Clone Of The Promoter Of Human ISL1 Gene And Mechanism Regulated By Dexamethasone And Hypoxia

Background: ISL1 takes part in lots of downstream genes as an important transcription factor.It is highly conversed in different species.The amino acid sequences which ISL1 codes are homologous in human and rat.ISL1 plays an important role in differentiation and development of heart and pancreas in embryo.It is still irreplaceable in islet and specified neuron cells after birth.Focused researching areas of ISL1 used to be target genes which ISL1 has an effect on.Domestic scholar has done some research on the promoter of mouse ISL1 gene and discovered its transcriptional starting site and cis-acting elements.The mechanism of human ISL1 gene expression is still unknown.In this study,we focused on the promoter and regulation mechanism of human ISL1 gene expression.Objectives: To clone a plasmid containing promoter of ISL1 gene and confirm whether dexamethasone and hypoxia regulate ISL1 gene expression.Methods: First,we forecasted DNA sequence of human ISL1 gene promoter by bioinformation method.The DNA sequence of genome in HEK293 cell was used as a template for PCR amplification to clone plasmids containing promoter sequences of human ISL1 gene.Second,we transferred these plasmids into HEK293 cell and measured their promoter activity by dual-luciferase reporter assay system and found the minimal functional fragment of human ISL1 gene promoter.Third,we used Genomatix to predict potential transcription factor binding sites and find HIF-1binding sites and Glucocorticoid response elements.Dual-luciferase reporter assay system and deleting mutant method was used to confirm DXMS influence on the promoter activity and its binding sites.RT-qPCR and Western blot were conducted toconfirm the effect from DXMS and hypoxia on mRNA and protein level of human ISL1 gene expression.Finally,we used Flow Cytometry to see whether DXMS or hypoxia induced apoptosis in HEK293 cell.Results:Plasmid sequencing confirmed successful construction of human ISL1 gene promoter plasmid.Bioinformational website predicted HIF-1 binding sites and Glucocorticoid response element(GRE)in the minimal functional fragment of human ISL1 gene promoter.DXMS could increase promoter activity of plasmids containing GRE.DXMS had no effect on the promoter without the sequence of GRE.The mRNA and protein level of ISL1 in HEK293 cell were relatively higher after put in enough DXMS.mRNA and protein level of ISL1 in HEK293 cells which were cultured in hypoxic condition for 24 hours higher than those in normal condition.The apoptosis rate of HEK293 is higher after put into some DXMS or calculated hypoxic condition for 24 hours than those without DXMS and in normal condition.Conclusion:The reporter plasmids containing human ISL1 gene promoter sequence were successfully constructed and they had relatively high promoter activity in HEK293 cells.DXMS could increase ISL1 gene promoter activity through GRE.DXMS and hypoxia could increase ISL1 gene expression in mRNA and protein level.DXMS and hypoxia could induce apoptosis in HEK293 cells.