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Application of miR-21 and target gene thereof in preparation of reagents or drugs diagnosing and/or controlling osteoarthritis

Osteoarthritis, mir-21 technology, applied in anti-inflammatory agents, disease diagnosis, drug combination, etc., can solve the imbalance between chondrocyte synthesis and catabolism, chondrocyte apoptosis, and silencing of PI3K/Akt/mTOR signaling pathway Questions such as ambiguity

Inactive Publication Date: 2018-08-03
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the silencing of PI3K / Akt / mTOR signaling pathway can lead to excessive apoptosis of chondrocytes, and then cause the imbalance of chondrocyte synthesis and catabolism; however, what factors lead to silencing of PI3K / Akt / mTOR signaling pathway is still unclear

Method used

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  • Application of miR-21 and target gene thereof in preparation of reagents or drugs diagnosing and/or controlling osteoarthritis
  • Application of miR-21 and target gene thereof in preparation of reagents or drugs diagnosing and/or controlling osteoarthritis
  • Application of miR-21 and target gene thereof in preparation of reagents or drugs diagnosing and/or controlling osteoarthritis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Detection of miRNA expression profile in cartilage tissue of patients with osteoarthritis and normal control group

[0049] Experimental objects: case group (knee cartilage of patients with osteoarthritis) and control group (knee cartilage of patients with amputation).

[0050] 1. Total RNA extraction

[0051] After informed consent was obtained from the subjects, the joint fluid was extracted through articular cavity puncture, placed in sterile, enzyme-free cryopreservation tubes, and stored in liquid nitrogen.

[0052] The total RNA was extracted by the Trizol one-step method, and the specific operation was as follows: take 300 μL of joint fluid, add 4 times the volume of Trizol solution (TRIzol Reagent, Invitrogen, USA, Cat. No. 15596-018), then add 200 μL of chloroform, shake the centrifuge tube vigorously, and mix well. Place at room temperature for 5-10min; centrifuge at 12000rpm for 15min and transfer the upper aqueous phase to another new centrifuge ...

Embodiment 2

[0072] Example 2: Bioinformatics method predicts that PIK3C2A gene is identified as the target gene of miR-21

[0073] Apply bioinformatics methods to predict the target gene, by comparing the matching degree of miRNA and the 3'-UTR of the target gene, especially the 6-8 bp seed sequence at the 5' end of miRNA. The prediction of miRNA target molecules is mainly based on the principle of base pairing, and the target gene is predicted according to the complementarity between the 3'-UTR of the target molecule and the miRNA through the bioinformatics database. False-positive and false-negative results are unavoidable if any target molecule database is used alone, so four independent microRNA target molecule databases are used, namely miRanda (http: / / www.microrna.org / ), TargetScan (http: / / www.targetscan.org / ), PicTar (http: / / pictar.mdc-berlin.de / ) and PITA (http: / / genie.weizmann.ac.il / pubs / mir07 / mir07), predicted miR-21 The target gene is PIK3C2A.

Embodiment 3

[0074] Example 3: Construction of PIK3C2A wild-type and mutant expression vectors

[0075] In order to further verify the theoretical target gene, the expression vectors of PIK3C2A wild type (wild type) and mutation prediction sites (mutant type, MT) were constructed, and then the expression vectors were inserted into the luciferase reporter gene vector (pmiR- REPORT, Ambion, USA), psi-CHECK-PIK3C2A WT and psi-CHECK-PIK3C2A MT were determined by DNA sequencing. Lipofectamine 3000 was used to transfect PIK3C2A3'-UTR WT and MT recombinant vectors into chondrocytes respectively, each vector blank group, transfected miR-21mimic group, mimic negative control group, miR-21inhibitor group, inhibitor negative control group, and continued to culture cells for 24 hours According to the instructions of Promega's dual luciferase detection kit, three replicate wells were set up in each group, and the fluorescence intensity of cells in each well of the 24-well plate was detected. The ratio...

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Abstract

The invention discloses application of miR-21 and a target gene thereof in preparation of reagents or drugs diagnosing and / or controlling osteoarthritis, relating to the technical field of biology. The reagent or drug diagnosing and / or controlling osteoarthritis is prepared from an inhibitor and / or inhibitor composition downregulating activity of miR-21 or blocking activity of miR-21, and the miR-21 inhibitor regulates a key transcription factor PIK3C2A gene related to differentiation and phenotype stability of cartilage cells in cartilage cells so as to stabilize the phenotype of the cartilage cells and prevent matrix outside the cartilage cells from degrading. Therefore, the invention provides application of miR-21 and / or antisense oligonucleotide thereof and / or target gene of miR-21 inpreparation of reagents or drugs diagnosing and / or controlling osteoarthritis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of miR-21 and its target gene in the preparation of reagents or medicines for diagnosing and / or preventing and treating osteoarthritis. Background technique [0002] Osteoarthritis (OA) is a chronic degenerative disease mainly involving weight-bearing joints (hip, knee and other joints). , destruction and bone hyperplasia, sclerosis of subchondral bone, cystic degeneration, clinically manifested as pain, joint deformity and dysfunction, etc., which seriously affect the quality of life of patients and bring heavy economic burden to families and society. At present, OA is one of the main diseases that affect the quality of life of middle-aged and elderly people. According to the survey, the prevalence rate of people over 60 years old can reach 50%, and that of people over 75 years old can reach 80%, and the disability rate can be as high as 53%. With the aging of the p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883A61K45/00A61P19/02A61P29/00G01N33/68
CPCA61K45/00A61P19/02A61P29/00C12Q1/6883C12Q2600/158G01N33/6893G01N2800/105
Inventor 陆军
Owner SOUTHEAST UNIV
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