摘要
目的探讨miR-20b是否直接靶向VEGF 3′-非编码区(3′-UTR)而调控其表达。方法采用miRanda软件预测VEGF 3′-UTR是否存在miR-20b的结合位点;使用PCR方法扩增RAW264.7细胞VEGF基因3′-UTR序列,经双酶切后克隆到pmiR-RB-Report^(TM)质粒海肾荧光素酶的下游,得到pmiR-RB-Report TM-VEGF 3′-UTR双荧光素酶重组质粒,使用电泳法和测序法进行验证;将重组质粒或空质粒与miR-20b模拟物或其对照共转染HeLa细胞,24h后检测相应荧光素酶活性。结果 VEGF 3′-UTR存在miR-20b的结合位点;获得了含VEGF 3′-UTR的双荧光素酶报告载体;重组质粒与miR-20b模拟物共转染组HeLa细胞的荧光素酶活性明显低于空质粒转染组(P<0.05)。结论成功构建小鼠VEGF基因3′-UTR双荧光素酶报告载体,miR-20b可以直接靶向VEGF 3′-UTR而负性调控其表达。
Objective To investigate whether miR-20 bdirectly targets VEGF 3′-untranslated region（3′-UTR）and regulates its expression.Methods MiRanda software was used to predict the binding sites of miR-20 bto VEGF 3′-UTR.3′-UTR sequence of VEGF gene was amplified using polymerase chain reaction（PCR）in RAW264.7cells.PCR products were cloned into the downstream of Renilla luciferase in pmiR-RB-Report TMplasmid to obtain pmiR-RB-Report TM-VEGF 3′-UTR recombinant vector.Electrophoresis and DNA sequencing methods were used to verify the recombinant plasmid.The recombinant plasmid or empty plasmid and miR-20 bmimic or its control were co-transfected into HeLa cells and 24 hlater,the corresponding luciferase activity was detected by Dual-LuciferaseReporter Assay System.Results The recombinant dual luciferase reporter vectors with VEGF 3′-UTR were acquired.There was a binding site of miR-20 bto 3′-UTR of VEGF gene.The luciferase activity in HeLa cells transfected with recombinant plasmid and miR-20 bmimics was significantly lower than in the empty plasmid group（P〈0.05）.Conclusion Mouse VEGF gene 3′-UTR dual luciferase reporter vector was successfully constructed.miR-20 bcould directly target VEGF gene 3′-UTR and negatively regulate its expression.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2016年第1期22-26,共5页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.81273273)
安徽省自然科学基金资助项目(No.1308085MH114)
