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MiR-223 is a miRNA with important functions in apoptosis, carcinogenesis, and inflammation, and it was demonstrated to be over-expressed in brain tissue after traumatic brain injury (TBI). However, few studies have focused on its role in protecting brain microvascular endothelial cells (BMECs). This study evaluated the protective effect of miR-223 on BMECs after stretch injury (SI). bEnd.3 cells (BMECs of mouse) were transfected with overexpressing and blocking lentivirus of miR-223, then were subjected to SI. After immunofluorescence assay, it was demonstrated that miR-223 overexpression significantly rescued the SI-induced loss of ZO-1 (Zonula Occludens 1, tight junction protein) (p < 0.01), while miR-223 blocking exacerbated the loss of ZO-1 (p < 0.05). Flow cytometry confirmed a significant increase in the proportion of apoptotic bEnd.3 cells after SI, and miR-223 overexpression reduced this proportion (p < 0.001). The result of Western blot revealed that miR-223 overexpression significantly reduced the expression of cleaved caspase-3 (cl-caspase 3) (p < 0.05) and RhoB (p < 0.01), while miR-223 blocking increased the expression of these proteins (p < 0.05, p < 0.001). Additionally, knockdown of RhoB significantly reduced the expression of cl-caspase 3 (p < 0.001). These findings suggested that miR-223 can alleviate SI-induced apoptosis of BMECs, and this anti-apoptotic effect is at least partially achieved by inhibiting the expression of RhoB. Moreover, miR-223 may play a role in maintaining the integrity of BBB during TBI. [ABSTRACT FROM AUTHOR]

Simple Summary: Bovine mammary epithelial cells (bMECs) are often used as cell models for mammary gland research. They are the most important cells for mammary gland function and the first line of defense for pathogen identification. MicroRNAs (miRNAs) are important regulatory factors involved in many physiological and pathological processes. Here, we examined a transcriptome profile of bovine mammary epithelial cell lines transfected with miR-375 inhibitor or negative control (NC) inhibitor, and further reveal the potential role of miR-375 in bMECs by differentially expressed mRNA analysis. We found that miR-375 potentially promotes inflammation in the mammary gland through the MAPK signaling pathway. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate post-transcriptional gene expression and several biological processes. Bovine mammary epithelial cells (bMECs) mediate critical immune responses in the mammary gland and the occurrence of mastitis. Current research focuses on miRNA regulation of bMECs, but the miR-375 regulatory mechanism in bMECs is unclear. This study explored the role of miR-375 by profiling the transcriptome of miR-375-silenced bMECs using RNA-seq and identifying differentially expressed mRNAs (DIE-mRNAs). There were 63 DIE-mRNAs, including 48 down-regulated and 15 up-regulated mRNAs between miR-375-silenced bMECs and the controls. The Kyoto encyclopedia of genes and genomes (KEGG) and Gene Ontology (GO) functional analysis showed that the DIE-mRNAs enriched nuclear receptor subfamily 4 group A member 1 (NR4A1) and protein tyrosine phosphatase non-receptor type 5 (PTPN5) anti-inflammatory genes of the mitogen-activated protein kinase (MAPK) signaling pathway. However, they showed an opposite trend to the expression of miR-375 silencing, suggesting that miR-375 promotes bMEC inflammation through the MAPK signaling pathway. The findings of this study provide a new reference for understanding the regulation of bMEC inflammation and cow mastitis. [ABSTRACT FROM AUTHOR]