PGC-1β调节大鼠ALAS-1基因表达机制的初步探讨

基础医学与临床 ›› 2010, Vol. 30 ›› Issue (6): 587-592.

PGC-1β调节大鼠ALAS-1基因表达机制的初步探讨

刘阳邵迪方福德常永生

中国医学科学院基础医学研究所北京协和医学院基础学院中国医学科学院基础医学研究所北京协和医学院基础学院中国医学科学院基础医学研究所北京协和医学院基础学院

收稿日期:2010-03-19 修回日期:2010-03-29 出版日期:2010-06-05 发布日期:2010-06-05
通讯作者: 常永生

Investigation of the mechanism of PGC-1β regulating transcription of rat ALAS-1

Yang LIU, Di SHAO, Fu-de FANG, Yong-sheng CHANG

Institute of Basic Medical Sciences, CAMS & PUMC IBMS, CAMS&PUMC IBMS, CAMS&PUMC

Received:2010-03-19 Revised:2010-03-29 Online:2010-06-05 Published:2010-06-05
Contact: Yong-sheng CHANG

摘要/Abstract

摘要： 目的检测PGC-1β在人肝癌细胞（Hep G2）和大鼠肝原代细胞中调节大鼠ALAS-1基因表达。方法在Hep G2细胞中瞬时转染PGC-1β，用双荧光报告系统，检测过表达PGC-1β时，大鼠ALAS-1启动子报告基因的活性变化。同时构建了5'端顺式元件系列截短和突变的ALAS-1启动子报告基因，检测并分析介导PGC-1β作用的转录因子结合元件。分离肝原代细胞并检测PGC-1β对ALAS-1转录的影响。构建干扰NRF-1基因的siRNA腺病毒，证明，NRF-1介导PGC-1β激活ALAS-1启动子转录的作用。结果在Hep G2细胞中，过表达PGC-1β显著促进ALAS-1表达。分别构建了FoxA2和NRF1结合元件突变的ALAS-1启动子，发现PGC-1β对NRF1突变的ALAS-1启动子的激活作用显著下降，而FoxA2作用不明显。在肝原代细胞中过表达PGC-1β促进了ALAS-1的转录。当用小RNA干扰NRF-1的表达后，则抑制了PGC-1β对ALAS-1转录的促进作用。结论在Hep G2 和大鼠肝原代细胞中，PGC-1β能够促进ALAS-1基因的表达，并且这种作用是通过NRF-1介导完成的。

关键词: PGC-1β, NRF-1, ALAS-1

Abstract: Objective we're trying to determine the cis-element in the regulation of rat ALAS-1 promoter by PGC-1β. Methods Transient transfection with PGC-1β was done in Hep G2, then dual-luciferase reporter assay was performed to investigate fold activity of rat ALAS-1 promoter when over-expressing PGC-1β. Reconstructing ALAS-1 promoter reporters including a series of 5'-deletions and cis-element mutations, dual-luciferase reporter assay was performed to assay NRF-1 binding sites in rat ALAS-1 promoter. We construct adenoviral PGC-1β and adenoviral siRNA of NRF1 and isolate rat primary hepatocytes and assay the effects of PGC-1β on the rat ALAS-1 transcription. Results Overexpression of PGC-1β stimulates the transcription of ALAS-1 in Hep G2 cells, while over-expressing NRF-1 alone had no stimulation. We constructed NRF-1 binding site mutated ALAS-1 promoter, the promoter activity was obviously diminished by overexpression of PGC-1β. PGC-1β promotes the transcription of ALAS-1 in the rat primary hepatocytes. When we use the siRNA to interfere the expression of NRF-1, the stimulating effect of PGC-1β on the ALAS-1 was attenuated. Conclusion PGC-1β promoting the transcription of ALAS-1 is mainly by NRF-1 in Hep G2 cells and rat primary hepatocytes.

Key words: PGC-1β, NRF-1, ALAS-1

刘阳邵迪方福德常永生. PGC-1β调节大鼠ALAS-1基因表达机制的初步探讨[J]. 基础医学与临床, 2010, 30(6): 587-592.

Yang LIU; Di SHAO; Fu-de FANG; Yong-sheng CHANG. Investigation of the mechanism of PGC-1β regulating transcription of rat ALAS-1[J]. Basic & Clinical Medicine, 2010, 30(6): 587-592.