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

Modulation of TLR-4/My D88 signaling cascade by mi R-21 is involved in airway immunologic dysfunction induced by cold air exposure

目的探讨mi R-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mi R-21,mi R-164,mi R-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mi R-21模拟物、mi R-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mi R-21的靶蛋白。利用lipofectamine2000分别将mi R-21模拟物、mi R-21模拟对照物、mi R-21抑制物、mi R-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/My D88蛋白水平。结果低温诱导气道上皮细胞内mi R-21水平提高(P<0.05),而mi R-164及mi R-155无显著变化。低温提高气道上皮细胞内mi R-21水平呈时间依赖性。双荧光素酶报告实验证实,TLR-4为mi R-21的直接靶蛋白。Western Blotting法检测结果显示转染mi R-21模拟物的BEAS-2B及16HBE细胞在常温培养下TLR-4/My D88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/My D88的蛋白水平(P<0.05);而使用mi R-21抑制物处理,可部分抑制低温诱导的TLR-4/My D88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mi R-21水平,降低TLR-4/My D88蛋白水平,影响气道上皮免疫功能。

Objective To investigate the role of mi R- 21 in airway immunologic dysfunction induced by cold air irritation.Methods Immortalized human airway epithelial cell lines BEAS- 2B and 16 HBE cells were cultured in air- liquid phases. The differential expressions of endogenous mi R- 21, mi R- 164, and mi R- 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 16 HBE cells together with mi R-21 mimic, mi R-21 mimic control, mi R-21 inhibitor, or mi R-21 inhibitor control. Following the transfection, dual luciferase reporter assay was performed to verify the action of mi R-21 on TLR- 4. mi R- 21 mimic, mi R- 21 mimic control, mi R- 21 inhibitor, and mi R- 21 inhibitor control were transfected via lipofectamine 2000 in BEAS- 2B or 16 HBE cells that were subsequently exposed to a temperature at 37 ℃ or cold irritation（30 ℃）, and the protein levels of TLR- 4/My D88 were detected by Western blotting. Results Cold irritation caused a timedependent up-regulation of mi R-21 in both BEAS-2B and 16 HBE cells（P0.05） without obviously affecting the expressions of mi R-164 and mi R-155. Dual luciferase reporter assay demonstrated a direct combination of mi R-21 and its target protein TLR-4. The synthesis levels of TLR-4/My D88 protein were decreased in mi R-21 mimic group even at a routine culture temperature（P0.05）, as also seen in cells with cold irritation（P0.05）. Treatment with the mi R- 21 inhibitor partially attenuated cold irritation- induced down- regulation of TLR- 4/My D88 protein（P0.05）. Conclusion Cold air irritation- induced airway immunologic dysfunction is probably associated with TLR-4/My D88 down-regulation by an increased endogenic mi R-21.