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Updated: Oct 17, 2025
Culturing Microglia from the Neonatal and Adult Central Nervous System
Published on: August 9, 2013
Downregulation of MicroRNA-145-5p in Activated Microglial Exosomes Promotes Astrocyte Proliferation by Removal of
Yong Ye1,2, Jie Hao3, Zhou Hong1,2
1Medical School of Nantong University, Nantong, 226001, Jiangsu, China.
Abstract:
In spinal cord injury, microglial activation plays an important role during the inflammatory process. Specifically, the cellular and molecular interactions between microglia and astrocytes are of critical importance. Cells can communicate with each other through the substances carried by exosomes, and overproliferated astrocytes would create a physical and chemical barrier that prevents neurite regeneration, thereby interfering with functional recovery. On the other hand, Smad3 is an important factor in the proliferation, migration, and apoptosis of astrocytes. In this study, supernatant and purified exosomes were collected from LPS-treated microglia and co-cultured with astrocytes. The results showed that astrocytic proliferation was promoted with higher levels of Smad3. Furthermore, miRNA sequencing analysis was performed on microglial exosomes after inflammation. The results revealed a differential expression of miR-145-5p in the exosomes. The Dual-Luciferase assay showed that miR-145-5p could bind to Smad3 mRNA and regulate the levels of Smad3 protein at the post-transcriptional level. Subsequently, exosomes were transfected with miR-145-5p mimics, and astrocytes after mechanical injury were cultured with these exosomes for 24 h. The levels of Smad3 and phosphor-Smad3 proteins were analyzed by western blot and qRT-PCR. CCK8 and flow cytometry showed lower proliferation of astrocytes after co-culturing with the exosomes transfected with the miR-145-5p mimic. This study finds that miR-145-5p was found to be a negative regulator of astrocyte proliferation, and that its downregulation promotes smad3 activity and thus astrocyte proliferation.
Insights
Microglia-derived exosomes regulate astrocyte proliferation after spinal cord injury. Exosomes carrying miR-145-5p inhibit astrocyte proliferation by downregulating Smad3, promoting functional recovery.
Area of Science:
- Neuroscience
- Cell Biology
- Regenerative Medicine
Background:
- Microglial activation and astrocyte interactions are crucial in spinal cord injury (SCI) inflammation.
- Overproliferated astrocytes impede neurite regeneration and functional recovery post-SCI.
- Smad3 is a key regulator of astrocyte proliferation, migration, and apoptosis.
Purpose of the Study:
- To investigate the role of microglial exosomes in regulating astrocyte behavior following SCI.
- To identify specific microRNAs within exosomes that influence astrocyte proliferation.
- To elucidate the mechanism by which microglial exosomes affect astrocyte Smad3 levels and proliferation.
Main Methods:
- Collection of exosomes from LPS-treated microglia and co-culture with astrocytes.
- miRNA sequencing of microglial exosomes to identify differentially expressed miRNAs.
- Dual-Luciferase assay to confirm the interaction between miR-145-5p and Smad3 mRNA.
- Transfection of exosomes with miR-145-5p mimics and subsequent co-culture with mechanically injured astrocytes.
- Analysis of Smad3 protein levels, astrocyte proliferation, and apoptosis using Western blot, qRT-PCR, CCK8, and flow cytometry.
Main Results:
- LPS-treated microglia-derived exosomes promoted astrocytic proliferation, correlating with higher Smad3 levels.
- miR-145-5p was identified as differentially expressed in microglial exosomes post-inflammation.
- miR-145-5p directly targets Smad3 mRNA, regulating Smad3 protein levels post-transcriptionally.
- Co-culture with miR-145-5p mimic-transfected exosomes significantly reduced astrocyte proliferation and Smad3/phosphor-Smad3 protein levels.
Conclusions:
- miR-145-5p acts as a negative regulator of astrocyte proliferation in the context of SCI.
- Downregulation of miR-145-5p in microglial exosomes enhances Smad3 activity, leading to increased astrocyte proliferation.
- Microglial exosomal miR-145-5p represents a potential therapeutic target for promoting functional recovery after spinal cord injury.
