Application of miR181s in preparing drug for treating acute enteritis

A technology of mir181s and 1.mir181s, applied in the field of biomedical materials, can solve the problem that the understanding of the mechanism of miRNA inhibiting translation is not completely clear, and achieve the effects of low immunogenicity, simple production and synthesis process, and low cost.

Inactive Publication Date: 2014-03-12
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mechanisms by which miRNAs inhibit translation are not fully understood in contrast to the mechanisms by which mRNA is cleaved

Method used

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  • Application of miR181s in preparing drug for treating acute enteritis
  • Application of miR181s in preparing drug for treating acute enteritis
  • Application of miR181s in preparing drug for treating acute enteritis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: miR181 binds to TNF-α3'-UTR and promotes the degradation of TNF-α mRNA

[0034] 1.1 Experimental method

[0035] 1.1.1 Construction of TNF-α3'-UTR full-length and its point mutation reporter plasmid

[0036] The full-length 3'-UTR target fragment of TNF-α was amplified from cDNA by PCR method, and the full-length fragment was used as a template to design primers to amplify the 3'-UTR fragment of miR-181 binding site mutation.

[0037] PCR amplification primers are as follows

[0038]

[0039] 1.1.2 Co-transfection of cells with plasmid and miRNA fragments and detection of reporter genes

[0040] The PCR products of T789 and T789 mutation obtained above were constructed into pGL3-basic plasmid (purchased by Invitrogen) by molecular cloning method. A549 cells were inoculated in a 24-well plate, and when the cell confluency reached 60%, each well was transfected with the above-mentioned 0.5 μg T789 or T789 mutant luciferase reporter plasmid, 0.05 μg pRL pl...

Embodiment 2

[0054] Example 2: In vivo distribution of cholesterol-modified miR181d

[0055] 2.1 Experimental method

[0056] 2.1.1 Modified miR‐181d (agomir‐181d)

[0057] miRNA is modified by cholesterol, thio, and 2-methoxy (called agomir, mainly used in animal experiments), the stability in vivo is increased, and it can be well absorbed by tissue cells without transfection reagents. In order to facilitate the study of the absorption and distribution of agomir-181d in mice, we introduced Cy5 fluorescent modification on the synthetic fragment ( Figure 6 ).

[0058] 2.1.2 Reverse transcription of miRNA (Stem‐loop method)

[0059] (1) Dilute the RNA to 5.5ng / μl with RNase free water.

[0060] (2) The reaction system is as follows:

[0061]

[0062] The reaction steps are as follows: 16°C for 10 minutes, 42°C for 40 minutes, 85°C for 2 minutes, 4°C forever

[0063] 2.2.3 qPCR of miRNA (Stem‐loop method)

[0064] (1) Dilute the cDNA template 20-100 times with DDW.

[0065] (2) Th...

Embodiment 3

[0078] Example 3: Anti-acute inflammatory bowel disease of cholesterol-modified miR181d

[0079] 3.1 Experimental method:

[0080]3.1.1 Modeling of TNBS-induced colitis in mice

[0081] BALB / c female mice aged 6-8 weeks were selected for the experiment, and the mice were anesthetized by intraperitoneal injection of sodium pentobarbital at a weight of 0.01ml / g. Draw out 100 μl of 2.5% TNBS with a syringe connected to an enema needle, carefully insert the needle into the anus of the mouse to a depth of 3‐4 cm, avoid hurting the intestinal wall during the process, and slowly push in the liquid medicine. The control group was given the same volume of 50% alcohol solution, and the needle was pulled out slowly after the enema was completed. In order to ensure that the sensitizing solution can fully interact with the intestinal tract, the mouse was fixed head down on a Stand on an inclined plane for 30 minutes. Keep the body temperature of the mouse, and put the mouse back into ...

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Abstract

The invention discloses application of microRNA-181s (miR-181s for short) in preparing a drug for treating acute enteritis. Specially, the effect of miR-181d is obvious. A fact that the small ribonucleic acid drug of miR-181d, which is modified with cholesterol, is concentratedly distributed in a small intestine is discovered through intravenous administration. The mechanism of action lies in that the small ribonucleic acid drug combines with complementary sequences in the TNF[alpha]3'-UTR area of a tumor necrosis factor to promote the degradation of TNF-[alpha]mRNA, so as to inhibit an acute enteritis reaction induced by TNBS. The small nucleic acid drug is obvious in effect, can be well concentrated in small intestine tissue, and enters cells to take effect. The small ribonucleic acid drug can also act on 3'-UTR of various inflammatory factors containing miR181d binding sequences to inhibit the expression of the inflammatory factors. Therefore, the small nucleic acid drug has high application value and good prospect for treating inflammatory bowel diseases.

Description

technical field [0001] The invention belongs to the technical field of biomedical materials, and relates to the use of a small molecular RNA, namely miR-181d, in the preparation of therapeutic drugs for acute enteritis. Background technique [0002] Inflammatory bowel disease (IBD) refers to a group of chronic intestinal inflammatory diseases, including ulcerative colitis (UC) and Crohn's disease (CD). Its etiology and pathogenesis have not been fully clarified, and it is known that the inflammatory response caused by the abnormal response of the intestinal mucosal immune system plays an important role in the pathogenesis of IBD. At present, it is believed that it is caused by the interaction of multiple factors, mainly including environmental changes, genetic polymorphisms, intestinal flora imbalance, initial and adaptive immune responses. Environmental factors such as smoking, diet, sanitary conditions, etc.; nearly 70 CD-related genes and 50 UC-related genes have been di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P1/00A61P29/00
Inventor 殷武曹丹
Owner NANJING UNIV
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