【作者】 杜娟；

【导师】 束进；

【作者基本信息】 江苏大学 ， 儿科学（专业学位）， 2023， 硕士

【摘要】 目的:通过构建野生型及突变型ADRB2基因启动子重组质粒,探讨转录因子E2F1对哮喘易感基因ADRB2的转录表达调控的影响。方法:以BEAS-2B细胞的DNA为模板,通过PCR扩增获得人ADRB2基因启动子区域1918 bp大小片段。将此片段插入萤光素酶报告基因载体pGL3-basic中,获得野生型ADRB2基因启动子萤光素酶报告质粒pGL-1879/+39(wt-E2F1)。通过双萤光素酶报告基因系统测定人ADRB2基因启动子片段在A549及BEAS-2B细胞中的活性,使用生物信息学手段预测其潜在的E2F1结合位点;并以pGL-1879/+39(wt-E2F1)为模板对关键调控区域的E2F1结合位点进行点突变获得突变型质粒(mut-E2F1)。将重组质粒与E2F1过表达或小干扰质粒共转染至A549及BEAS-2B细胞后,分别检测野生型及突变型质粒萤光素酶活性以确定E2F1对哮喘易感基因ADRB2启动子的转录调控作用。通过实时荧光定量PCR(Quantitative Real-time PCR,RT-qPCR)及免疫印迹实验(Western blot)观察过表达和敲低E2F1后对哮喘易感基因ADRB2 mRNA及蛋白表达水平的影响探索E2F1对其表达调控作用。利用染色质免疫沉淀(Chromatin Immunoprecipitation,ChIP)技术验证E2F1能否在体内与人ADRB2基因启动子相结合。结果:1.构建人ADRB2基因启动子的重组质粒pGL-1879/+39(wt-E2F1),双荧光素酶检测发现,在A549细胞中,pGL-1879/+39(wt-E2F1)的启动子活性是pGL3-basic的491倍(**P<0.01);在BEAS-2B细胞中,其启动子活性是pGL3-basic的140倍(***P<0.001),且预测到该片段内含有多个转录因子E2F1结合位点。2.ChIP qPCR实验发现,相较于Ig G,E2F1的结合位点富集明显(***P<0.001),提示转录因子E2F1可与人ADRB2基因启动子相结合。3.在A549及BEAS-2B细胞中,与pc DNA3.1组相比,pc DNA-E2F1组ADRB2重组质粒pGL-1879/+39(wt-E2F1)的启动子萤光素酶活性分别增加了2.46倍和1.73倍(**P<0.01,***P<0.001);E2F1小干扰处理后,重组质粒pGL3-1879/+39(wt-E2F1)在A549、BEAS-2B细胞中的启动子活性分别降低了45.7%及43.3%(***P<0.001);对野生型质粒pGL-1879/+39(wt-E2F1)进行点突变后,相较于wt-E2F1,过表达及小干扰处理E2F1后,突变质粒mut-E2F1的活性变化无统计学意义。4.与pc DNA3.1组相比,过表达E2F1后BEAS-2B细胞中ADRB2基因的mRNA相对表达量增加了1.23倍(**P<0.01);与si-NC组相比,小干扰处理E2F1后ADRB2基因的mRNA相对表达量降低了36%(**P<0.01)。5.与pc DNA3.1组相比,过表达E2F1后BEAS-2B细胞中ADRB2基因蛋白表达量增加了61%(*P<0.05);与si-NC组相比,小干扰处理E2F1后其蛋白表达量降低了49%(**P<0.01)。结论:转录因子E2F1可能通过与哮喘易感基因ADRB2启动子结合,调控该基因启动子活性、mRNA及蛋白表达水平,为哮喘等疾病的治疗提供更多潜在可能性。更多还原

【Abstract】 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.更多还原