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Application of miR-216a and target gene thereof in vascular senescence and atherosclerotic heart disease

A technology of mir-216a, 1.mir-216a, which is applied in the fields of clinical medicine, biology, and laboratory medicine to achieve the effects of inhibiting inflammation, promoting inflammatory response, and improving endothelial function

Inactive Publication Date: 2018-11-06
FUWAI HOSPITAL CHINESE ACAD OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The role of MiR-216a in vascular endothelial cell senescence and endothelial inflammatory response has not been reported, and whether miR-216a can be used as a serological marker for atherosclerotic plaque stability and coronary heart disease and other cardiovascular diseases remains to be elucidated

Method used

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  • Application of miR-216a and target gene thereof in vascular senescence and atherosclerotic heart disease
  • Application of miR-216a and target gene thereof in vascular senescence and atherosclerotic heart disease
  • Application of miR-216a and target gene thereof in vascular senescence and atherosclerotic heart disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Establishment of HUVECs aging model and detection of miR-216a expression during endothelial cell aging

[0062] 1. HUVECs (human umbilical vein endothelial cells) culture

[0063] After the umbilical cord is isolated, soak it in 4°C physiological saline and process it within 2 hours. The umbilical cord was removed, and the umbilical vein was perfused with PBS to wash twice. The umbilical cord was perfused with 0.1% Collagenase I solution (Cat#C0130-100MG, Sigma-Aldrich, SaintLouis, MO, USA), tied at both ends, placed in a beaker in a water bath at 37°C, and incubated for 20 minutes. Take a 50mL tube to recover the digestive solution, wash it once with PBS, shake it up and down to let the cells fall off, and then recover the PBS. Centrifuge at 1000g for 5 minutes, remove the supernatant, add ECM containing 5% FBS to resuspend the cells, and transfer to a T25 cell culture flask. After incubation at 37°C for 12 hours, the supernatant medium was removed, washed...

Embodiment 2

[0082] Example 2 Long-term stable overexpression of miR-216a induces endothelial cell senescence

[0083] In order to establish cell lines stably overexpressing miR-216a, PDL4HUVECs were infected with pre-miR-216a recombinant lentivirus (Ubi-EGFP-MCS-IRES-puromycin) and negative control (negative control, NC) virus vector (Genechem, Shanghai, China). After 3 days, 400 ng / mL puromycin (Cat. #8833, Sigma-Aldrich, Saint Louis, MO, USA) was administered for selection. When the infection efficiency reaches more than 95% (that is, the number of cells expressing green fluorescent protein exceeds 95%), the next step of the experiment is performed. Cell lines stably expressing miR-216 and its negative control were serially passaged and PDLs were calculated.

[0084] After 15 days of stable overexpression of miR-216a, PDL20HUVECs showed obvious senescence-related phenotypes such as hypertrophy and growth arrest as the number of passages increased ( figure 2A), it was found that miR-...

Embodiment 3

[0086] Example 3 Effects of miR-216a on endothelial adhesion, proliferation and migration

[0087] The present invention uses Lipofectamine 3000 (Cat.#L3000c015, Invitrogen, Carlsbad, CA, USA) transient transfection reagent to transfect miR-216a inhibitor and negative control (negative control, NC) (Cat.#B01001, GenePharma, Suzhou, China ), the final transfection concentration was 50nM.

[0088] 3.1 Monocyte-endothelial cell adhesion assay

[0089] The present invention uses the mononuclear-endothelial adhesion test to detect the adhesion ability of PDL8, PDL20 stably overexpressing miR-216a cell lines and naturally aging PDL44 cells transfected with miR-216a inhibitors. Endothelial cells were seeded in 24-well culture plates, and adhesion experiments were performed when the cell confluence reached 90%. Collect THP-1 cells and resuspend to 1×10 in RPMI-1640 medium 6 cells / mL, add 5 μL CellTracker to 1 mL THP-1 cells TM CM-Dil red fluorescent dye (Cat. #C7000, Invitrogen, ...

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Abstract

The invention discloses a biomarker miR-216a and target genes Smad3 and Smad7 relating to vascular endothelial senescence and inflammatory reaction, coronary atherosclerosis of the aged and atherosclerotic plaque stability, and verifies up-regulated expression of the miR-216a in a tissue or blood sample of a patient with atherosclerosis and high specificity expression of the miR-216a in people with unstable atherosclerotic plaque. The invention also discloses the application of the miR-216a and an inhibiting agent in preparation of products for evaluating individual atherosclerotic plaque stability and coronary heart disease occurring risk. The atherosclerotic plaque stability and development are evaluated and predicated by evaluation diagnosis in the early stage of coronary atherosclerosis of the aged through the miR-216a; the miR-216a is of important significance to early treatment and medical cost of coronary atherosclerosis and provides a treatment target and an important basis toclinical applications such as gene therapy and drug therapy.

Description

technical field [0001] The invention relates to the fields of laboratory medicine, clinical medicine and biotechnology, in particular to the application of miR-216a and its target gene in vascular aging and atherosclerotic heart disease. Background technique [0002] Atherosclerosis and its cardiovascular complications such as acute myocardial infarction, unstable angina and ischemic stroke seriously threaten human health. Aging is an independent risk factor for atherosclerosis. Endothelial inflammatory response and dysfunction caused by aging of vascular endothelial cells are the initial stages of atherosclerosis. In vivo studies further demonstrated that endothelial cells in human atherosclerotic plaques exhibit a marked senescence phenotype compared with normal vessels. However, the molecular mechanisms underlying endothelial cell aging and dysfunction are not fully understood. Genetic, epigenetic and environmental factors jointly affect vascular aging and inflammatory...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883A61K31/713A61P9/10A61P29/00
CPCA61P9/10A61P29/00A61K31/713C12Q1/6883C12Q2600/178C12Q2600/158
Inventor 张伟丽杨淑均陈宇米雪楠杨云云李健
Owner FUWAI HOSPITAL CHINESE ACAD OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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