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Applications of mechanical sensitive miR-138-5p as drug target in skeleton muscle system diseases

A muscle system and drug technology, applied in the fields of molecular biology and medicine, to achieve the effect of enhancing differentiation ability

Inactive Publication Date: 2015-12-23
NORTHWESTERN POLYTECHNICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far there is no report on the effect of miR-138 on osteoblast differentiation and bone formation under the mechanical stimulation condition of unloading.

Method used

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  • Applications of mechanical sensitive miR-138-5p as drug target in skeleton muscle system diseases
  • Applications of mechanical sensitive miR-138-5p as drug target in skeleton muscle system diseases
  • Applications of mechanical sensitive miR-138-5p as drug target in skeleton muscle system diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: R screening verifies miRNAs that are significantly altered under mouse hindlimb unloading conditions. The specific steps are:

[0030] (1) Construction of hindlimb unloading mouse model: Balb / c mice were selected and hung on the ring of the tail suspension device with a paper clip, and the hindlimb unloading was carried out by tail suspension of the mouse spine at an angle of 30° to the ground. During the experiment, the animals had free access to food and water, and the mice were sacrificed 28 days later. The subsequent femur was scanned by MicroCT, and the parameters related to bone formation were analyzed and tested by MicroviewV2.1.2.

[0031] (2) Extraction of load-bearing bone RNA: Take the femur and tibia samples of Balb / c experimental mice, peel off the clean muscle, and grind the bone tissue with liquid nitrogen. After the grinding is sufficient, transfer it to a 1.5ml EP tube and add 1ml TRIzol (Invitrogen Company) , and then shaker to mix. Centr...

Embodiment 2

[0040] Example 2: Realtime PCR and Western blot were used to detect the effects of three-dimensional rotation and hindlimb unloading conditions on the expression of MACF1 in MC3T3-E1 preosteoblasts and mouse bone tissue. Specific steps are as follows:

[0041] (1) RNA extraction and realtime PCR detection: After MC3T3-E1 pre-osteoblast three-dimensional rotation for 48 hours, the cells were lysed with Trizol, and the total RNA of each group of cells was extracted by the chloroform-isopropanol method. For the bone tissue RNA extraction method, see above. Extraction method of bone RNA. The extracted mRNA was reverse transcribed into cDNA using TAKARA kit (DRR037A). The reverse transcription conditions are: 37°C for 15 minutes; 85°C for 15s.

[0042] The cDNA of each group was used as a template, GAPDH was used as an internal reference, and Realtime PCR was used to detect the gene expression of MACF1. The primer sequences of MACF1 and GAPDH used are as follows:

[0043] Table...

Embodiment 3

[0047] Embodiment 3: utilize TargetScan database ( http: / / www.targetscan.org / ) predicted miR-138 and MACF1 The targeting effect of , the species is mouse, the gene is MACF1, and miR-138 is the target miRNA.

[0048] Figure 4 showed that antagomir-138-5p treatment inhibited the expression of miR-138-5p ( Figure 4 A), promoting the expression of osteogenic differentiation-related genes ALP and ColIα1 ( Figure 4 B), increase ALP activity ( Figure 4 C), and significantly promote the formation of mineralized nodules ( Figure 4 D).

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Abstract

The present invention discloses applications of mechanical sensitive miR-138-5p as a drug target in skeleton muscle system diseases. The technical scheme comprises that the microtubule microfilament cross-linking factor 1 involving microRNA occurring in osteoporosis is regulated, and the antisense nucleotide antagomir-138-5p of the miR-138-5p is used for treating osteoporosis or other skeleton muscle system diseases, wherein the antagomir-138-5p reverses the osteoblast bone formation inhibition caused by the mechanical unloading condition at the cell and animal levels, the antagomir-138-5p promotes osteoblast differentiation and bone formation through the targeting condition MACF1, the miR-138-5p of MACF1 is regulated to become the therapeutic target of the osteoporosis, and the antisense nucleotide antagomir-138-5p of the miR-138-5puse is used as the osteoporosis treatment drug.

Description

technical field [0001] The invention belongs to the fields of molecular biology and medicine, and in particular relates to the application of a mechanically sensitive miR-138-5p as a drug target in diseases of the musculoskeletal system. Background technique [0002] MicroRNAs (miRNAs) are a group of evolutionarily conserved single-stranded RNA molecules with a short chain length (about 22 nucleotides), which can inhibit the translation of target genes or degrade target genes by binding to the 3' untranslated region of target genes. Gene. So far, 1424 microRNAs have been identified in human cells, and each microRNA has a regulatory effect on a large number of target genes. About 50% of human protein-coding genes are regulated by microRNAs. MicroRNA participates in complex biological processes, such as early development, cell proliferation, apoptosis, cell death, fat metabolism, etc., regulates many target gene organs, and is widely distributed in organisms. Osteoblasts, as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K31/7088A61P19/10A61P19/08
Inventor 骞爱荣陈志浩亚瑟·阿尔法特印崇胡丽芳赵帆李迪杰张琰李润芝苏佩红马小莉仇伍霞
Owner NORTHWESTERN POLYTECHNICAL UNIV
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