Method for attacking miRNAs through free radicals to cause oxidative damage to RNAs

A technology of oxidative damage and free radicals, applied in the field of RNA oxidative damage, can solve the problem of limited oxidative modification of microRNA

Inactive Publication Date: 2015-09-30
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, many studies have shown that oxidatively modified messenger RNA molecules have a significant impact on various functions of cells, but the research on microRNA oxidative modification is still very limited.
At present, there is no report on effective oxidative modification of miRNA. In vitro or in vivo, the attack of ROS can lead to guanine-specific oxidative modification of microRNA, which plays an important role in the development of nucleic acid drugs.

Method used

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  • Method for attacking miRNAs through free radicals to cause oxidative damage to RNAs
  • Method for attacking miRNAs through free radicals to cause oxidative damage to RNAs
  • Method for attacking miRNAs through free radicals to cause oxidative damage to RNAs

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Fenton's reagent oxidation system causes miRNA oxidative damage and produces 8-oxoguanine

[0031] Cu in this example + / Fe 2+ with H 2 o 2 The Fenton’s reagent oxidation system has a very strong oxidation ability, and is the main reaction for organisms to produce OH (hydroxyl radicals); this reaction system is used to oxidize chemically synthesized double-stranded miRNA mimics samples (hsa-let-7a-1: 5'-UGAGGUAGUAGGUUGUAUAGUU-3'SEQ ID NO:12), namely miRNA (80μg) and 0.5mM ferric citrate (Fe 3+ ) or copper sulfate (Cu 2+ ), 5mM ascorbic acid (Asc) and 2mM H 2 o 2 , in 10mM NaH 2 PO 4 / Na 2 HPO 4 Buffer (pH 7.4) system, incubated at 37°C for 1h. After the incubation, 10 μl of 100 mM deferoxamine methanesulfonate (iron ion scavenger) or dicyclohexanone oxalyldihydrazone (copper ion scavenger) was added to terminate the reaction. Add 1 / 10 volume of 3M NaAc, 1 / 10 volume of 1 μg / ml glycogen and 2.5 volumes of ethanol to the above solution, and precipita...

Embodiment 2

[0033] Example 2: H 2 o 2 Oxidative damage of miRNA during apoptosis induced in myocardial H9c2 cells, producing 8-oxoguanine

[0034] This embodiment is to detect H 2 o 2 The degree of miRNA oxidative damage in the process of inducing myocardial H9c2 cell apoptosis (which can be obtained from American Type Culture Collection); 2 o 2 H9c2 cells were treated, and cells were collected after 3h for reactive oxygen species (Reactive oxygen species, ROS) detection (which can be obtained from Invitrogen), and flashPAGE TM The fractionator system (Ambion, Inc) was used to extract miRNA, and Northwestern Blot analysis was performed as described, and the results are shown in figure 2 a and figure 2 b.

[0035] The results of this example show that with 0mM H 2 o 2 Treatment groups were control, 25 μM, 50 μM, 100 μM and 200 μM H 2 o 2 The intracellular ROS levels of the treatment groups increased by 2.25±0.19, 3.43±0.31, 5.54±0.46, 6.72±0.57 (p2 o 2 The increase multiples ...

Embodiment 3

[0036] Example 3: Iron overload induces miRNA oxidative damage in a myocardial injury model, producing 8-oxoguanine

[0037] The iron overload (iron overload) in the cardiomyocytes of this embodiment can cause a significant increase in the intracellular ROS level, and cause damage to myocardial function and increased apoptosis of cardiomyocytes. Obtained from the Experimental Animal Center of the Institute of Zoology, Chinese Academy of Sciences) intraperitoneal injection of iron dextran to quickly establish an iron overload model, the dose is 12 mg / day, five days a week, and sacrificed after injection for four weeks; found that the iron overload ( In the Fe) group, blue iron particles were deposited in the cytoplasm, but in the placebo control (Plecebo) group, no iron particles were found in the cells, confirming the successful establishment of the iron overload model in mice ( image 3 a); At the same time, the levels of ROS and apoptosis in the Fe group were significantly h...

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Abstract

The invention relates to a method for attacking miRNAs through free radicals to cause oxidative damage to the RNAs. According to the method, 8-oxoguanine is generated through oxidation of guanine 8-bit carbon in an oxidative stress state so that the miRNAs undergo oxidative damage. A damage mode is selected from one or more of the following modes: (1) the oxidative damage is caused to the miRNAs through a Fenton reagent oxidation system; (2) the oxidative damage is caused to the miRNAs in the process of using H2O2 to process and induce apoptosis; (3) the oxidative damage is caused to the miRNAs in a Fe overloading induced myocardial injury animal model; and (4) the oxidative damage is caused to the miRNAs in a myocardial ischemia-reperfusion injury animal model. According to the method, the miRNA subtype variety of the miRNAs undergoing the oxidative damage in the process of using H2O2 to induce the myocardial apoptosis is determined, and functional differences are expressed through the generation and degradation of the oxidative damage miRNAs in myocardial cells, location characteristics, the synergistic effect with other biomacromolecule and oxidative damage miRNA regulatory genes. The method is simple in process and obvious in damage effect, the applied range is wide, and the method has application environment friendliness.

Description

Technical field: [0001] The invention relates to a method for causing RNA oxidative damage by free radicals attacking microRNA, and belongs to the technical field of biological applications. Background technique: [0002] At present, the molecular mechanism of apoptosis under oxidative stress is the core issue of the theory of free radicals and oxidative damage. Small non-coding nucleotide molecules—microRNA (microRNA, miRNA) is a newly discovered non-coding RNA with a length of about 22 nt (19-25 nt) that is conservative in evolution in recent years. The principle of complementary pairing acts on mRNA, thereby affecting the protein translation process and causing gene silencing, and its role is reflected in the regulation of gene expression at the post-transcriptional level. At present, more and more evidences show that miRNA plays a key role in various biological processes, and miRNA has become an important tool in life science research and a new method for the treatment ...

Claims

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Application Information

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IPC IPC(8): A61K33/34A61K31/327A61K31/721A61K33/26
Inventor 李培峰高洁丁素玲周露玙
Owner QINGDAO UNIV
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