pcDNA-DEST47-miR-29b重组质粒的构建及其对VEGF表达的靶向调控作用  

Construction of pcDNA-DEST47-miR-29b and its regulatory effects on expression of VEGF

作　　者：单鸿阁 高辉[1] 张海芹[1] 董娟聪[1] 金顺子[1] 

机构地区：[1]吉林大学公共卫生学院卫生部放射生物学重点实验室,长春130021 [2]延边大学医学院,吉林延吉133000

出　　处：《长春中医药大学学报》2014年第4期722-725,共4页Journal of Changchun University of Chinese Medicine

基　　金：国家自然科学基金项目(30870584;81371890);教育部高等学校博士学科点专项科研基金项目(20120061110063)

摘　　要：目的构建has-miR-29b重组表达载体,并探讨其对VEGF表达的靶向调控作用。方法利用生物信息学方法预测has-miR-29b与VEGF-3'UTR的结合位点;将PCR扩增的miR-29b前体序列和pcDNA-DEST47载体经酶切后连接,构建pcDNA-DEST47-miR-29b表达载体;采用荧光素酶检测法和Western blot法证实miR-29b对VEGF蛋白表达的抑制作用。结果 psiCHECK2-VEGF荧光素酶报告质粒和pcDNA-DEST47-miR-29b两种质粒共转染组的荧光素酶活性明显低于单独转染的空载体组和Lu-VEGF组(P<0.01),同时发现其他种类miRNA(Let-7g)不能抑制VEGF荧光素酶活性,而miR-29b明显抑制VEGF荧光素酶活性;转染pcDNADEST47-miR-29b质粒后在Jurkat细胞中VEGF蛋白的表达明显受抑制。结论本研究成功构建pcDNADEST47-miR-29b重组质粒;miR-29b与VEGF具有靶向关系,且miR-29b参与其调控作用。Objective To construct the recombinant expression vector of has-rniR-29b and to explore its effect on expression of VEGF. Methods Bioinformatics was used to analyze the potential binding sites of has-miR-29b and VEGF-3＇UTR. The precursor of miR-29b was amplified by PCR and the PCR products were cloned into digestion pcDNA-DEST47 vector to con- struct pcDNA- DEST47-miR-29b expression vector. The effect of miR-29b interaction with the 3＇-UTR of VEGF on luciferase activity was detected with a dual 1 uciferase assay system and the expression level of VEGF protein affected by miR-29b was detected by Western blotting. Results The luciferase activity was significantly lower in cells co-transfected with psiCHECK2-VEGF luciferase reporter plasmid and pcDNA-DEST47-miR-29b compared with empty vector group and Lu- VEGF group （ P 〈 0.01 ）. The lueiferase activity was not inhibited in the other types of miRNA （Let-Tg）, whereas miR-29b could negatively regulate the luciferase activity by interacting with the 3＇-UTR of VEGF. VEGF protein expression was signif- icantly inhibited in Jurkat cells transfeeted with pcDNA-DEST47-miR-29b plasmid. Conclusion peDNA- DEST47-miR- 29b plasmid were successfully constructed; miR-29b targetedly regulates the expression of VEGF.

关 键 词：miR-29b VEGF 肿瘤 调控 

分 类 号：R332[医药卫生—人体生理学]