Runt相关转录因子1调控上皮间质转化在卷烟烟雾诱导的气道上皮细胞中的作用

Role of Runt-related transcription factor 1 in regulating epithelial-mesenchymal transition in cigarette smoke extract stimulated rat airway epithelial cells

目的通过研究Runt相关转录因子1(Runt-related transcription factor 1,RUNX1)在经卷烟烟雾提取物(cigarette smoking extract,CSE)刺激的大鼠气道上皮细胞中的表达,探讨RUNX1对上皮–间质转化(epithelial-mesenchymal transition,EMT)的调控作用。方法采用酶消化法提取大鼠的原代支气管上皮细胞,并用不同浓度的CSE进行刺激,采用CCK-8检测细胞的活性,探索合适的CSE处理浓度。用CSE处理细胞后,构建RUNX1干扰和过表达载体,转染入细胞,使RUNX1基因沉默或过表达,用免疫细胞化学法检测RUNX1蛋白的表达,用蛋白印迹法检测RUNX1、核因子κB(nuclear factor-κB,NF-κB)、转录因子Snail、上皮细胞标志物E-钙黏蛋白(E-cadherin,E-cad)以及间质细胞标志物波形蛋白(vimentin,VIM)的表达。结果支气管上皮细胞的活力可被CSE降低,且降低程度与CSE的浓度成正比。通过CSE的诱导,原代大鼠支气管上皮细胞E-cad表达水平显著降低(P<0.05),RUNX1、NF-κB、Snail、VIM的表达水平明显升高(均P<0.05)。干扰RUNX1基因后,E-cad表达上调(P<0.05),NF-κB、Snail、VIM的表达下调(均P<0.05)。而过表达RUNX1后,E-cad的表达下调(P<0.05),NF-κB、Snail、VIM的表达上调(均P<0.05)。结论CSE可促进气道上皮细胞RUNX1的表达,RUNX1通过NF-κB/Snail通路调控EMT的进程。

Objective To study the expression of human Runt-related transcription factor 1(RUNX1)in rat airway epithelial cells stimulated by cigarette smoking extract(CSE),and explore the role of RUNX1 in regulating epithelial-mesenchymal transition(EMT).Methods Primary rat bronchial epithelial cells were cultured by enzyme digestion and stimulated with different concentrations of CSE.The viability of cells was detected by CCK-8 to explore the appropriate concentration of CSE.After the cells were treated with CSE,the Runx1 interference and overexpression vectors were constructed and transfected into the cells to silence or overexpress the Runx1 gene.Immunocytochemical method was used to detect RUNX1 expression and Western blot analysis was used to detect the expression of RUNX1,nuclear factor-κB(NF-κB),Snail,E-cadherin,and vimentin.Results The survival rate of bronchial epithelial cells could be reduced by CSE,and the degree of reduction was directly positively correlated to the concentration of CSE.After CSE stimulation,the expression level of E-cadherin in primary rat bronchial epithelial cells decreased significantly(P<0.05);the expression levels of RUNX1,NF-κB,Snail and vimentin significantly increased(P<0.05).After interfering with RUNX1gene,the expression level of E-cadherin was up-regulated(P<0.05),and the expression levels of NF-κB,Snail and vimentin were down-regulated(P<0.05).After overexpression of RUNX1 gene,the expression level of E-cadherin decreased(P<0.05),and the expression levels of NF-κB,Snail and vimentin increased(P<0.05).Conclusions CSE promotes the expression of RUNX1 in rat airway epithelial cells.RUNX1 might regulate EMT process by involving in the regulation of NF-κB/Snail expression.