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Lung adenocarcinoma exosome specific miRNAs, and target gene and applications thereof

A technology of lung adenocarcinoma and exosomes, applied in the field of lung adenocarcinoma diagnosis, to achieve the effect of blocking absorption, promoting malignant transformation, and reducing content

Active Publication Date: 2018-11-13
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At this stage, no mature miRNA molecular markers have been used in the clinical diagnosis of lung adenocarcinoma technology and products

Method used

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  • Lung adenocarcinoma exosome specific miRNAs, and target gene and applications thereof
  • Lung adenocarcinoma exosome specific miRNAs, and target gene and applications thereof
  • Lung adenocarcinoma exosome specific miRNAs, and target gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Screening of lung adenocarcinoma exosome-specific miRNA

[0050] 1. Extraction and identification of exosomes

[0051] The culture supernatant of lung adenocarcinoma cell line A549 was collected. Centrifuge at 3000g for 15 minutes at 4°C, collect the supernatant and place it in a new EP tube. Centrifuge at 16000g for 1 hour at 4°C, collect the supernatant and put it in a new EP tube. Centrifuge at 120,000g for 2 hours at 4°C and discard the supernatant. Resuspend the exosome pellet in PBS.

[0052] Take 10 μl of exosome suspension, drop it on the carbon-containing copper grid, add 1% uranyl acetate after drying, let it stand for 5 minutes, rinse with PBS for 2 minutes, absorb excess liquid with filter paper, wash it three times and then dry it. The morphology of exosomes was observed under a transmission electron microscope with a voltage of 80kV, and the results were as follows: figure 1 Figure A shows.

[0053] The particle size distribution of exosom...

Embodiment 2

[0060]Example 2 Exosomes containing specific miRNA promote transformation of normal fibroblasts into tumor-associated fibroblasts (CAF)

[0061] The present invention found that exosomes containing the three specific miRNAs screened in the examples can be absorbed by normal fibroblasts and promote the transformation of normal fibroblasts into tumor-associated fibroblasts (CAFs), showing morphological changes of CAFs, markers High expression of sex molecules and enhanced migration ability.

[0062] In this example, exosomes secreted by A549 cells (including hsa-miR-22-3p, hsa-miR-21-5p, hsa-miR-21-3p) were labeled with green fluorescent protein, and these exosomes were compared with human lung After the fibroblasts were incubated with MRC5, exosomes were found to enter the interior of the fibroblasts ( Figure 4 Figure A), showing that these exosomes can be absorbed by fibroblasts; the cell morphology of fibroblasts that absorbed exosomes changed from the original scattered di...

Embodiment 3

[0064] Example 3 Exosome-specific miRNA of the present invention promotes the key gene FOXN3 of malignant transformation of normal fibroblasts

[0065] 1. The present invention found that after normal fibroblasts absorbed the exosomes secreted by tumor cells containing the specific miRNA of the present invention, the expression of the gene FOXN3 decreased, and FOXN3 was the target molecule of this group of specific miRNA.

[0066] The exosomes secreted by lung adenocarcinoma obtained in Example 1 were used to incubate fibroblast MRC5, and the expression of FOXN3 was detected by qPCR method. It was found that after absorbing exosomes, the expression of FOXN3 in MRC5 was significantly reduced ( Figure 6A ); the dual fluorescent reporter system is an effective detection system for determining the target gene of miRNA, and the 3 specific miRNAs screened according to Example 1 may be designed and synthesized in the 3' UTR binding region of the mRNA of FOXN3, and connected into a fl...

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Abstract

The invention provides lung adenocarcinoma exosome specific miRNAs, and a target gene and applications thereof. High flux sequencing technology is combined with systematic bioinformatics analysis methods, the specific high-content exosome miRNAs related with lung adenocarcinoma is obtained through screening; the RNA sequences of the lung adenocarcinoma exosome specific miRNAs are represented by SEQ ID No.1-3. It is found that the common target gene of the miRNAs is FOXN3. The target gene is capable of inhibiting fibroblast migration capability, reduction of the expression amount of the targetgene is capable of promoting entering of fibroblast into tumor tissues, and promoting tumor growth. The lung adenocarcinoma exosome specific miRNAs and the target gene thereof can be taken as lung adenocarcinoma diagnosis markers and can be used for preparation of diagnostic kits. An inhibitor of the miRNAs or an expression promoter of the target gene can be used for preparing medicines used for treating lung adenocarcinoma, or medication guidance in treatment of lung adenocarcinoma; and the clinical application value is relatively high, and the application prospect is promising.

Description

technical field [0001] The invention relates to the technical field of lung adenocarcinoma diagnosis, in particular to the application of lung adenocarcinoma exosome-specific miRNA and its target gene in the diagnosis of lung adenocarcinoma. Background technique [0002] The mortality rate of lung cancer ranks first in the cancer mortality rate of residents in my country. Lung adenocarcinoma is a type of lung cancer. The age of onset is relatively low, and women and non-smokers are relatively more common. Because there are no obvious clinical symptoms in the early stage, and hematogenous metastasis is prone to occur , leading to a late diagnosis. And because the genetic characteristics of tumor cells in the advanced stage of the disease are not yet clear, resulting in a single treatment method, limited to surgery, radiotherapy and chemotherapy, and targeted drugs only target specific types of tumor cells such as EGFR-positive cells, and are prone to drug resistance. lead to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/6886A61K45/00A61P35/00
CPCA61K45/00A61P35/00C12N15/113C12N2310/141C12Q1/6886C12Q2600/106C12Q2600/158C12Q2600/178
Inventor 米双利张健李蒙李阳阳李倩
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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