Screening And Validation Of Transcription Factors In The Core Promoter Region Of New Adipocytokine Omentin

Background: Omentin is a new type of adipokine,also known as Intelectin-1(ITLN-1).ITLN-1 is not only closely related to obesity,diabetes,tumors and other diseases.Studies have shown that ITLN-1 has anti-inflammatory and anti-atherosclerotic effects,and serum ITLN-1 level in patients with unstable carotid plaques due to ischemic stroke was significantly lower than the stable plaque group.There are significant differences in ITLN-1 levels between individuals.ITLN-1 is expressed to varying degrees in human endothelial cells,reticulocytes,and tissues such as colon,ovary,lung,and placenta,but not in subcutaneous fat.Therefore,studying the transcriptional regulation mechanism of human ITLN-1 gene will have important research significance.DNA pull down is a technology based on short DNA fragments with high affinity sites for DNA-binding proteins and developed with the help of a biotin / streptavidin purification system,which is widely used in the research of DNA-protein interactions.Objective: In order to further explore the transcriptional regulation mechanism of ITLN-1 during expression,we used the ITLN-1 core promoter sequence,which was determined in previous experiments,as a DNA probe for DNA pull down experiments.The protein bound to this sequence was enriched,and the transcription factor was screened based on the bioinformatics analysis.The effect of the transcription factor on the expression of ITLN-1 was initially determined by small interference RNA and double luciferase reporter gene experiments.Methods: 1.Design primers based on the core promoter region of ITLN-1 and label them with biotin;obtain the target gene by PCR technology,purify and recover it by agarose gel electrophoresis,and obtain the target probe;DNA pull down technology was used to pull down nuclear proteins.The products were silver-stained after SDS-PAGE,and differential bands were selected for mass spectrometric detection.The bioinformatics website was used to analyze the data to screen out transcription factors.2.Design the siRNA based on the transcription factors that were initially selected,specifically knock them down,and test the impact of the transcription factor knockdown on ITLN-1 gene expression;3.Construct the transcription factor into the plasmid and transfer it into 293 T cells together with the plasmid carrying the ITLN-1 core promoter region.After 48 h,the effect of overexpression of this transcription factor on the transcription of the ITLN-1 core promoter region was determined by double-luciferase activity test.Results: 1.Compared with the negative probe,the target probe synthesized based on the ITLN-1 core promoter region pulls down 72 differential proteins by DNA pull down.Through enrichment analysis,we know that the main biological functions of 32 proteins are related to nucleic acid binding.KDM5 A,ENO1,HSP90AA1,PNN,PRPF6,and CHD5 are closely related to transcription through database search.We chose KDM5 A,which is prominent in transcription studies,for further research.Western blot was used to detect the expression of KDM5 A in DNA pull down products;2.Transfect HEK293 cells with two KDM5A-specific siRNAs(siKDM5A-1,siKDM5A-2),and found that the interference efficiency of siKDM5A-1 is more than 70%,and the interference efficiency of siKDM5A-2 is less than 70%;When siKDM5A-1 was used to knock down KDM5 A,ITLN-1 expression was up-regulated;3.Transfect 293 T cells with different combinations of pGL3_basic,pcDNA3.1,pRL,pcDNA3.1-KDM5 A,and pGL3_ITLN1-1.Cells were lysed 48 h after transfection for double luciferase reporter gene detection.The cells co-transfected with pcDNA3.1-KDM5 A and pGL3_ITLN1-1 had significantly higher RLU values than the other groups.This indicates that KDM5 A overexpression promotes transcription of the ITLN-1 core promoter region.Conclusions: 1.For the core region of ITLN-1 promoter,we successfully synthesized the target probe with biotin labeling,and enriched the transcription factors bound in this region.Six transcription-related proteins including KDM5 A,ENO1,HSP90AA1,PNN,PRPF6,and CHD5 were screened;2.After knocking down KDM5 A in HEK293 cells,the expression of ITLN-1 at the mRNA level is up-regulated.Overexpression of KDM5 A can promote the transcription of the core promoter region of ITLN-1;3.KDM5 A,as a histone demethylase,is involved in gene silencing of ITLN-1,but its non-demethylase-dependent characteristics enhance the transcription of ITLN-1 core promoter.