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miRNA applied method for inhibiting migration of endothelial cell

An endothelial cell, mir-155 technology, applied in the field of applying miRNA to inhibit the migration of endothelial cells in inflammatory state, can solve the problem that the pathogenesis of atherosclerosis is not fully understood, and achieve the effect of preventing cardiovascular disease, low cost, and inhibiting migration.

Inactive Publication Date: 2012-11-07
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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Problems solved by technology

So far, the pathogenesis of atherosclerosis is still not fully understood

Method used

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  • miRNA applied method for inhibiting migration of endothelial cell
  • miRNA applied method for inhibiting migration of endothelial cell
  • miRNA applied method for inhibiting migration of endothelial cell

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Embodiment 1

[0021] 1. Screening method of miR-155 target gene

[0022] Using TargetScan, PicTar and other software to search for possible target genes of miR-155, it was found that the 3'untranslated region (3'UTR) of the ETS-1 gene contained two miR-155 target sites (attached figure 1 ). ETS-1 related to endothelial cell inflammation was selected as a candidate gene.

[0023] 2. Reporter gene validation of ETS-1 as a target gene of miR-155

[0024] Construct pGL3-ETS1-3'UTR recombinant plasmid, co-transfect tool cell 293T with miR-155 mimic, freeze and thaw cells once 24 hours after transfection, lyse cells with special lysate, 100 μl per well, pipette evenly, and fully lyse Samples are available for use. Using Promega’s luciferase detection kit, add 50 μl of LARII and 5 μl of lysate into a 1.5ml centrifuge tube and mix well, then detect firefly luciferase with a microplate reader; add 50 μl of Stop&Glo solution to inactivate firefly luciferase, Renilla luciferase was detected with...

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Abstract

The present invention relates to an miRNA applied method for inhibiting migration of endothelial cells. The method comprises steps of: (1) screening an miR-155 target gene and validating by a reporter gene, and using a recombinant angiotensin II to stimulate endothelial cells for simulation of inflammatory response of the endothelial cells in an atherosclerosis state; (2) using an miR-155 simulant to increase expression of the miRNA in the inflammatory endothelial cells; (3) detecting inhibition of the miR-155 simulant on expression of an angiotensin II induced ETS-1 protein by using a Western blot method; (4) detecting migration of the inflammatory endothelial cells by using a single-layer endothelial cell scratching method. The method provided by the invention has advantages of simple process and low cost, employs the miR-155 to effectively inhibit migration activity of the inflammatory endothelial cells, and at the same time provides effective intervention targets for prevention of coronary heart disease.

Description

technical field [0001] The invention belongs to the application field of miRNA, and in particular relates to a method for using miRNA to inhibit migration of endothelial cells in an inflammatory state. Background technique [0002] Cardiovascular disease is the number one killer of human beings, and its high morbidity and mortality have always attracted worldwide attention. Therefore, actively exploring the pathogenesis of atherosclerosis will play an important role in the treatment and prognosis of coronary heart disease. So far, the pathogenesis of atherosclerosis is still not fully understood. For a long time, the theory of inflammatory response of endothelial cells has been generally accepted by the scientific community. However, in recent years, studies have found that the migratory activity of endothelial cells is greatly enhanced when they are in a state of inflammatory response, which directly leads to positive remodeling of atherosclerotic blood vessels and promot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N15/113C12Q1/02G01N33/68
Inventor 朱霓秦永文赵仙先袁文俊
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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