Abstract
Of the known regulators of atherosclerosis, miRNAs have been demonstrated to play critical roles in lipoprotein homeostasis and plaque formation. Here, we generated a novel animal model of atherosclerosis by knocking in LDLRW483X in C57BL/6 mice, as the W483X mutation in LDLR is considered the most common newly identified pathogenic mutation in Chinese familial hypercholesterolemia (FH) individuals. Using the new in vivo mouse model combined with a well-established atherosclerotic in vitro human cell model, we identified a novel atherosclerosis-related miRNA, miR-23a-3p, by microarray analysis of mouse aortic tissue specimens and human aortic endothelial cells (HAECs). miR-23a-3p was consistently downregulated in both models, which was confirmed by qPCR. Bioinformatics analysis and further validation experiments revealed that the TNFα-induced protein 3 (TNFAIP3) gene was the key target of miR-23a-3p. The miR-23a-3p-related functional pathways were then analyzed in HAECs. Collectively, the present results suggest that miR-23a-3p regulates inflammatory and apoptotic pathways in atherogenesis by targeting TNFAIP3 through the NF-κB and p38/MAPK signaling pathways.
Highlights
Of the known regulators of atherosclerosis, differentially expressed miRNA (miRNA) have been demonstrated to play critical roles in lipoprotein homeostasis and plaque formation
An in vitro model was established by treating human aortic endothelial cells (HAECs) with oxidized LDL, a well-known atherogenic factor [6, 22]. oxLDL is known to initiate atherogenesis by stimulating endothelial cells to overexpress cell surface adhesion molecules [e.g., intercellular adhesion molecule (ICAM)1] [22,23,24] and inflammatory cytokines, such as E-selectin [25, 26], and to induce endothelial cell apoptosis by activating caspase-3 and caspase-9 [27]
The inflammatory cytokines, E-selectin and ICAM-1, were significantly increased under 50 g/ml oxLDL treatment for 24 and 48 h, respectively, compared with untreated cells (Fig. 2C, D). These results suggested that oxLDL activated HAECs and initiated the inflammatory process that led to apoptosis
Summary
Of the known regulators of atherosclerosis, miRNAs have been demonstrated to play critical roles in lipoprotein homeostasis and plaque formation. Using the new in vivo mouse model combined with a well-established atherosclerotic in vitro human cell model, we identified a novel atherosclerosis-related miRNA, miR-23a-3p, by microarray analysis of mouse aortic tissue specimens and human aortic endothelial cells (HAECs). MiRNAs have been shown to play critical roles in processes such as lipoprotein homeostasis (e.g., miR-122, miR-223, and miR-148a) [7,8,9,10], endothelial cell inflammation and plaque progression. The most frequently used in vivo mouse models are APOE / and LDLR / mice [14, 19, 24, 30] These models differ in how plasma lipoprotein clearance is dysregulated, both show enhanced atherosclerosis. Approximately 46% of the variants are single missense mutations of LDLR [33], suggesting that a LDLR-null or missense mutation model may be more applicable, mimicking the majority of human hypercholesterolemia cases
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