Establishment of a reporter gene system for activity detection of hepatocyte nuclear factor 4α / 第二军医大学学报

ABSTRACT

Objective To establish a luciferase reporter gene system for detecting the activity of hepatocyte nuclear factor 4α (HNF4α), so as to screen the small molecule compounds regulating the activity of HNF4α. Methods HNF4α was purified by affinity chromatography. The direct interaction of DNA fragment or small molecule compounds to the HNF4α was determined by protein thermal shift assay. The constructing recombinant plasmid pGL3-NINJ1-3p or pGL3-NINJ1-9p, which contained three copies or nine copies of the HNF4α response element in the Ninjurin 1 (NINJ1) promoter, was transfected into hepatoma carcinoma cells. The transcriptional activity of HNF4α was detected by dual-luciferase reporter gene assay. The expressions of HNF4α and its down-stream genes were analyzed in hepatoma carcinoma cells treated with small molecular compound luteolin or alverine by real-time quantitative PCR. The changes of HNF4α transcriptional activity of cells treated with luteolin or alverine were estimated by luciferase reporter gene assay. Results Protein thermal shift confirmed that the HNF4α response element in NINJ1 promoter bound to HNF4α protein. In the hepatoma carcinoma cells with overexpression of HNF4α, both pGL3-NINJ1-3p and pGL3-NINJ1-9p could detect the alteration of the transcriptional activity of HNF4α, and pGL3-NINJ1-9p was more sensitive than pGL3-NINJ1-3p (P<0.01). Luteolin and alverine, both directly interacting with HNF4α, down-regulated and up-regulated the expression of HNF4α target genes, respectively. Moreover, pGL3-NINJ1-9p could validate the effect of luteolin or alverine on the transcriptional activity of HNF4α. Conclusion We successfully establish a detection system for HNF4α activity in hepatoma carcinoma cells by the reporter gene vector pGL3-NINJ1-9p. This system is a tool for screening small molecule compounds that regulate HNF4α activity.