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Plasma exosome circRNA marker for diagnosis and prognosis monitoring of gastric cancer and application of plasma exosome circRNA marker

An exosome and marker technology, which is applied in DNA/RNA fragments, recombinant DNA technology, and the determination/examination of microorganisms, etc., can solve the problems of few tumor markers and lack of specificity of gastric cancer diagnostic markers.

Pending Publication Date: 2022-07-22
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the existing clinical diagnostic markers for gastric cancer such as CEA, CA199, CA724, etc. lack specificity, and there are few tumor markers that can truly assist clinical diagnosis. Therefore, it is urgent to develop a rapid and accurate detection of exosomal circRNA in plasma. Methods

Method used

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  • Plasma exosome circRNA marker for diagnosis and prognosis monitoring of gastric cancer and application of plasma exosome circRNA marker
  • Plasma exosome circRNA marker for diagnosis and prognosis monitoring of gastric cancer and application of plasma exosome circRNA marker
  • Plasma exosome circRNA marker for diagnosis and prognosis monitoring of gastric cancer and application of plasma exosome circRNA marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Extraction of plasma exosomal circRNA

[0045] (1) Collection and preparation of plasma

[0046] 1) Collect 8ml of cubital venous blood with an EDTA anticoagulant blood collection tube, and immediately after blood collection, gently invert and mix 5 times to fully mix;

[0047] 2) After standing at room temperature for 30min, centrifuge with a horizontal centrifuge at 3000rpm / min for 10min and preliminarily separate the upper plasma;

[0048] 3) Use a pipette to transfer the plasma to an RNase free imported EP tube, and centrifuge again with a horizontal centrifuge at 3000g / min for 15min to remove residual cell debris;

[0049] 4) Use a pipette to transfer and dispense the centrifuged upper plasma into new RNase free imported EP tubes (260 μl / tube); store at -80°C for later use or place at 4°C for immediate use.

[0050] (2) Isolation and extraction of plasma exosomes

[0051] 1) Take out the plasma from -80°C and thaw at 4°C, use a pipette to draw 250 μl into a new ...

Embodiment 2

[0070] Detection and determination of plasma exosome hsa_circ_0006853

[0071] (1) Preparation of cDNA

[0072] 20μl reverse transcription system: 10X RT Mix 2μl, Random hexamers (50ng / nl) 1μl, HiScriptEnzyme Mix 2μl, Total RNA≤1ug and balance of RNase free ddH 2 0.

[0073] Reverse transcription program: 37 °C for 15 min, 85 °C for 5 s, and stored at 4 °C.

[0074] The reaction product can be placed at 4°C for immediate use in qPCR reaction, or at -20°C for half a year, or stored at -80°C to avoid repeated freezing and thawing.

[0075] cDNA can be expressed as RNase free ddH based on the expression level of the gene in the sample. 2 O appropriately diluted or used directly in the qPCR reaction system.

[0076] (2) Real-time fluorescence quantitative PCR

[0077] 20 μl qPCR reaction system: 10 μl of 2XAceQ qPCR SYBR Green MasterMix, 0.4 μl of Forward Primer (10 μM), 0.4 μl of Reverse Primer (10 μM), 2 μl of cDNA and balance of RNase free ddH 2 O.

[0078] Perform real-...

Embodiment 3

[0083] The value of plasma exosome hsa_circ_0006853 as a diagnostic marker in gastric cancer

[0084](1) The present invention includes 59 cases of gastric cancer patients who went to Zhenjiang First People's Hospital and Jiangsu University Affiliated Hospital (Zhenjiang Jiangbin Hospital) and Aoyang Hospital Affiliated to Jiangsu University Gastrointestinal Surgery from July 2021 to January 2022 , to collect basic data such as age, gender, tumor size, location, degree of differentiation, lymph node metastasis, distant metastasis, nerve involvement, TNM stage, histological type, CEA, CA199, CA242, etc. Preoperative and postoperative cubital veins were collected and analyzed. Plasma exosomal hsa_circ_0006853 levels were detected. At the same time, 65 healthy people and 44 atrophic gastritis patients were included in the same period, matched by age and sex. The three groups of samples were collected, sampled, packaged, and stored in the same conditions at the same time, and the...

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Abstract

The invention provides a plasma exosome circRNA marker for diagnosis and prognosis monitoring of gastric cancer and application of the plasma exosome circRNA marker, and belongs to the technical field of molecular diagnosis. According to the invention, the hsacirc0006853 is found in the gastric cancer plasma exosome for the first time, and the diagnostic value of the hsacirc0006853 on the gastric cancer is determined. The invention further provides an extraction kit of the plasma exosome circRNA, the exosome circRNA can be conveniently and accurately extracted and detected from plasma, and analysis and application of the circRNA in the plasma exosome in a laboratory and clinically are facilitated. Experiments prove that the marker disclosed by the invention can be used for effectively diagnosing and prognostically monitoring gastric cancer, and can be used for identifying atrophic gastritis patients to a certain extent, so that an important basis is provided for clinical early diagnosis and treatment targets.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a plasma exosome circRNA marker used for the diagnosis and prognosis monitoring of gastric cancer and its application. Background technique [0002] Gastric cancer (GC) is the most common malignant tumor in the world. The occurrence and development of gastric cancer is a multifactorial, multicellular, and multistep process. Due to the lack of specific symptoms in the early stage of gastric cancer, the vast majority of confirmed cases are diagnosed. At advanced stage, the prognosis is poor, with extensive metastasis and high recurrence. And the existing treatment methods such as chemotherapy and radiotherapy not only have strong side effects but also are prone to drug resistance. Therefore, it is an important problem to be solved urgently to further explore non-invasive related molecular markers that are helpful for the early diagnosis and prognosis of gas...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/178C12Q2600/158
Inventor 许文荣王冬丽钱晖李荣张帆邱双洋黄晓娟
Owner JIANGSU UNIV
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