Xu Rui, Li Qi, Zhou Xiang-dong, Perelman Juliy M, Kolosov Victor P
Department of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.
Zhonghua Yi Xue Za Zhi. 2012 Dec 11;92(46):3291-5.
To explore the variation of small ubiquitin-related modification (SUMO) level of glucocorticoid receptors (GRs) exposed to acrolein stimulation as well as the influence of the variation on mucus hypersecretion.
The recombinant plasmid was constructed by inserting small ubiquitin-like modifier 1 (SUMO1) cDNA into eukaryotic expression plasmid pcDNA3.1-EGFP with a flag sequence marker. The recombinant plasmid pcDNA3.1-EGFP-flag-SUMO1 was identified by enzyme digestion analysis. The 16HBE cells were cultured and randomized divided into 9 following groups: A. control; B. acrolein stimulated; C. dexamethasone incubated; D. acrolein stimulated and dexamethasone incubated; E. SUMO1 transfected; F. pcDNA3.1-EGFP vector transfected; G. SUMO1 transfected and acrolein stimulated; H. SUMO1 transfected, acrolein stimulated and dexamethasone incubated; I. pcDNA3.1-EGFP vector transfected, acrolein stimulated and dexamethasone incubated. Each group consisted of 5 parallel wells and experiments were repeated for 4 times for statistical analysis. The transfection efficiency was evaluated by the expression of enhanced green fluorescent protein (EGFP) via fluorescence microscope. The SUMO modification level of GRs was measured with co-immunoprecipitation and Western blot. The transcription level of mucin (MUC) 5AC was evaluated with reverse transcription-polymerase chain reaction (RT-PCR). The MUC5AC secreted in supernatant was measured by enzyme-linked immunosorbent assay (ELISA).
The SUMO modification level was much lower in group B (0.079 ± 0.023) than that in group A (0.446 ± 0.068) (t = -23.13, P = 0.000) and in group D (0.574 ± 0.018) than that in group C (0.843 ± 0.028) (t = -36.34, P = 0.000). The transcription level of MUC5AC was significantly lower in group H (0.330 ± 0.063) than group D (0.617 ± 0.190) (q = -7.80, P = 0.000). Through ELISA, there was no significance between groups G and B in the term of secretion level of MUC5AC. While the secretion level of MUC5AC was much lower in group H ((0.416 ± 0.092) µg/L) than group D ((0.663 ± 0.104) µg/L) and group G ((0.740 ± 0.343) µg/L) (q = -7.31, -9.59; P = 0.001, 0.000).
Acrolein decreases the SUMO modification of GRs and reduces the inhibitory effect of dexamethasone on the transcription of MUC5AC.
探讨丙烯醛刺激下糖皮质激素受体（GRs）的小泛素相关修饰（SUMO）水平变化及其对黏液高分泌的影响。
将小泛素样修饰物1（SUMO1）cDNA插入带有flag序列标记的真核表达质粒pcDNA3.1-EGFP中构建重组质粒。通过酶切分析鉴定重组质粒pcDNA3.1-EGFP-flag-SUMO1。培养16HBE细胞并随机分为以下9组：A.对照组；B.丙烯醛刺激组；C.地塞米松孵育组；D.丙烯醛刺激+地塞米松孵育组；E.SUMO1转染组；F.pcDNA3.1-EGFP载体转染组；G.SUMO1转染+丙烯醛刺激组；H.SUMO1转染、丙烯醛刺激+地塞米松孵育组；I.pcDNA3.1-EGFP载体转染、丙烯醛刺激+地塞米松孵育组。每组设5个平行孔，实验重复4次进行统计学分析。通过荧光显微镜观察增强绿色荧光蛋白（EGFP）的表达评估转染效率。用免疫共沉淀和蛋白质免疫印迹法检测GRs的SUMO修饰水平。用逆转录-聚合酶链反应（RT-PCR）评估黏蛋白（MUC）5AC的转录水平。用酶联免疫吸附测定（ELISA）法检测上清液中分泌的MUC5AC。
B组（0.079±0.023）的SUMO修饰水平显著低于A组（0.446±0.068）（t=-23.13，P=0.000），D组（0.574±0.018）的SUMO修饰水平显著低于C组（0.843±0.028）（t=-36.34，P=0.000）。H组（0.330±0.063）的MUC5AC转录水平显著低于D组（0.617±0.19）（q=-7.80，P=0.000）。通过ELISA法检测，G组和B组的MUC5AC分泌水平无显著差异。而H组（（0.416±0.092）μg/L）的MUC5AC分泌水平显著低于D组（（0.663±0.104）μg/L）和G组（（0.740±0.343）μg/L）（q=-7.31，-9.59；P=0.001，0.000）。
丙烯醛降低GRs的SUMO修饰水平，并减弱地塞米松对MUC5AC转录的抑制作用。
