南方医科大学学报

南方医科大学学报 ›› 2016, Vol. 36 ›› Issue (01): 98-.

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miR-21 靶向TLR-4/MyD88信号通路介导冷空气诱导的气道免疫失调

许瑞，黄华萍，韩忠，李敏超，周向东   

  • 出版日期:2016-01-20 发布日期:2016-01-20

Modulation of TLR-4/MyD88 signaling cascade by miR-21 is involved in airway
immunologic dysfunction induced by cold air exposure

  • Online:2016-01-20 Published:2016-01-20

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摘要： 目的探讨miR-21在冷空气诱导的气道上皮免疫功能失调中的作用及其可能的潜在分子机制。方法气-液双相法培养人
永生化气道上皮细胞BEAS-2B及16HBE，通过逆转录荧光定量PCR法检测不同时间梯度冷暴露的细胞中内源性miR-21，
miR-164，miR-155的水平改变。通过构建TLR-4 3’UTR端及其结合位点突变质粒，并与miR-21模拟物、miR-21抑制物以及对
照共转染BEAS-2B及16HBE细胞，进行双荧光素酶标记试验证实TLR-4为miR-21的靶蛋白。利用lipofectamine2000分别将
miR-21模拟物、miR-21模拟对照物、miR-21抑制物、miR-21抑制对照物转染给BEAS-2B及16HBE细胞，并给予常温（37 ℃）培
养或冷暴露（30 ℃），以Western Blot法检测各实验组TLR-4/MyD88蛋白水平。结果低温诱导气道上皮细胞内miR-21水平提
高（P<0.05），而miR-164及miR-155无显著变化。低温提高气道上皮细胞内miR-21水平呈时间依赖性。双荧光素酶报告实验
证实，TLR-4为miR-21的直接靶蛋白。Western Blotting法检测结果显示转染miR-21模拟物的BEAS-2B及16HBE细胞在常温
培养下TLR-4/MyD88 蛋白水平显著低于对照组（P<0.05），低温刺激可降低气道上皮细胞内TLR-4/MyD88 的蛋白水平（P<
0.05）；而使用miR-21抑制物处理，可部分抑制低温诱导的TLR-4/MyD88的蛋白水平下调（P<0.05）。结论低温刺激可能通过
提高气道上皮细胞内miR-21水平，降低TLR-4/MyD88蛋白水平，影响气道上皮免疫功能。

Abstract: Objective To investigate the role of miR-21 in airway immunologic dysfunction induced by cold air irritation.
Methods Immortalized human airway epithelial cell lines BEAS-2B and 16HBE cells were cultured in air-liquid phases. The
differential expressions of endogenous miR-21, miR-164, and miR-155 in the cells induced by cold air exposure for different
time were detected by real-time PCR. The reporter plasmid containing wild-type or mutated 3’UTR of TLR-4 were constructed
and co-transfected into BEAS-2B cells or 16HBE cells together with miR-21 mimic, miR-21 mimic control, miR-21 inhibitor, or
miR-21 inhibitor control. Following the transfection, dual luciferase reporter assay was performed to verify the action of
miR-21 on TLR-4. miR-21 mimic, miR-21 mimic control, miR-21 inhibitor, and miR-21 inhibitor control were transfected via
lipofectamine 2000 in BEAS-2B or 16HBE cells that were subsequently exposed to a temperature at 37 ℃ or cold irritation
(30 ℃), and the protein levels of TLR-4/MyD88 were detected by Western blotting. Results Cold irritation caused a timedependent
up-regulation of miR-21 in both BEAS-2B and 16HBE cells (P<0.05) without obviously affecting the expressions of
miR-164 and miR-155. Dual luciferase reporter assay demonstrated a direct combination of miR-21 and its target protein
TLR-4. The synthesis levels of TLR-4/MyD88 protein were decreased in miR-21 mimic group even at a routine culture
temperature (P<0.05), as also seen in cells with cold irritation (P<0.05). Treatment with the miR-21 inhibitor partially attenuated
cold irritation-induced down-regulation of TLR-4/MyD88 protein (P<0.05). Conclusion Cold air irritation-induced airway
immunologic dysfunction is probably associated with TLR-4/MyD88 down-regulation by an increased endogenic miR-21.