Serum exosome miRNA biological marker and kit for early diagnosis of gastric cancer

A technology of biomarkers and exosomes, applied in the field of medicine, achieves good specificity and sensitivity, and good clinical application value

Inactive Publication Date: 2017-05-24
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, circulating miRNAs as diagnostic markers have certain limitations because they are easily affected by miRNAs released from damaged cells.

Method used

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  • Serum exosome miRNA biological marker and kit for early diagnosis of gastric cancer
  • Serum exosome miRNA biological marker and kit for early diagnosis of gastric cancer
  • Serum exosome miRNA biological marker and kit for early diagnosis of gastric cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The collection of embodiment 1 serum sample

[0033]A total of 310 serum samples were collected, including 150 histopathologically confirmed gastric cancer patients and 160 healthy controls. All serum samples were obtained from the First Affiliated Hospital of the Third Military Medical University of the Chinese People's Liberation Army, and written informed consent was signed with the participating patients and approved by the hospital ethics committee. Hemolytic, lipemic, and other abnormal sera were excluded.

[0034] The final study included 204 serum exosome samples, including 100 patients with gastric cancer and 104 normal controls.

[0035] The relationship between the expression of miR-19b and miR-106a and the clinical characteristics of gastric cancer patients is shown in Table 1. The expression of candidate miRNAs was correlated with age (miR-19b, p=0.598; miR-106a, p=0.111), gender (miR-19b, p=0.114; miR-106a, p=0.074), lymphatic metastasis (miR-19b, p=0.6...

Embodiment 2

[0038] Example 2 Extraction of exosome miRNA

[0039] The blood was collected in a serum coagulation tube, then centrifuged at 400g for 15 minutes, and the serum was collected and stored at -80°C for later use. Serum exosomes were extracted according to the serum exosome extraction reagent instructions (SBI, EXOQ20A-1). The isolated exosomes were then resuspended with 200ul PBS, and the morphology of the exosomes was detected by transmission electron microscopy. Serum exosomal miRNA was extracted according to the mirVana PARIS Kit (Ambion, #AM1556) reagent instructions. Because there is no recognized endogenous miRNA in serum as an internal reference, an additional 50 nM cel-miR-39 (Ribo) was used as a reference. by Qubit TM 3.0 Fluorometer to detect the concentration of the extracted RNA. At the same time, serum exosomes were lysed with cell lysis buffer, centrifuged at 12000rpm for 15min, the supernatant was collected, and the protein concentration was determined by BCA ...

Embodiment 3

[0041] Example 3 Preliminary screening experiment for detection of exosomal miRNA by RT-qPCR

[0042] Detection of each miRNA in serum exosomes using hsa-miR-19b-3p, hsa-miR-106a-5p, hsa-miR-30a-3p, hsa-miR-17-5p and cel-miR-39 specific probes expression.

[0043] According to the instructions of the reverse transcription reagent (ABI, #4427975), add 16.7ng (1.67μL) of total RNA to the 5μL reaction reverse transcription reaction system for each sample. Then according to Premix Ex Tq TM (Takara, #RR390B) and each miRNA-specific probe reagent instruction manual for RT-qPCR reactions. Mann-Whitney t test method was used to analyze the experimental data, and a P value less than 0.05 and a multiple of change greater than 1.5 times were considered statistically significant.

[0044] We compared the expression levels of these four miRNAs in the serum exosomes of 20 gastric cancer patients and 20 normal controls, and found that hsa-miR-19b-3p and hsa-miR-106a-5p were significantly ...

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Abstract

The invention relates to a serum exosome miRNA biological marker and a kit for early diagnosis of gastric cancer. The serum exosome miRNA biological marker consists of miR-19b and miR-106a. The serum exosome miRNA biological marker uses serum exosomes miRNA, especially two miRNA (hsa-MiR-19b-3p united hsa-miR-106a-5p) closely relevant with a gastric cancer serve as gastric-cancer early diagnosis markers, and the marker is superior to clinic means such as an existing gastroscope in early diagnosis and has better specificity and sensitivity. The kit consists of a specific amplification primer and a general PCR amplification reagent, has a good clinical application value in early diagnosis of the gastric cancer and provides a new thinking and method for improvement of the early diagnosis level of the gastric cancer.

Description

technical field [0001] The invention belongs to the technical field of medicine, in particular to a serum exosome miRNA biomarker and a kit for early diagnosis of gastric cancer. Background technique [0002] With increasing morbidity and mortality, cancer has become a major cause of harm to public health worldwide. According to the latest statistics, gastric cancer is still the second leading cause of cancer death in China. Although significantly improved surgical techniques combined with radiotherapy and chemotherapy can prolong the survival of patients with early gastric cancer, the prognosis of patients with advanced gastric cancer is still not optimistic. Therefore, there is an urgent need for more reliable and convenient early diagnosis methods for gastric cancer. At present, gastroscopy is still the gold standard for the standard diagnosis of gastric cancer. Due to the trauma and limitations of this method, it is not suitable for every patient. In addition, althoug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/158C12Q2600/178
Inventor 王宁郑新川汪莉娜刘鑫
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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