摘要
目的构建用于鉴定microRNA(miRNA)靶基因的报告基因系统并进行功能验证,为研究动脉粥样硬化的发生发展机制奠定良好基础。方法在pGL3-control载体的荧光素酶基因下游克隆位点插入DNA甲基转移酶3B(DNMT3B)3′非编码区序列,经酶切、测序验证是否插入及正确性。生物信息学分析DNMT3B与miRNA-125b的靶向关系,并用miRNA-125b过表达载体与所构建载体共转染人血管平滑肌细胞,双荧光素酶报告基因系统检测荧光素酶活性。结果成功构建了DNMT3B3′非编码区报告基因载体,经酶切和测序鉴定正确;共转染细胞24h后,双荧光素酶报告基因系统检测荧光素酶活性,结果显示与空载体对照组相比,构建载体组荧光素酶活性下降了54.8%(P<0.01)。结论成功构建了DNMT3BmRNA 3′非编码区报告基因载体,且提示miRNA-125b靶向调控了DNMT3B。
Objective To construct a reporter gene system for the identification of microRNA target genes and verify its func-tion,and lays a good foundation for studying the mechanisms of the occurrence and development of atherosclerosis.Methods Put-ting the DNA methyltransferase 3B（DNMT3B）3′untranslation region into the cloning site downstream of the luciferase gene of the pGL3-Control vector,and the correctness was validated by restriction enzyme digestion and sequencing.The bioinformatics was used to analysis the relationship between DNMT3B and miRNA-125b and the miRNA-125b overexpression vector were co-transfected with the constructed vectors into the human vascular smooth muscle cells,and the luciferase activity was detected by dual luciferase reporter assay system.Results The DNMT3B 3′untranslation region reporter vector was built successfully,and the correctness was verified by the restriction enzyme digestion and sequencing;after co-transfected 24 h,compared with the empty vector control group,the luciferase activity of the constructed vector group decreased significantly by 54.8%（P〈0.01）.Conclusion The DN-MT3B 3′untranslation region reporter vector built successfully,which suggest that miRNA-125b may target-regulate DNMT3B.
出处
《重庆医学》
CAS
CSCD
北大核心
2014年第13期1587-1590,共4页
Chongqing medicine
基金
国家自然科学基金资助项目(81200118
81260063)
教育部新世纪优秀人才计划基金资助项目(NCET-10-0916)
宁夏区自然基金资助项目(NZ12174)
