关键词: miRNAs, 内毒素, 水通道蛋白1, 大鼠肠微血管内皮细胞

Abstract: Objective To investigate the influences of miRNAs on lipopolysaccharide (LPS)-induced aquaporin 1 (AQP1) expression in rat intestinal microvascular dothelial cell. Methods The rat intestinal microvascular endothelial cells were separated from newborn rat intestine by pipetting and digesting with trypsin. Immunofluorescence was used to verify the separated cells. The expression levels of AQP1 and miRNAs were determined by quantitative real time PCR (qRT-PCR). The miRNAs array combined with online softwares was used to identify the miRNAs which targeted AQP1. Transient transfection was performed to further verify the target miRNAs. Results Immunofluorescence staining showed vWF positive in intestinal microvascular endothelial cells. After treated with LPS (0, 0.1, 1, 10 mg/L), the mRNA expression level of AQP1 were decreased with the increased concentration of LPS. Compared with control cells, there were significant differences of AQP1 mRNA expression levels in 1 and 10 mg/L LPS-treated cells（P<0.05 and P<0.001）. Compared with the control group, 22 miRNAs were up-regulated (≥2 fold) and 9 miRNAs were down-regulated（<2 fold）in LPS-treated group. miR-423-5p, miR-874-5p, miR-361-3p, miR-219a-5p, miR-27b-3p, miR-133b and miR-666 could bind to the promoter region of AQP1. miR-874-5p, miR-361-3p, miR-133b and miR-666 were up-regulated in LPS-treated intestinal microvascular endothelial cells as compared with the control cells. Furthermore, the mRNA expression level of AQP1 was decreased after transfection of miR-133b or miR-666 mimics in rat intestinal microvascular endothelial cells compared with cells transfected with control sequence. Conclusions LPS could up-regulate the expression level of miR-133b and miR-666 to inhibit the expression of AQP1, thus affect the intestinal mucosal permeability.

Key words: MiRNAs, LPS, Aquaporin 1, Rat intestinal microvascular endothelial cell