NDRG2基因通过去泛素化酶Zc3h12d在NF-κB转导通路中的调控机制

Mediated mechanism of NDRG2 in the regulation of neutrophil nuclear transcription factor-κB signaling pathway through the deubiquitylating enzyme of Zc3h12d

目的:研究NDRG2基因与去泛素化酶Zc3h12d在NF-κB转导通路中的调控机制。方法:采用LPS刺激人支气管气道上皮16HBE细胞株来构建炎性刺激时气道黏液高分泌模型,用瞬时转染过表达NDRG2基因和Zc3h12d si RNA干扰下调16HBE细胞,将细胞分成LPS+瞬时转染过表达NDRG2组(A组)、LPS+瞬时转染过表达NDRG2+Zc3h12d si RNA组(B组)、LPS+瞬时转染Zc3h12d si RNA组(C组)、LPS+转染空白载体组(D组),以未做任何处理的16HBE细胞作为对照组(E组)。反转录PCR检测气道黏蛋白(mucin,MUC)5AC m RNA的表达水平,ELISA法检测IL-1β、IL-6等炎症因子及MUC5AC的分泌水平,Western印迹检测Zc3h12d和NF-κB p65的分泌水平,激光共聚焦法检测16HBE细胞内MUC5AC表达。结果:与B组比较,A组炎症因子及MUC5AC表达明显减少,差异有统计学意义(P<0.05);而B组与C组比较,差异无统计学意义(P>0.05)。与D组比较,A组炎症因子及MUC5AC的表达明显降低,差异均有统计学意义(均P<0.05)。D组与E组比较,LPS刺激后炎症因子及MUC5AC分泌增多,差异均有统计学意义(均P<0.05)。结论:NDRG2基因可以通过Zc3h12d而实现对NF-κB转导通路的调控。

Objective： To explore the role NDRG2/Zc3h12d in the regulation of neutrophil nuclear transcription factor-κB signaling pathway and the underlying mechanisms. Methods： Sixteen HBE cells were incubated with lipopolysaccharide （LPS） to establish airway mucus hypersecretion model, which was transfected with NDRG2 or Zc3h12d siRNA. The cells were divided into 5 groups： a LPS＋NDRG2 siRNA （Group A）, a LPS＋ NDRG2 and Zc3h12d siRNAs （Group B）, a LPS＋Zc3h12d siRNA （Group C）, a LPS＋ empty plasmid （Group D）, and a negative control group （Group E）. The reverse transcription-polymerase chain reaction （RT-PCR） was used to measure the expression level of mucin （MUC） 5AC mRNA. The levels of MUC5AC and the inflammatory factors were examined by the enzyme-linked immunosorbent assay （ELISA）. The NF-κB p65 and the Zc3h12d protein levels were measured by Western blot. The MUC5C expression was further examined by laser confocal method. Results： Compared with Group B, the levels of MUC5AC mRNA and protein in Group A were decreased （P〈0.05）, there was no significant difference in the MUC5AC mRNA and MUC5AC protein levels between the Group B and the Group C （P〉0.05）. Compared with Group D, the MUC5AC （mRNA and protein） and inflammatory factor levels in the Group A were significantly decreased （P〈0.05）; compared with Group E, after incubation with LPS, the levels of MUC5AC （mRNA and protein） and inflammatory factors in the Group D was increased, with significantly difference （P〈0.05）. Conclusion： NDRG2 can regulate the function of NF-κB signaling pathway through the deubiquitylating enzyme of Zc3h12d.