含p53保守结合位点的microRNA表达载体的构建及应用

目的:构建含p53保守结合位点的microRNA(miRNA)表达载体,促进相关miRNA在具有野生型p53蛋白细胞中的高效表达。方法:改构miRNA表达载体pCMV-miR,在其多克隆位点前插入p53保守结合位点,分别将miR-138、miR-34a和miR-21前体序列pre-miR-138、pre-miR-34a和pre-miR-21插入上述改构的载体pCMV/p53-miR,将构建的pCMV/p53-miR-138、pCMV/p53-miR-34a和pCMV/p53-miR-21表达载体转染具有野生型p53的HeLa细胞和不表达p53的H1299细胞,分析p53对上述miRNA表达调控的影响。结果:转染改构的miRNA表达载体后,HeLa细胞中miR-138、miR-34a和miR-21的表达水平明显提高,它们对应的已知靶基因Cyclin D3、CDK2和PTEN的表达同时被显著下调。结论:在p53转录调控作用下,具有p53保守结合位点的miRNA表达载体能够更加有效地提高miRNA的表达水平;构建的载体不但可用于促进相关miRNA的表达,也能用于miRNA是否受p53调控的检测。

Constructing and Application of microRNA Expression Vector Con taining p53 Consensus Sequence

Objective： To construct microRNA（miRNA） expression vectors containing p53 consensus sequence （p53CON） and promote miRNA expression in cells with wild-type p53 expression. Methods： Modifying commer cial miRNA expression vector pCMV-miR by inserting p53CON to construct pCMV/p53-miR-138, pCMV/p53-miR- 34a and pCMV/p53-miR-21 expression vectors, miRNA expression vectors were transfected into HeLa cells（p53 wild-type） and H1299 cells（lack expression of p53）, then analyzed the miRNA expression. Results： The expres sion levels of miR-138, miR-34a and miR-21 were significantly increased in HeLa cells, which correspond to their target genes Cyclin D3, CDK2 and PTEN mRNA and protein levels extremely decreased. Conclusion： The miRNA expression vectors with p53CON can efficiently promote the expression of miRNA with p53 binding site and can be used in the detection of p53-dependent miRNA expression.