人延伸体复合物Elp3亚基表达及功能研究

The Expression and Function of Human Elongator Complex Protein 3

目的将人延伸体复合物(elongator)的中心亚基elp3(elongtor complexprotein3)基因导入293T细胞,观察它对293T细胞生长特性以及基因转录活性的影响。方法用脂质体将pFlAGCMV4-Elp3真核表达载体导入293T细胞,通过G418持续筛选阳性克隆,建立elp3的稳定表达细胞株。通过RT-PCR、免疫荧光法检测外源性elp3基因的表达。MTT法测定细胞生长曲线、流式细胞仪法测定细胞周期及凋亡情况。瞬时转染不同组合的质粒后,收集细胞用荧光素酶报告基因法检测转染前后荧光素酶活性的变化。结果Elp3可以抑制293T细胞生长,并使细胞出现G1期阻滞,未引起293T细胞凋亡。elp3能激活转录,但作用较弱。elp4也可激活转录,过量表达elp3基因与elp4基因时,可明显激活转录,说明两者有协同激活转录作用。Elp3与转录因子TFⅡF亚基RAP30基因过量表达时,两者也可协同激活转录。结论Elp3对293T细胞的生长有明显抑制作用,可诱导G1期细胞阻滞,具有转录激活功能,并可分别与Elp4亚基、RAP30亚基协同激活转录。

Objective The core subunit elp3 of human elongator complex was introduced in the 293T cells to observe its effects on the growth and transcription activity of 293 cells. Methods pFIAGCMV4-EIp3 plasmid was stably transfected into 293T cell. The expression of Elp3 gene was conformed by RT-PCR and immunofluorescence. MTT assay was applied to detect the growth of 293T cells. Flow cytometry was used to determine the cell cycle distribution and cell apoptosis. Luciferase reporter gene assay was applied for the detection of transcription activity. Results Over-expression of Elp3 inhibited the growth of 293T cell and arrested the cells in Gl phase of the cell cycle, but did not induce apoptosis. Elp3 could activate transcription. EIp4 gene could also activate transcription by immediate transfection, and over-expression of the two genes could synergistically activate transcription. Transcription factor F is important for transcription and its two subunits, and RAP30 and RAP74. RAP30, could activate transcription by immediate transfection. Over-expression of Elp3 gene and RAP30 gene can synergistically activate transcription. Conclusion Elp3 can inhibit the growth of 293T cell and arrest the cells in G1 phase. It can activate transcription and activate transcription in the presence of Elp4 or RAP30.