miRNA and application of miRNA in preparation of product for diagnosing and treating posterior capsule opacification

A post-onset cataract technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, gene therapy, etc., can solve the problems of iris hemorrhage, high cost, scope of action, targeting, etc.

Inactive Publication Date: 2014-05-07
SHANDONG EYE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Once PCO occurs, it is usually treated with Nd:YAG laser posterior capsulotomy, which is not only expensive, but also has complications such as retinal detachment, macular edema, and iris hemorrhage.
In terms of drug treatment, the implantation of sustained-release drug devices in the eye is currently an ideal drug delivery method for the prevention and treatment of after-onset cataracts. However, in drug sustained-release devices, the control of drug concentration effects, range of action, and targeting still need to be improved.

Method used

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  • miRNA and application of miRNA in preparation of product for diagnosing and treating posterior capsule opacification
  • miRNA and application of miRNA in preparation of product for diagnosing and treating posterior capsule opacification
  • miRNA and application of miRNA in preparation of product for diagnosing and treating posterior capsule opacification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Screening of has-miR-204-5p and determination of target gene SMAD4

[0018] 1. Sample RNA extraction and detection

[0019] Biological material source: lens capsule of normal people and post-cataract patients

[0020] Total RNA including miRNA was extracted according to the instructions of TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN).

[0021] 1) Collect the crystal capsule and add 1mL TRIzol (10μL β-mercaptoethanol) reagent, after cracking, put it at room temperature

[0022] Set for 5min.

[0023] 2) Add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 2-3 minutes, and centrifuge at 12,000×g for 15 minutes at 4° C.

[0024] 3) Remove the supernatant, add 1.5 times the volume of absolute ethanol, and mix well.

[0025] 4) Transfer the solution and precipitate obtained in step 3 to a 2mL RNeasy Mini spin column, cover the tube gently, centrifuge at ≥8000xg for 15s at room temperature, discard the waste liquid ...

Embodiment 2

[0045] Example 2: Determination of has-miR-204 target gene SMAD4

[0046] According to bioinformatics analysis, it was found that has-miR-204 binds to the 2880-2887bp of the 3'-UTR region of the SMAD4 gene, such as figure 2 As shown in A.

[0047] Amplify the 3'-UTR region and Mut-SMAD4-3'-UTR region of SMAD4 gene, the sequence is 359bp, add BglII in the upstream and MluI restriction site in the downstream respectively, the sequence is shown in Table 1, and the pGL3-Basic Vector (Promega) as the vector backbone, pGL3-SMAD4, pGL3-SMAD4-3'-UTR and Mut-pGL3-SMAD4-3'-UTR vectors were constructed.

[0048] As a chemically synthesized mature miRNA mimic, miRNA mimic can mimic endogenous miRNA to function after transfection into cells. As a chemically synthesized mature miRNA inhibitor, miRNA inhibitor can specifically inhibit the function of miRNA after transfection into cells. Therefore, under in vitro culture conditions, miRNA mimic and miRNA inhibitor are used to simulate the...

Embodiment 3

[0051] Example 3: has-miR-204-5p inhibits the occurrence of EMT in a PCO model cultured in vitro

[0052] 1. Detection of EMT marker gene expression from PCO patient materials

[0053] In line with the relevant national policies and regulations, and on the basis of the consent of the sampling subjects, the capsular bag materials of patients with post-cataract PCO and normal people were selected, and Western blotting was performed to detect the expression of EMTmarker genes E-cadherin, a-SMA, and Vimentin. It was found that Compared with normal people, the expression of E-cadherin was significantly down-regulated, and the expression of a-SMA and Vimentin were up-regulated, such as image 3 shown.

[0054] 2. Up-regulation of has-miR-204-5p expression inhibits EMT

[0055] In accordance with the relevant national policies and regulations, and on the basis of the consent of the sampling subjects, the pouch materials of PCO patients and normal people were selected, and TGF- Aft...

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Abstract

The invention aims to provide a miRNA and an application of miRNA in preparation of a product for diagnosing and treating posterior capsule opacification. The miRNA is miR-204-5p and the RNA sequence is SEQ ID NO:1. A miRNA gene product can be used for adjusting the expression of an SMAD4 gene, blocking a TGF (Transforming Growth Factor)-beta/Smads signal channel and inhibiting epithelial-mesenchymal transition (EMT), and has the potential of preparing the diagnosing and treating product for the posterior capsule opacification PCO.

Description

[0001] The present invention claims that the filing date is December 27, 2012, the application number is 201210579895x, and the title of the invention is: the priority of the invention application for the screening of cataract-related miRNA. technical field [0002] The invention belongs to the related technical field of gene diagnosis and treatment, and specifically relates to the application of miR-204-5p in the preparation and detection of post-cataract diagnosis and treatment products. Background of the invention [0003] Post-onset cataract (PCO), also known as posterior capsule opacity, refers to the opacity of the posterior lens capsule after extracapsular cataract extraction, or after partial cortical absorption of traumatic cataract. It is the most common complication after cataract extraction, with an incidence rate of 30% to 50% in adults and 100% in children. With the development of phacoemulsification, post-cataract has become the main factor affecting visual re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P27/12
Inventor 黄钰森王晔赵晓雯
Owner SHANDONG EYE INST
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