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

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.