γ-GCS基因抑制元件E-box的实验研究

Experimental study of E-box, a transcriptional suppressor element in γ-GCS gene

目的 明确大鼠肺泡上皮细胞γ谷氨酰半胱氨酸连接酶催化亚单位编码基因(GCLC基因)负调控区域( -745^-705)上的功能元件E box作用。方法 利用迁移率变动试验(EMSA)和超级迁移率变动试验证实大鼠肺泡上皮细胞GCLC基因负调控区域的E- box元件能与转录因子USF1 /USF2特异性的结合;通过共转染USF1 /USF2表达质粒(pCMV -USF1 /pCMV- USF2)和GCLC -luc确认E- box元件在基因调控中的作用;最后通过构建USF1 /USF2的逆转录病毒载体,将表达USF1 /USF2的重组逆转录病毒感染肺泡上皮细胞, 用Western印迹检测细胞GCLC蛋白表达受影响的情况,进一步确定E box元件与USF作用在GCLC基因表达中的最终作用。结果 EMSA和超级迁移率变动试验证实GCLC基因负调控区域的功能元件是E- box,且该元件与USF特异结合参与GCLC基因的调控;共转染USF1 /USF2表达质粒的细胞GCLC基因表达明显下降;Western印迹证实感染重组USF1 /USF2逆转录病毒使肺泡上皮细胞的GCLC蛋白表达明显下降。结论 E -box元件通过与转录因子USF作用抑制GCLC基因表达,E box元件是γGCS基因的重要的抑制元件。

Objective To validate that the E-box element in the -745-705 region of rat γ-GCS catalytic subunit gene (GCLC gene), already found to be a negative regulatory region, is an important transcriptional suppressor element. Methods Electrophoretic mobility shift assays (EMSA) and antibody supershift assay were used to confirm the specific binding of USF, a transcription factor that binds the regulatory sequence -745--705, and E-box element. Rat alveolar epidermal cells of the line CCL-149 were cultured and then divided into 4 groups: group 1 to be transfected with GCLC-Luc, group 2 to be co-transfected with pGCLC-Luc and pCMV-USF1, group 3 to be co-transfected with pGCLC-Luc and pCMV-USF2, and group 4 to be co-transfected with pGCLC-Luc and blank vector pCMV. Recombinant retrovirus vector PLXSN-USF1 and recombinant retrovirus vector PLXSN-USF2 were constructed. Another CCL-149 cells were divided into 4 groups: group A to be infected with recombinant USF1 virus, group B to be infected with recombinant USF2 virus, group C to be infected with blank vector, and group D not to be infected with any plasmid. Western blotting was used to detect the concentrations of GCLC protein. Results EMSA showed that only the probes with complete E-box element could be bound by the USFs. Supershift assay showed that the transcription factor binding the probe was USF. The luciferase activity of the CCL-149 cells co-transfected with pCMV-USF1/USF2 and GCLC-Luc were decreased by 66.4% and 63.2% respectively in comparison with those transfected with GCLC-Luc only (both P<0.05) and decreased by 54.5% and 61.1% respectively in comparison with those of the cells of the blank vector group (both P<0.05). Western blotting showed that the expression of GCLC protein of the CCL-149 cells transfected with PLXSN-USF1 and PLXSN-USF2 were decreased in comparison with that of the control cells. Conclusion The interaction between E-box and USF suppresses the expression of GCLC gene. E-box is an important transcriptional suppressor element of γ-GCS gene.