Study On The Regulation Of FASN Gene Transcription By SCAP/SREBP1

In order to elucidate the transcriptional mechanism of dairy cow SCAP / SREBP regulating fatty acid synthase gene in liver cells,the coding sequence of SCAP gene was cloned from the liver tissue c DNA of Holstein cow and the pc DNA3.1-SCAP expression vector was constructed.The FASN luciferase reporter gene vector was constructed by PCR from the cow genomic DNA to construct three different fragment lengths.The subcellular localization of SCAP and SREBP1 protein was observed by laser scanning confocal microscope with SCAP and SREBP1 by immunofluorescence.The expression of SCAP gene m RNA was detected by real-time fluorescence quantitative PCR.The protein expression of SCAP and nuclear SREBP1 was detected by Western blotting.The FASN gene promoter plasmid was transfected into liver cells and transfected with pc DNA3.1-SREBP1 plasmid,pc DNA3.1-SCAP plasmid and cotransfection of pc DNA3.1-SREBP1 and pc DNA3.1-SCAP plasmid as double fluorescent The enzyme assay detects the activity of the FASN promoter.Later,FASN gene promoter plasmid and pc DNA3.1-SREBP1 plasmid were transfected into liver cells and transfected with different doses of pc DNA3.1-SCAP plasmids as the treatment,dual luciferase assay FASN promoter activity.The pc DNA3.1-SREBP1 plasmid,the pc DNA3.1-SCAP plasmid and the pc DNA3.1-SREBP1 and pc DNA3.1-SCAP plasmids were transfected into liver cells respectively for processing,and the m RNA of SREBP1/FASN was detected by real-time fluorescence quantitative PCR The effect of different treatment factors on intracellular lipid droplets was analyzed by Nile red staining.The results showed that the CDS region of the cloned SCAP gene was 3836 bp.The results of confocal laser scanning microscopy showed that SCAP localized in the cytoplasm.SCAP and SREBP1 overexpression enhanced the expression of SCAP and SREBP1 in liver cells.Three FASN gene promoter vectors,PGL3-FASN1-3,were constructed and their lengths were 177 bp,255bp and 399 bp,respectively.Sequence analysis showed that both FASN2 and FASN3 contained SRE binding sites.The results of dual luciferase assay showed that the promoter activity of FASN was increased with the length of FASN.Compared with the control group transfected with p CDNA3.1 vector,the activity of FASN promoter in SREBP1 group was significantly increased,The co-treatment of SCAP and SREBP1 cell FASN promoter activity was also significantly increased.After co-transfecting SCAP and SREBP1 plasmids,SCAP can increase the activity of PGL3-FASN promoter in a dose-dependent manner.Overexpression of SCAP/ SREBP1 can increase the m RNA expression of FASN in liver cells and increase the intracellular lipid droplets significantly.This study shows that SCAP/SREBP pathway can increase the lipid droplet synthesis in liver cells by promoting the transcriptional expression of FASN gene.