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A kind of circRNA inhibits the application in the field of angiogenesis

An angiogenesis and drug technology, applied in the fields of biochemistry, molecular biology and medicine, which can solve the problem of unclear circRNA

Active Publication Date: 2020-03-24
易舟(上海)生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large number of studies have shown that it can influence tumor and angiogenesis by acting on downstream miRNAs to affect mRNA expression (Li, C.-Y., Biomedicine & Pharmacotherapy, 2017 95, 1514–1519.), but the specific role of circRNA is still unclear

Method used

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  • A kind of circRNA inhibits the application in the field of angiogenesis
  • A kind of circRNA inhibits the application in the field of angiogenesis
  • A kind of circRNA inhibits the application in the field of angiogenesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Table 1 Primer Sequence

[0025]

[0026] Circ-0007411 interference fragment and overexpression plasmid construction: circ-0007411 interference fragment design: The siRNA sequence shown in Table 1 was synthesized in Suzhou Gemma Company. Overexpression vector construction: Synthesize the complete linear sequence of circ-0007411, anneal the sequence into a double-stranded DNA fragment, and insert it into the LV-Circ vector through multiple cloning sites. The recombinant plasmid is identified by sequencing, and the Control negative control is the LV without the inserted sequence -Circ empty vector. Take 10 μl of transfection reagent Lipofectamine2000 and mix with 100 μl of serum-free and antibiotic-free opti-MEMI medium to form a solution. Mix 10 μl of diluted siRNA with 100 μl of serum-free and antibiotic-free opti-MEMI medium to prepare a solution. After the above two solutions were placed at room temperature for 5 minutes, they were thoroughly mixed, and after sta...

Embodiment 2

[0031] Make a well-shaped mark on the bottom of a 35mm culture dish with a marker pen in advance, and inoculate and culture human retinal microvascular endothelial cells. When the cell density is close to 100%, use a 10μl pipette tip to mark the scratch according to the well-shaped mark. After washing with PBS, replace the medium, and at 0h , 24h to take pictures, and use ImageJ to measure the edge distance change. The results of the cell scratch test showed that when the cell scratch spacing in each group was approximately equal at the initial time, after 24 hours, figure 1 It shows that the wound healing distance of 7411OE group is (134.4±45.4) μm, and the scratch healing distance of 7411-2 group is (330.0±23.1) μm. The healing speed of the 7411OE group was significantly lower than that of the other groups, and the difference was statistically significant (t=8.25, P<0.01). The healing speed of the 7411-2 group was significantly higher than that of the other groups, and the ...

Embodiment 3

[0033] After each group of Example 1 was transfected according to the conditions, 600 μl of DMEM medium was added to each well of a 24-well plate, and at the same time, it was placed in a transwell chamber with a pore size of 8.0 μm, and 200 μl of 1×10 4 Culture of individual retinal microvascular endothelial cells in FBS-free medium. After 48 hours, discard the medium in the upper and lower chambers, wipe the cells in the upper chamber with a cotton swab, add 600 μl of 0.4 g / L paraformaldehyde to the 24-well plate, fix at room temperature for 30 minutes, discard the paraformaldehyde, wash the back of the chamber twice with PBS, and use a 24-well plate Add 600 μl of crystal violet, stain at room temperature for 20 minutes, wash the back of the chamber twice with PBS after aspirating, observe and take pictures under a microscope, and randomly select 5 fields of view for cell counting.

[0034] Such as figure 2 As shown, the number of outward migration of human retinal microva...

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Abstract

The invention discloses application of circRNA in the field of angiogenesis inhibition, and belongs to the fields of biochemistry, molecular biology and medicine. The invention discovers that hsa_circ_0007411 can continuously act on eye vascular endothelial cells, change the structure of angiogenesis and inhibit angiogenesis. By applying the hsa_circ_0007411 to the eye microvascular endothelial cells, 90 percent of angiogenesis can be effectively inhibited, and angiogenesis of neovascularization-related diseases can be effectively inhibited. The effect of hsa_circ_0007411 on cell proliferationand migration is verified, thereby proving that hsa_circ_0007411 can remarkably inhibit cell proliferation and migration, has a remarkable inhibition effect on the proliferation of endothelial cells,and has important guiding significance for diseases related to the proliferation of vascular endothelial cells.

Description

[0001] The invention relates to an application in the field of inhibiting angiogenesis by circRNA, which belongs to the fields of biochemistry, molecular biology and medicine. Background technique [0002] Angiogenesis refers to the formation of new blood vessels from the development of existing capillaries or postcapillary veins, mainly including: degradation of vascular basement membrane in the activation phase; activation, proliferation and migration of vascular endothelial cells; reconstruction of new blood vessels And the vascular network, is a complex process involving many kinds of molecules from many kinds of cells. Angiogenesis is a complex process of coordinated action of pro-angiogenic factors and inhibitory factors. Under normal circumstances, the two are in a state of balance. Once this balance is broken, the vascular system will be activated to cause excessive angiogenesis or inhibit the vascular system to degenerate blood vessels. The mechanism of angiogenesis i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/7105A61K48/00A61P9/14A61P35/00A61P27/02A61P25/00A61P9/10
CPCA61K31/7105A61P9/10A61P9/14A61P25/00A61P27/02A61P35/00
Inventor 肖振
Owner 易舟(上海)生物医药有限公司
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