The Activity Of The Carbamoyl Phosphate Synthase 1 Promoter In Human Liver-derived Cells Is Dependent On Hepatocyte Nuclear Factor 3-beta

Objective: Carbamoyl phosphate synthase 1(CPS1)is the rate-limiting enzyme in the first step of the urea cycle and an indispensable enzyme in the metabolism of human liver.There is a close relationship between CPS1 gene abnormality and hepatocellular carcinoma.Gene regulation of the hepatocyte lineage is mainly at the transcriptional level,and all kinds of LETFs or heterologous nuclear receptors play a vital role.However,promoter studies on the low expression of key enzymes in the liver cell urea metabolic pathway are few.CPS1 epigenetic regulation involves promoter analysis,and the role of LETFs in the regulation is not yet fully elucidated.In this study,CPS1 gene promoter was used to study the relationship between the liver enriched transcription factor and CPS1 transcription regulation.Methods: The CPS1 full-length promoter was constructed and transfected with various LETFs.The relationship between LETFs and the transcription of CPS1 promoter was detected by Dual-Luciferase Reporter Assay.The recombinant plasmids were constructed from the truncated fragments of CPS1 promoter,and the positive regulatory transcription factors were respectively transfected with the truncated fragments to detect the changes of fluorescence activity.Using the transcription factor binding site prediction software to predict the region with the most positive changes in activity,and to construct the site mutation recombinant vector,transfection,and detect the change of fluorescence activity.Biotin probes and primers were synthesized at the site and EMSA and Ch IP were respectively used to verify the existence of bingding site in vitro and in vivo.The over expression and interference of transcription factors can be used to prove the relationship between transcription factors and the transcription activity of CPS1 gene,and the expression of protein level and m RNA level can be proved.Finally,the detection of urea production in liver derived cells to further demonstrate the regulation of transcription factors and CPS1 gene expression.Results: LETFs were co transfected with the full-length promoter of recombinant plasmid p GL4-2086 in Hep G2 and BEL-7404 cells,Dual-Luciferase Reporter Assay found the transcriptional activity of CPS1 were significantly increased in two kinds of cells after transfected with transcription factor HNF3?.Then all recombinant promoter fragments were,respectively,cotransfected with HNF3?,demonstrating that the fluorescence activity of p GL4-70 was significantly higher than that of transient transfection after cotransfection with HNF3?.The experimental data indicate that the essential binding sites of the HNF3? may exist in the oligonucleotide-70 nt to +73 nt.Softwares were used to analyse the potential HNF3? binding sites in the nucleotides-70 nt to +73 nt.And two sites with high comprehensive evaluation were found,and mutant primers were designed to construct the mutant vectors.Two mutant vectors and normal p GL4-70 were transfected into Hep G2 respectively,Dual-luciferase activity assay showed that the binding site 2 corresponding to p GL4-70-HNF3?mut2 fluorescence activity was significantly lower than that of p GL4-70,which indicated that the binding site 2was effective.After the synthesis of the probe and primer according to site 2,it was found that there was an obvious gel blocking phenomenon in the bands of biotin probe in EMSA and the DNA-transcription factor complexes could be‘super-shifted' by the addition of antibodies.The addition of cold probe has obvious competitive effect,and the mutant probe has no competitive effect.The bands amplified by the specific antibody group were similar to those in the Input group in Ch IP assay,but there was no amplification band in the negative control group and the no antibody group.The results of EMSA and Ch IP confirmed the interaction between HNF3? and CPS1 gene in vivo and in vitro.Over expression and interference experiments showed that the fluorescence activity of the CPS1 promoter increased with the overexpression of p FLAG-HNF3?,showing a concentration dependent.And the fluorescence intensity of transfected HNF3? si RNA group was significantly lower than that in the negative control group NC.In addition,CPS1 also showed the same trend in protein level and m RNA level.With the increase of NH4 CL concentration,the amount of urea increased in different degrees after co-transfected with p FLAG-HNF3? in Hep G2 and BEL-7404 cells in the urea production experiment.The transcription factor HNF3? could promote the metabolism of ammonia by regulating the expression of CPS1.Conclusion:1.Both CPS1 and HNF3? were tissue-specific,and the expression levels in liver-derived cells were significantly higher than that in non hepatocytes.2.Liver enriched transcription factor HNF3? is closely related to the regulation of CPS1 transcription.HNF3? promotes the transcription of CPS1 and enhances the expression of CPS1 gene.3.HNF3? can promote the metabolism of ammonia by regulating the expression of CPS1,which is expected to provide a suitable target for the construction of a new type of ammonia detoxification liver cells.