CN110951734A - RNA inhibitor and application thereof - Google Patents
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- CN110951734A CN110951734A CN201911293361.9A CN201911293361A CN110951734A CN 110951734 A CN110951734 A CN 110951734A CN 201911293361 A CN201911293361 A CN 201911293361A CN 110951734 A CN110951734 A CN 110951734A
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Abstract
The invention provides an RNA inhibitor and application thereof, which is an miRNA-17-5p inhibitor and has an miRNA-17-5p inhibition sequence. The miRNA-17-5p inhibitor can be used for inhibiting the activation and proliferation of astrocytes after spinal cord injury. The invention applies that oligodendrocytes generate a large amount of exosomes carrying miR-17-5p after spinal cord injury, and the exosomes can be ingested by astrocytes, thereby promoting the activation characteristic of the astrocytes, designing an antagonist of miR-17-5p, realizing the purpose of relieving excessive proliferation of the astrocytes, and providing an effective treatment target point for the treatment of spinal cord injury.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to an RNA inhibitor and application thereof.
Background
Spinal Cord Injury (SCI) is a serious central nervous system injury disease involving millions of patients worldwide, whose quality of life and work is severely affected. According to statistics, only less than 1% of SCI patients can completely recover the nerve function, most of the patients need long-time hospitalization and rehabilitation, and full-time or part-time nursing personnel, so that the patients cannot normally work, heavy burden is brought to families and society, and the medicine treatment and the surgical operation treatment of the SCI at home and abroad do not obtain satisfactory clinical treatment effects. Therefore, it is of great importance to study the mechanism and treatment of SCI. Astrocytes are the most abundant and abundant cells in the Central Nervous System (CNS). Astrocytes can switch to an activated phenotype after SCI, called reactive astrocytes, characterized by increased cell soma, increased expression of intermediate silk proteins such as Glial Fibrillary Acidic Protein (GFAP) and Nestin (Nestin), reactive astrocytes essential in acute injury site healing and tissue remodeling processes, which eventually interact with other cell types such as fibroblasts or other Glial cells to form Glial scars. At the same time, reactive astrocytes secrete inhibitory extracellular matrix molecules, such as Chondroitin Sulfate Proteoglycans (CSPGs), inhibiting axon growth and regeneration. The formation of glial scars prevents axonal regeneration and functional recovery, and is believed to be the primary cause of poor regenerative capacity in the mammalian CNS. Research proves that miRNA plays a regulating role in the occurrence, growth and normal activities of spinal cord, and miRNA is shown to participate in the regulation roles of subsequent injury, nerve regeneration, neural plasticity and the like of SCI. Potential targets for post-SCI deregulated miRNAs include genes encoding genes involved in inflammation, oxidation and apoptosis, which play important roles in the pathogenesis of spinal cord injury, suggesting that aberrant expression of miRNAs may be associated with the pathogenesis of spinal cord injury. miR-17 is one of miR-17-92 cluster members, and besides the function of promoting the generation of tumors, gene chip results show that miR-17 is one of miRNA which is abnormally changed after SCI. The key miRNA influencing the activation of astrocytes after spinal cord injury is searched for and can be used as an effective target point for treating spinal cord injury, and the miRNA has important medical significance and profound social significance.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an RNA inhibitor and application thereof, and the application of the miRNA-17-5p inhibitor can relieve astrocyte activation and reproduction after spinal cord injury, and has important medical significance and profound social significance.
In order to solve the above technical problems, the embodiment of the present invention provides an RNA inhibitor, which is an miRNA-17-5p inhibitor and has an miRNA-17-5p inhibitory sequence.
Wherein, the miRNA-17-5p inhibitory sequence is as follows:
5’-3”CUACCUGCACUGUAAGCACUUUG。
the invention also provides application of the RNA inhibitor in inhibiting the activation and proliferation of astrocytes after spinal cord injury.
The invention also provides a method for alleviating astrocyte activation after spinal cord injury by using the RNA inhibitor, which comprises the following steps:
(1) firstly, giving a miRNA-17-5p inhibition sequence to prepare a miRNA-17-5p inhibitor;
(2) collecting exosomes secreted by oligodendrocytes;
(3) and co-incubating the collected exosomes and the damaged astrocytes to realize that the astrocytes take up the exosomes and reduce the over-activation of the damaged astrocytes.
The specific operation method of the step (3) comprises the following steps: subjecting astrocytes to a reaction at 2X 103One/well was inoculated in a 96-well plate previously coated with polylysine and placed at 37 ℃ in 5% CO2Culturing for 48h in an incubator, after the cells enter a logarithmic growth phase, sucking out a culture medium, adding antago-control-Exo exosomes and antagomiR-17-Exo exosomes, after incubating for 48h in each group of 6 multiple holes, sucking out the culture medium, adding 100ul of mixed solution into each hole, incubating for 4h in a constant temperature shaking table at 37 ℃, and measuring the light absorption value with the wavelength of 450nm by using a microplate reader.
Further, adding miR-17-5p inhibitor control into OLN-93 cells to extract exosomes from antago-control-Exo; the antagomiR-17-Exo is prepared by adding a miR-17-5p inhibitor into OLN-93 cells and then extracting exosomes.
Wherein the mixed solution is formed by mixing a base catalyst and a CCK-8 solution according to the volume ratio of 9: 1.
Wherein, the miRNA-17-5p inhibitory sequence in the step (1) is as follows:
5’-3”CUACCUGCACUGUAAGCACUUUG。
the technical scheme of the invention has the following beneficial effects: the invention proves that the miRNA-17-5p inhibitor can reduce the activation and reproduction of astrocytes after spinal cord injury, and has important medical significance and profound social significance.
Drawings
FIG. 1 is a schematic diagram of the uptake of exosomes secreted by oligodendrocytes by astrocytes in the present invention;
FIG. 2 is a graph showing the results of measuring the cell viability of astrocytes by CCK-8 according to the present invention;
FIG. 3 is a schematic diagram of the upregulation of miR-17-5p expression following spinal cord injury in the present invention;
FIG. 4 is a schematic diagram of cell localization of miR-17-5p after spinal cord injury detection by combining immunofluorescence and in situ hybridization techniques in the invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.
The overactivation of astrocytes after spinal cord injury is the main cause of the formation of glial scar, can inhibit axon growth and regeneration, and is an important factor for hindering the repair after spinal cord injury. Oligodendrocytes produce a large number of miR-17-5 p-carrying exosomes following spinal cord injury.
The relevant detection is carried out by a rat spinal cord impact injury model. Fast Blue detects the change of tissue morphology after spinal cord injury (figure 3A), qPCR detects the expression change of miR-17-5p after spinal cord injury, the result shows that the expression of miR-17-5p after injury is gradually up-regulated (figure 3B), in-situ hybridization detects the expression of miR-17-5p after injury, and the result shows that the expression of miR-17-5p after injury is gradually up-regulated (figure 3C).
FIG. 4 is a schematic diagram showing the cell localization of miR-17-5p after spinal cord injury detection by combining immunofluorescence with in situ hybridization. In fig. 4, SPL: detecting the level of spinal cord tissue after spinal cord injury; OL: and detecting miR-17-5p in the oligodendrocyte of the primary culture.
The exosome carrying the miR-17-5p can be taken up by astrocytes, so that the activation of the astrocytes is promoted. As shown in FIG. 1, which is a schematic diagram showing that exosomes secreted by oligodendrocytes can be taken up by astrocytes, exosomes secreted by CM-Dil-labeled OLN-93 were incubated with astrocytes at 37 ℃ for 24h, and analyzed by observation through a fluorescence microscope, and the green color is an astrocyte-specific marker GFAP, and the experimental group red-labeled OLN-93-Exo was found to be mainly present in cytoplasm. Bar = 50 μm in fig. 1.
Example 1
The invention provides an RNA inhibitor which is an miRNA-17-5p inhibitor and has an miRNA-17-5p inhibition sequence.
The miRNA-17-5p inhibition sequence is as follows:
5’-3”CUACCUGCACUGUAAGCACUUUG。
example 2
The invention provides an application of miRNA-17-5p inhibitor in inhibiting activation and reproduction of astrocytes after spinal cord injury.
A method of using a miRNA-17-5p inhibitor to reduce astrocyte activation following spinal cord injury, comprising the steps of:
(1) firstly, giving a miRNA-17-5p inhibition sequence to prepare a miRNA-17-5p inhibitor;
the miRNA-17-5p inhibition sequence is as follows:
5’-3”CUACCUGCACUGUAAGCACUUUG。
(2) collecting exosomes secreted by oligodendrocytes;
(3) co-incubating the collected exosomes and the damaged astrocytes to realize that the astrocytes take up the exosomes and reduce the over-activation of the damaged astrocytes; the specific operation method comprises the following steps: subjecting astrocytes to a reaction at 2X 103One/well was inoculated in a 96-well plate previously coated with polylysine and placed at 37 ℃ in 5% CO2Culturing for 48h in an incubator, after the cells enter logarithmic growth phase, sucking out a culture medium, adding antago-control-Exo exosomes (the antago-control-Exo is that the OLN-93 cells are added with miR-17-5p inhibitor for comparison and then exosomes are extracted) and antagomiR-17-Exo exosomes (the antagomiR-17-Exo is that the OLN-93 cells are added with miR-17-5p inhibitor for contrast and then exosomes are extracted), after incubating for 48h in each group of 6 multiple pores, sucking out the culture medium, adding 100ul mixed liquor (90 ul medium and 10ul CCK-8 solution) into each pore, incubating for 4h at constant temperature of 37 ℃ by using a shaker, measuring the absorbance value at the wavelength of 450nm by using a microplate reader, detecting the cell viability of astrocytes by using CCK-8, and obtaining the exosomes secreted by the damaged oligodendrocytes after miR-17-5p inhibitor pretreatment, acting on astrocytes can alleviate overactivation of injured astrocytes.
The invention applies that oligodendrocytes generate a large amount of exosomes carrying miR-17-5p after spinal cord injury, and the exosomes can be ingested by astrocytes, thereby promoting the activation characteristic of the astrocytes, designing an antagonist of miR-17-5p, realizing the purpose of relieving excessive proliferation of the astrocytes, and providing an effective treatment target point for the treatment of spinal cord injury.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> university of southeast Tong
<120> RNA inhibitor and application thereof
<141>2019-12-16
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>23
<212>RNA
<213>miRNA
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cuaccugcac uguaagcacu uug 23
Claims (8)
1. An RNA inhibitor, which is an miRNA-17-5p inhibitor and has an miRNA-17-5p inhibitory sequence.
2. The RNA inhibitor of claim 1, wherein the miRNA-17-5p inhibitory sequence is:
5’-3”CUACCUGCACUGUAAGCACUUUG。
3. use of an RNA inhibitor according to claim 1 or 2 for inhibiting the activation and proliferation of astrocytes following spinal cord injury.
4. A method of reducing astrocyte activation following spinal cord injury using an RNA inhibitor according to claim 1 or 2, comprising the steps of:
(1) firstly, giving a miRNA-17-5p inhibition sequence to prepare a miRNA-17-5p inhibitor;
(2) collecting exosomes secreted by oligodendrocytes;
(3) and co-incubating the collected exosomes and the damaged astrocytes to realize that the astrocytes take up the exosomes and reduce the over-activation of the damaged astrocytes.
5. The method according to claim 4, wherein the specific operation method of step (3) is as follows: subjecting astrocytes to a reaction at 2X 103One/well was inoculated in a 96-well plate previously coated with polylysine and placed at 37 ℃ in 5% CO2Culturing for 48h in an incubator, after the cells enter a logarithmic growth phase, sucking out a culture medium, adding antago-control-Exo exosomes and antagomiR-17-Exo exosomes, after incubating for 48h in each group of 6 multiple holes, sucking out the culture medium, adding 100ul of mixed solution into each hole, incubating for 4h in a constant temperature shaking table at 37 ℃, and measuring the light absorption value with the wavelength of 450nm by using a microplate reader.
6. The method according to claim 5, wherein antago-control-Exo is an exosome extracted from OLN-93 cells after miR-17-5p inhibitor control is added; the antagomiR-17-Exo is prepared by adding a miR-17-5p inhibitor into OLN-93 cells and then extracting exosomes.
7. The method of claim 5, wherein the mixture is prepared by mixing culture medium and CCK-8 solution at a volume ratio of 9: 1.
8. The method according to claim 4, wherein the miRNA-17-5p inhibitory sequence of step (1) is:
5’-3”CUACCUGCACUGUAAGCACUUUG。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113528642A (en) * | 2021-07-09 | 2021-10-22 | 四川大学 | New application of reagent for detecting CDR1as expression level |
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| CN106011173A (en) * | 2016-01-20 | 2016-10-12 | 北京希普生国际生物医学研究院 | Preparation method of human oligodendrocyte precursor cell inhibiting nerve secondary injury, kit and application thereof |
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| PEIWEI HONG ET AL.: "Functional Requirement of Dicer1 and miR-17-5p in Reactive Astrocyte Proliferation after Spinal Cord Injury in the Mouse", 《MIRNA FUNCTION IN REACTIVE ASTROGLIOSIS》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113528642A (en) * | 2021-07-09 | 2021-10-22 | 四川大学 | New application of reagent for detecting CDR1as expression level |
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