[Inhibitory Effect of MiR-429 on Expressions of ZO-1, Occludin, and Claudin-5 Proteins to Improve the Permeability of Blood Spinal Cord Barrier ]

Overview

Journal Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi

Specialty General Surgery

Date 2020 Sep 15

PMID 32929911

Citations 1

Authors

Rui Sun

Deshui Yu

Affiliations

Soon will be listed here.

Abstract

Objective: To explore the feasibility and mechanism of inhibiting miR-429 to improve the permeability of the blood spinal cord barrier (BSCB) , and provide a new gene therapy target for enhancing the spinal cord microenvironment.

Methods: First, the immortalized human brain microvascular endothelial cell line (hCMEC/D3) was transfected with the anti-miR-429 antagonist (antagomiR-429) and its negative control (antagomiR-429-NC), respectively. The miR-429 expression of hCMEC/D3 cells was observed by fluorescence microscopy and real-time fluorescence quantitative PCR to verify the transfection efficiency of antagomiR-429. Then the effect of miR-429 on BSCB permeability was observed . The experiment was divided into 4 groups. The blank control group (group A) was constructed of normal hCMEC/D3 cells and Ha-sc cells to prepare the BSCB model, the hypoxia-induced group (group B), the hypoxia-induced+antagomiR-429-NC group (group C), and the hypoxia-induced+antagomiR-429 group (group D) were constructed of normal, antagomiR-429-NC transfected, and antagomiR-429 transfected hCMEC/D3 cells and Ha-sc cells to prepare the BSCB models and hypoxia treatment for 12 hours. The permeability of BSCB was measured by horseradish peroxidase (HRP) permeability. Real-time fluorescence quantitative PCR, Western blot, and immunofluorescence staining were used to observe the expressions of ZO-1, Occludin, and Claudin-5.

Results: The antagomiR-429 and antagomiR-429-NC were successfully transfected into hCMEC/D3 cells under a fluorescence microscope, and the transfection efficiency was about 90%. Real-time fluorescence quantitative PCR results showed that the relative expression of miR-429 in antagomiR-429 group was 0.109±0.013, which was significantly lower than that of antagomiR-429-NC group (0.956±0.004, <0.05). HRP permeability measurement, real-time fluorescence quantitative PCR, and Western blot results showed that the HRP permeability of groups B and C were significantly higher than those of groups A and D ( <0.05), and the relative expressions of ZO-1, Occludin, and Claudin-5 proteins and mRNAs were significantly lower in groups B and C than in groups A and D ( <0.05) and in group D than in group A ( <0.05); there was no significant difference between groups B and C ( >0.05). Immunofluorescence staining showed that the immunofluorescence of ZO-1, Occudin, and Claudin-5 at the cell membrane boundary in group D were stronger than those in groups B and C, but not as strong as that in group A.

Conclusion: Inhibition of miR-429 expression can promote the expressions of ZO-1, Occludin, and Claudin-5 proteins in microvascular endothelial cells, thereby improving the increased permeability of BSCB due to hypoxia.

Citing Articles

Blood-Spinal Cord Barrier: Its Role in Spinal Disorders and Emerging Therapeutic Strategies.

Chopra N, Menounos S, Choi J, Hansbro P, Diwan A, Das A NeuroSci. 2024; 3(1):1-27.

PMID: 39484675 PMC: 11523733. DOI: 10.3390/neurosci3010001.

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