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。
Objective To determine the cis-element in the regulation of rat ALAS-1 promoter by PGC-1β.MethodsTransient 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 constructed adenoviral PGC-1β and adenoviral siRNA of NRF1 and isolated rat primary hepatocytes and examined the effects of PGC-1β on the rat ALAS-1 transcription.Results Overexpression of PGC-1β stimulated the transcription of ALAS-1 in Hep G2 cells,while over-expressing NRF-1 alone had no stimulation effect.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 used 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 transcription of ALAS-1 is mainlyexplained by NRF-1 in Hep G2 cells and rat primary hepatocytes.
出处
《基础医学与临床》
CSCD
北大核心
2010年第6期587-592,共6页
Basic and Clinical Medicine
基金
国家重点基础研究发展计划(973计划)(2004CB518602和2006CB503909)
国家高技术研究发展计划(863计划)(2006AA02Z192)
国家自然科学基金(30700386和30721063)
