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目的检测PGC-1α和ERRα在人肝癌细胞(HepG2)中对小鼠SHP转录的影响,鉴定ERRα调节SHP启动子利用的顺式元件。方法在HepG2细胞和人胚肾细胞(293A)中瞬时转染ERRα和/或PGC-1α,双荧光酶报告基因实验检测小鼠SHP启动子的活性。构建5′删切和顺式元件突变的启动子报告基因,结合HepG2细胞中的双荧光酶报告基因实验,检测ERRα作用的结合位点。结果在HepG2和293A细胞中,共表达ERRα和PGC-1α能促进SHP转录。分析SHP启动子序列发现5个潜在的ERRα结合位点。通过删切启动子5′端,筛选出其中3个元件参与SHP的转录调节。进一步突变以上3个结合元件,鉴定到其中2个位点参与PGC-1α协同ERRα调节SHP转录的过程,另一个位点参与了SHP基本水平的转录。结论在HepG2细胞中,除核受体FXR、ERRγ之外,发现ERRα可以作为调节SHP基因转录的新途径,但需共激活因子PGC-1α同时存在。
Objective To detect the effect of PGC-1α and ERRα on SHP transcription in mouse hepatoma cells (HepG2) and to identify the cis-elements that ERRα regulates the utilization of SHP promoter. Methods ERRα and / or PGC-1α were transiently transfected into HepG2 cells and human embryonic kidney cells (293A). The double-luciferase reporter assay was used to detect the activity of mouse SHP promoter. Construction of a promoter and reporter gene for 5 ’deletion and cis-element mutation, combined with double luciferase reporter assay in HepG2 cells, detected the binding site of ERRα. Results Coexpression of ERRα and PGC-1α in HepG2 and 293A cells promoted SHP transcription. Analysis of the SHP promoter sequence revealed five potential ERRα binding sites. By deleting the 5 ’end of the promoter, three of them were screened for transcriptional regulation of SHP. Further mutating more than three binding elements, two sites were identified, in which PGC-la cooperated with ERRa to regulate SHP transcription and the other involved in basic SHP transcription. Conclusion In addition to the nuclear receptors FXR and ERRγ, ERRα was found to be a new pathway regulating the transcription of SHP gene in HepG2 cells. However, co-activator PGC-1α was required to coexist.