markers in the preparation of a kit for evaluating gastric cancer risk, characterized in that, The plasma exosome circRNA marker is hsa_circ_0000513; the nucleotide sequence of the marker hsa_circ_0000513 is shown in SEQ ID NO:

1.

2. Use of a primer pair for detecting the plasma exosomal circRNA marker described in claim 1 in the preparation of a kit for evaluating the risk of gastric cancer and / or a reagent for evaluating the risk of gastric cancer, characterized in that, The primer pair is the upstream and downstream primers for detecting the content of hsa_circ_0000513; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.3.

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