基础医学与临床 ›› 2019, Vol. 39 ›› Issue (1): 42-46.

• 研究论文 • 上一篇    下一篇

转染miRNAs降低脂多糖诱导的大鼠肠微血管内皮细胞水通道蛋白1表达

赵承承,陈建泉,吕纯业   

  1. 南京市江宁医院
  • 收稿日期:2018-01-11 修回日期:2018-04-29 出版日期:2019-01-05 发布日期:2018-12-28
  • 通讯作者: 吕纯业 E-mail:chunyel@163.com
  • 基金资助:
    江苏省卫生厅课题

Transfection of miRNAs reduces LPS-induced aquaporin 1 expression in rat intestinal microvascular endothelial cells

  • Received:2018-01-11 Revised:2018-04-29 Online:2019-01-05 Published:2018-12-28

摘要: 目的 探讨大鼠肠微血管内皮细胞miRNAs对脂多糖(LPS)诱导的水通道蛋白1(AQP1)表达的影响。方法 用机械吹打法及胰蛋白酶消化法从大鼠空肠分离微血管内皮细胞,用免疫荧光染色鉴定细胞;用荧光实时定量PCR(RT-qPCR)检测AQP1和miRNAs的表达;miRNAs芯片分析结合在线软件筛选调控AQP1表达的miRNAs,瞬时转染miRNAs模拟体及对照序列进行进一步验证。结果 肠微血管内皮细胞vWF免疫荧光染色为阳性。用(0、0.1、1和10 mg/L)LPS处理细胞后,AQP1 mRNA表达水平随LPS浓度增加呈下降趋势,其中1和10 mg/L LPS处理细胞后AQP1 mRNA表达水平与对照相比有明显差异(P<0.05和P<0.001);与对照组相比,LPS处理组有22个表达上调2倍以上的miRNAs,9个下调2倍以上的miRNAs;软件预测显示芯片结果中的miR-423-5p、miR-874-5p、miR-361-3p、miR-219a-5p、miR-27b-3p、miR-133b和miR-666可与AQP1启动子区互补配对;miR-874-5p、miR-361-3p、miR-133b和miR-666在LPS处理后的肠微血管内皮细胞中表达上调;转染miR-133b和miR-666模拟体后可使AQP1 mRNA表达水平下调。结论 LPS可通过上调miR-133b和miR-666表达水平,间接抑制AQP1的表达,继而影响肠黏膜通透性。

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

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