The RBP4 Promoter Cloning And The Effect Of VDR/VD₃ On The RBP4 Expression

Retinol binding protein 4?RBP4?is a type of hydrophobic transport protein,which is mainly expressed in the liver.It is responsible for transporting retinol and its active metabolites from the liver to target tissues in the body.RBP4 dysfunction plays an important role in the pathological process characterized by metabolic syndrome.So exploring the molecular mechanisms that regulate the expression of RBP4 can prevent or alleviate the health problems caused by RBP4 changes.Because the VDR/VD₃ complex can not only play the role of transcription factors in the regulation of target genes in the cell,but also perform function as a steroid hormone.It is speculated that VDR/VD₃ may be involved in the regulation of RBP4.In order to clarify the possible regulatory role of VDR/VD₃ on the expression of RBP4,it is planned to conduct an analysis of the expression regulation of RBP4by VDR/VD₃ on the basis of RBP4 promoter cloning and bioinformatics analysis.The results will provide a direct regulation process between the RBP4 and VDR/VD_3.Meanwhile,the experimental data also lays a theoretical foundation for the research of RBP4 gene regulation mechanism.Therefore,this study first used the genomic sequence of the human RBP4 gene in NCBI was used as a template to amplify the RBP4 gene promoter fragment.The promoter was analyzed the cis-acting elements and transcription factor binding sites by using bioinformatics.And then,a systematic deletion method was used to construct a series of RBP4 promoter luciferase reporter gene vectors.the dual luciferase reporter gene technology was used to determine the RBP4 promoter regulatory region.Finally,in the case of treatment with overexpressed VDR and active VD₃,real-time quantitative PCR?qPCR?,dual luciferase activity detection,enzyme linked immunosorbent assay?ELISA?and immunofluorescence?IF?method were adopted to research the regulation of nuclear transcription factor VDR/VD₃complex on RBP4 at the transcription and protein level,respectively.The experimental results indicated that the obtained sequence of 2065 bp upstream of the human RBP4 transcription site had VDR binding sites at-1027,-812,and-618,respectively.Double luciferase analysis revealed that the core regulatory region of the human RBP4 gene promoter is located in the region of-720^-415 upstream of the transcription start site.At the same time,the constructed VDR expression vector can significantly increase the VDR mRNA level in cells?P<0.01?.However,compared with the negative control group,the mRNA and protein expression levels of RBP4 did not change significantly,over-expressing VDR and adding active VD₃.The result indicated that the expression of RBP4 isn't regulated directly by VDR.However,in the three regions upstream of the RBP4 transcription start site-91/+98,-209/+98,and-415/+98,VDR significantly increased the activity of the RBP4 promoter in HEK293T cells?P<0.05?.