Screening and biological role analysis of long non-coding RNA in exosomes of the brain extracellular space in mice with traumatic brain injury
10.3760/cma.j.cn501098-20240912-00557
- VernacularTitle:创伤性脑损伤小鼠脑细胞间隙外泌体中长链非编码RNA的筛选及生物学作用分析
- Author:
Ju ZHOU
1
;
Ruiting ZHAO
;
Ye TIAN
;
Hengjie YUAN
Author Information
1. 海南省妇女儿童医学中心药学部，海口 570100
- Publication Type:Journal Article
- Keywords:
Brain injuries;
Exosome;
Long non-coding RNA
- From:
Chinese Journal of Trauma
2025;41(2):192-200
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To screen differentially expressed long non-coding RNA (lncRNA) in exosomes of the brain extracellular space in mice with traumatic brain injury (TBI) and explore their biological roles in brain injury repair.Methods:Twenty-four C57BL/6J male mice were randomly divided into TBI group and control group with 12 in each group. The TBI group was treated with drill craniostomy followed by hydraulic impact while the control group was treated with drill craniostomy only. The brain tissue samples were collected at 3 hours after TBI. The exosomes from the brain extracellular space were isolated with papain digestion combined with an exosome isolation kit, the appearance of the exosomes was observed by phosphotungstic acid negative staining under a transmission electron microscope, and western blot was used to identify the exosomal marker proteins cluster of differentiation 63 (CD63), tumor susceptibility gene 101 (TSG101) and heat shock protein 70 (HSP70). After exosome identification, RNA libraries were constructed and subjected to library quality control, whole transcriptome sequencing was performed to detect, characterize and annotate lncRNA. Subsequently, the RNA high-throughput sequencing results were verified by randomly selecting five differentially expressed lncRNA. Moreover, the accuracy of the RNA high-throughput sequencing results was verified by qRT-PCR. The differentially expressed lncRNA were screened afterwards. Finally, bioinformatics analysis of differentially expressed lncRNA was performed, i.e., gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis were performed based on differentially expressed lncRNA-related genes.Results:(1) Identification of exosomes: the exosomes were cup-shaped with clear and intact membranes and diameters of 30-100 nm. Western blot confirmed the presence of typical exosome markers of the exosomes, such as CD63, TSG101, and HSP70, as well as astrocyte marker glial fibrillary acidic protein (GFAP), all of which were found in the brain tissue. (2) Validation of lncRNA high-throughput sequencing data accuracy: the 4/5 sequencing accuracy indicated that the sequencing results of lncRNAs were largely reliable. (3) Screening of differentially expressed exosomal lncRNAs after TBI: compared with the control group, a total of 442 lncRNA were significantly differentially expressed in the TBI group, including 255 up-regulated and 187 down-regulated lncRNA. (4) Bioinformatics analysis of differentially expressed lncRNAs after TBI: GO analysis showed that the differentially expressed lncRNA mainly involved in cell localization, cognition, learning or memory, were closely related to the composition of the nucleus and organelles and the binding processes of proteins, oxygen, and kinases. KEGG pathway analysis showed that upregulated lncRNA were mainly enriched in the mitogen activated protein kinase (MAPK) signaling pathway, while downregulated lncRNA were mainly enriched in the endocytosis pathway. The pathway relation network showed that MAPK signaling pathway played a key role in downstream regulation.Conclusions:There are significant differences in lncRNA expression profiles of the exosomes in the extracellular spaces of the brain cells in mice with TBI, and the differentially expressed lncRNA may mediate the process of neuronal repair, body prognosis and learning ability recovery after TBI by participating in MAPK and endocytosis signaling pathways.
