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Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures
Published on: July 25, 2022
MiR-367 alleviates inflammatory injury of microglia by promoting M2 polarization via targeting CEBPA
Hui Pei1, Qian Peng2, Shewei Guo3
1Department of Emergency Intensive Care Unit, the First Affiliated Hospital of Zhengzhou University, No.1, East Jianshe Road, Erqi District, Zhengzhou, 450052, Henan Province, China.
Abstract:
MiR-367 was reported to regulate inflammatory response of microglia. CCAAT/enhancer-binding protein α (C/EBPA) could mediate microglia polarization. In this study, we explored the possible roles of miR-367 and CEBPA in intracerebral hemorrhage (ICH). ICH and normal specimens were obtained from the tissue adjacent to and distant from hematoma of ICH patients, respectively. Microglia were isolated and identified by immunofluorescence. The isolated microglia were treated with erythrocyte lysate and randomly divided into 8 groups using different transfection reagents. The transfection efficiency of miR-367 was determined by qRT-PCR. The expressions of M1 and M2 microglia markers were detected by Western blotting. The relationship between CEBPA and miR-367 was confirmed by dual luciferase reporter system. Flow cytometry was performed to determine the level of apoptosis in the cells transfected with miR-367 and CEBPA in erythrocyte lysate-treated microglia. We found that miR-367 expression level was downregulated in ICH specimens. Erythrocyte lysate-treated microglia was successfully established using erythrocyte lysate, as decreased miR-367 expression was observed. Overexpression of miR-367 could significantly decrease the expressions of MHC-ІІ, IL-1β, and Bax, reduced apoptosis rate, and increased the expressions of CD206, Bal-2, and Arg-1 in erythrocyte lysate-treated microglia. CEBPA was proved to be a direct target for miR-367, which could inhibit microglia M2 polarization and increase apoptosis rate. However, in the presence of both CEBPA and miR-367 mimic, the protein and mRNA expressions of CEBPA were decreased, leading to promoted microglia M2 polarization and a decreased apoptosis rate. MiR-367 regulates microglia polarization by targeting CEBPA and is expected to alleviate ICH-induced inflammatory injury.
Insights
MicroRNA-367 (miR-367) plays a key role in regulating microglia polarization and apoptosis following intracerebral hemorrhage (ICH). Targeting CCAAT/enhancer-binding protein α (C/EBPA) with miR-367 may alleviate ICH-induced inflammatory injury.
Area of Science:
- Neuroscience
- Molecular Biology
- Immunology
Background:
- Microglia play a crucial role in the inflammatory response and polarization.
- CCAAT/enhancer-binding protein α (C/EBPA) is implicated in mediating microglia polarization.
- Intracerebral hemorrhage (ICH) involves complex inflammatory processes mediated by microglia.
Purpose of the Study:
- To investigate the roles of microRNA-367 (miR-367) and C/EBPA in intracerebral hemorrhage (ICH).
- To elucidate the regulatory mechanism of miR-367 on microglia polarization and apoptosis in the context of ICH.
- To explore the therapeutic potential of miR-367 in alleviating ICH-induced inflammatory injury.
Main Methods:
- Isolation and characterization of microglia from ICH and normal human specimens.
- Establishment of an in vitro model of ICH using erythrocyte lysate-treated microglia.
- Assessment of miR-367 and C/EBPA expression, microglia polarization markers (M1/M2), apoptosis, and their regulatory relationship using qRT-PCR, Western blotting, dual luciferase reporter assay, and flow cytometry.
Main Results:
- miR-367 expression was downregulated in ICH specimens and erythrocyte lysate-treated microglia.
- Overexpression of miR-367 reduced M1 markers (MHC-II, IL-1β) and apoptosis (Bax), while increasing M2 markers (CD206, Bcl-2, Arg-1) in treated microglia.
- C/EBPA was identified as a direct target of miR-367; miR-367 inhibited M2 polarization and increased apoptosis by targeting C/EBPA, but co-transfection reversed these effects, promoting M2 polarization and reducing apoptosis.
Conclusions:
- miR-367 regulates microglia polarization and apoptosis by targeting C/EBPA in the context of ICH.
- miR-367 plays a protective role by promoting M2 microglia polarization and reducing apoptosis.
- Targeting the miR-367/C/EBPA axis holds promise for alleviating inflammatory injury in intracerebral hemorrhage.
