Transcription Regulation Of Transcription Factor E2F1 On The Asthma Susceptible Gene ADRB2

Objective:To investigate the effect of transcription factor E2F1 on the expression regulation of asthma susceptibility gene ADRB2 by constructing recombinant plasmids of wild and mutant type ADRB2 gene promoter.Methods:Using BEAS-2B cell DNA as template,the 1918 bp fragment of human ADRB2 gene promoter region was amplified by PCR.The fragment was inserted into the luciferase reporter plasmid pGL3-basic to obtain the wild-type ADRB2 gene promoter luciferase reporter plasmid pGL-1879/+39(wt-E2F1).The activity of human ADRB2 gene promoter in A549 and BEAS-2B cells was determined by dual luciferase reporter system,and the potential transcription factor binding sites were predicted by biological methods.pGL-1879/+39(wt-E2F1)was used as a template to obtain a mutant plasmid(mut-E2F1)by point mutation of E2F1 binding sites in key regulatory regions.After the recombinant plasmid was co-transfected into A549 and BEAS-2B cells with E2F1 overexpression or small interference plasmid,the luciferase activity of wild-type and mutant plasmids was detected to determine the regulatory effect of E2F1 on ADRB2 gene promoter.The effects of overexpression and knockdown of E2F1 on human ADRB2 mRNA expression and protein level were observed by real-time quantitative PCR(RT-qPCR)and Western blot.Moreover,chromatin immunoprecipitation(ChIP)was used to determine whether E2F1 binds to the human ADRB2 promoter in vivo.Results:1.The recombinant plasmid pGL-1879/+39(wt-E2F1)of human ADRB2 gene promoter was constructed,and the activity of pGL-1879/+39(wt-E2F1)promoter in A549 cells was 491 times that of pGL3-basic(**P<0.01).In BEAS-2B cells,the promoter activity was 140 times that of pGL3-basic(***P<0.001),and multiple E2F1 binding sites were predicted in this segment.2.ChIP qPCR showed that compared with Ig G,the binding site of E2F1 was significantly enriched(***P<0.001),suggesting that transcription factor E2F1 could bind to human ADRB2 gene promoter.3.In A549 and BEAS-2B cells,compared with pc DNA3.1 group,the promoter luciferase activity of ADRB2 recombinant plasmid pGL-1879/+39(wt-E2F1)in pc DNA-E2F1 group was increased 2.46 times and 1.73 times,respectively(**P<0.01,***P<0.001);After E2F1 treatment,the promoter activity of recombinant plasmid pGL3-1879/+39(wt-E2F1)in A549 and BEAS-2B cells was decreased by 45.7% and43.3%,respectively(***P<0.001).After point mutation of wild-type plasmid pGL-1879/+39(wt-E2F1),there was no statistical significance in the activity of mutated plasmid mut-E2F1 compared with wt-E2F1 after overexpression and small interference treatment of E2F1.4.Compared with pc DNA3.1 group,the mRNA relative expression of ADRB2 gene in BEAS-2B cells after E2F1 overexpression was increased by 1.23 times(**P<0.01).Compared with the si-NC group,the mRNA relative expression level of ADRB2 gene in E2F1 treated with small interference was decreased by 36%(**P<0.01).5.Compared with pc DNA3.1 group,the expression of ADRB2 gene protein in BEAS-2B cells after E2F1 overexpression was increased by 61%(*P<0.05);Compared with the si-control group,the protein expression level of E2F1 was decreased by 49% after small interference treatment(**P<0.01).Conclusion:Transcription factor E2F1 may regulate the activity of the gene promoter,mRNA expression and protein level by binding with the asthma susceptibility gene ADRB2 promoter,which provides more possibilities for the treatment of asthma and other diseases.