Abstract:

To construct a recombinant eukaryotic expression plasmid of microRNA let7a2 and express it in lung cancer A549 cells. Methodslet7a2 precursor sequence was amplified by RTPCR using RNA from A549 cells and was cloned into the pSilencer4.1 CMV neoexpression vector to produce the recombinant pSilencer4.1let7a2. Then the recombinant pSilencer4.1let7a2 was transfected into lung cancer A549 cells, and the prelet7a2 expression was verified by RTPCR. According to the miRBase Targets database, the target sequence of let7a2 was synthesized and inserted to the pMIRReport luciferase reporter vector to construct pMIRReportlet7a2T, which was cotransfected with pSilencer4.1let7a2 into A549 cells, and the relative luciferase activity was detected. The effect of let7a2 on A549 cell proliferation was tested by MTT. ResultsThe results of DNA sequencing showed that sequences of pMIRReportlet7a2T and pSilencer5.1let7a2 were correct. RTPCR prelet7a2 was overexpressed in A549 cells after transfection of pSilencer4.1let7a2. Cotransfection of pSilencer4.1let7a2 and pMIRReportlet7a2T showed that mature let7a2 had biological activity. MTT results indicated that let7a2 inhibited proliferation of the transfected A549 cells. ConclusionThe eukaryotic expression plasmid of let7a2 was successfully constructed and effectively expressed in A549 cells.

Key words: Lung neoplasms, Gene expression regulation, miRNA, Let7a2