KIF3A通过结合Wnt/β-catenin通路中的β-arrestin参与哮喘气道上皮的炎症调控

Kinesin family member 3A is involved in inflammatory regulation of asthmatic airway epithelial by binding to β-arrestin in the Wnt/β-catenin pathway

目的研究KIF3A参与哮喘气道炎症调控的可能机制,为哮喘治疗提供新的靶点和方向。方法选取20只雌性6~8周龄C57BL/6小鼠建立哮喘模型,随机分为哮喘组与对照组(n=10)。Western blot和免疫组化(IHC)检测两组小鼠KIF3A的表达;在体外分别用磷酸盐缓冲液(phosphate buffer solution, PBS)和屋尘螨(house dust mite, HDM)干预人支气管上皮细胞16HBE,Western blot和RT-PCR检测KIF3A的表达;构建含有KIF3A的过表达组(LV-KIF3A-45894-J2,LV-KIF3A)和RNA干扰序列的慢病毒载体组(LV-KIF3A-RNAi-81183-1,RNAi),用含有空载的慢病毒和阴性对照序列的慢病毒载体作为对照,分别感染16HBE;免疫共沉淀检测过表达组中KIF3A与β-arrestin的结合,同时Western blot和RT-PCR检测各组细胞β-catenin的表达水平,RT-PCR检测KIF3A基因差异表达对趋化因子CCL-17和CCL-26表达的影响。结果哮喘组的KIF3A表达水平较对照组减少(P<0.05)。HDM干预16HBE后的KIF3A蛋白和mRNA表达水平较对照组有下降趋势,但无统计学差异(P>0.05)。慢病毒载体感染16HBE 72 h且经流式分选绿色荧光阳性细胞后,RT-PCR和Western blot结果显示LV-KIF3A组和RNAi组的KIF3A蛋白和mRNA表达分别增多(P<0.05)和下降(P<0.05)。免疫共沉淀证实KIF3A与β-arrestin相结合。LV-KIF3A组和RNAi组的β-catenin蛋白总量无明显差异;与对照组相比,RNAi组的β-catenin mRNA表达显著下降(P<0.000 1),LV-KIF3A组则无统计学差异。RT-PCR结果显示,与对照组相比,RNAi组的趋化因子CCL-17和CCL-26的mRNA水平增加(P<0.001),LV-KIF3A组则降低(P<0.05)。结论 KIF3A与Wnt/β-catenin通路中的β-arrestin结合,通过调节趋化因子CCL-17和CCL-26的表达而参与哮喘气道上皮炎症的调控。

Objective To investigate the possible mechanism of kinesin family member 3 A(KIF3 A) taking part in the regulation of asthma airway inflammation, so as to provide a new target and direction for asthma treatment. Methods Female C57 BL/6 mice were randomly divided into asthma group(n=10) and control group(n=10). Western blotting and immunohistochemistry(IHC) were performed to detect the expression of KIF3 A in the 2 groups. After human bronchial epithelial 16 HBE cells were intervened using phosphate buffer solution(PBS) and house dust mite(HDM), Western blotting and RT-PCR were adopted to detect the KIF3 A expression. Moreover, the lentiviral vectors of KIF3 A overexpression( LV-KIF3 A-45894-J2, LV-KIF3 A) and knock-down(LV-KIF3 A-RNAi-81183-1, RNAi) were constructed and then transfected into 16 HBE cells, and the blank lentiviral vectors and those containing negative control sequence served as controls, respectively. The binding of KIF3 A to β-arrestin in the overexpressed cells was detected by immunoprecipitation, while the expression level of β-catenin in each group of cells was determined by Western blotting and RT-PCR. The effect of differential expression of KIF3 A on the expression of chemokines CCL-17 and CCL-26 was also determined. Results In animal models, the expression of KIF3 A was significantly lower in the asthma mice than the control mice(P<0.05). In vitro study indicated that HDM intervention decreased the expression of KIF3 A at protein and mRNA levels in 16 HBE cells, though there was no statistical difference(P>0.05). The RT-PCR and Western blotting results showed that the expression of KIF3 A were increased in the LV-KIF3 A transfected cells(P<0.05) and decreased in the RNAi treated cells(P<0.05). Immunoprecipitation confirmed the binding of KIF3 A to β-arrestin. No significant difference was found in the total amount of β-catenin protein between the LV-KIF3 A transfected cells and the RNAi treated cells. Compared with the corresponding cells, the mRNA level of β-catenin was significantly lower in the RNAi treated cells(P<0.0001), but no such difference was seen in the LV-KIF3 A transfected cells. The RT-PCR results suggested that the levels of chemokines CCL-17 and CCL-26 were increased in the RNAi treated cells(P<0.001), while decreased in the LV-KIF3 A transfected cells(P<0.05) as compared with their control cells. Conclusion KIF3 A binds to β-arrestin in the Wnt/β-catenin pathway, and is involved in asthmatic airway epithelial inflammation by regulating the expression of chemokines CCL-17 and CCL-26.