膜联蛋白A2对肺炎支原体诱导的人气道上皮细胞EGFR/NF-κB信号转导及黏蛋白表达的影响

Effect of annexin A2 on EGFR/NF-κB signal transduction and mucin expression in human airway epithelial cells treated with Mycoplasma pneumoniae

摘要:

目的 探讨沉默膜联蛋白A2（AnxA2）对肺炎支原体（MP）处理后人气道上皮细胞H292表皮生长因子受体（EGFR）/核因子κB（NF-κB）信号转导及黏蛋白表达的影响。方法 将H292细胞分为对照组、MP组、NC-siRNA+MP组和AnxA2 siRNA+MP组。MP组上皮细胞采用5 μg/mL MP抗原孵育2 h。NC-siRNA+MP组和AnxA2 siRNA+MP组细胞分别转染NC-siRNA和AnxA2 siRNA 24 h后用MP抗原刺激2 h。MTT法检测各组细胞活性；实时荧光定量PCR（qRT-PCR）检测各组细胞中AnxA2 mRNA的表达水平；Western blot法检测各组细胞AnxA2、磷酸化EGFR（p-EGFR）、磷酸化p65 NF-κB（p-p65 NF-κB）的表达水平；酶联免疫吸附试验检测黏蛋白5AC（MUC5AC）和黏蛋白5B（MUC5B）的分泌。结果 MP组和NC-siRNA+MP组细胞活性低于对照组（P  < 0.05），AnxA2 siRNA+MP组细胞活性高于MP组和NC-siRNA+MP组，但仍低于对照组（P  < 0.05）。MP组和NC-siRNA+MP组AnxA2 mRNA和蛋白表达水平均明显高于AnxA2 siRNA+MP组（P  < 0.05）。相比于对照组，MP组和NC-siRNA+MP组中p-EGFR、p-p65 NF-κB、MUC5AC和MUC5B的蛋白表达水平均明显升高（P  < 0.05），AnxA2 siRNA+MP组上述蛋白表达水平则低于MP组和NC-siRNA+MP组，但仍高于对照组（P  < 0.05）。结论 AnxA2通过介导人气道上皮细胞EGFR/NF-κB信号活化和黏蛋白表达参与MP抗原诱导的气道病变。

Abstract:

Objective To investigate the effect of annexin A2 (AnxA2) on epithelial growth factor receptor (EGFR)/nuclear factor-κB (NF-κB) signal transduction and mucin expression in human airway epithelial H292 cells treated with Mycoplasma pneumoniae (MP). Methods H292 cells were divided into control group, MP group, NC-siRNA+MP group, and AnxA2 siRNA+MP group. The cells in the MP group were incubated with 5 μg/mL MP antigen for 2 hours. The cells in the NC-siRNA+MP and AnxA2 siRNA+MP groups were transfected with NC-siRNA and AnxA2 siRNA for 24 hours, followed by MP antigen stimulation for 2 hours. The MTT method was used to measure cell viability; quantitative real-time PCR was used to measure the mRNA expression of AnxA2; Western blot was used to measure the protein expression of AnxA2, phosphorylated EGFR (p-EGFR), and phosphorylated p65 NF-κB (p-p65 NF-κB); ELISA was used to measure the secretion of mucin 5AC (MUC5AC) and mucin 5B (MUC5B). Results The MP and NC-siRNA+MP groups had lower cell viability than the control group (P  < 0.05). The AnxA2 siRNA+MP group had higher cell viability than the MP and NC-siRNA+MP groups and lower cell viability than the control group (P  < 0.05). The MP and NC-siRNA+MP groups had signifcantly higher mRNA and protein expression of AnxA2 than the AnxA2 siRNA+MP group (P  < 0.05). Compared with the control group, the MP and NC-siRNA+MP groups had significant increases in the protein expression of p-EGFR, p-p65 NF-κB, MUC5AC, and MUC5B (P  < 0.05); the AnxA2 siRNA+MP group had lower protein expression than the MP and NC-siRNA+MP groups, but higher protein expression than the control group (P  < 0.05). Conclusions AnxA2 is involved in the airway lesion induced by MP antigen via mediating EGFR/NF-κB signaling activation and mucin expression in human airway epithelial cells.