Construction of the shRNA expression vectors targeting cyclin D1 and investigation of the inhibitory effect of proliferation on A549 cells

Objective To construct Pgenesil-1 small hairpin RNA(shRNA) expression vectors targeting human cyclin D1 gene.To investigate their silencing effect in the A549 cells and the inhibitory effect of proliferation on A549 cells.MethodsThree designed DNA sequences with small hairpin structure were cloned into the transcripted carrier Pgenesil-1 in order to construct recombinant, and the recombinant was transformed into DH5a strains.Then the sequence analysis was carried out after the recombinant plasmid was identified by enzyme restriction.The recombinant vectors were transfected into the A549 cells.At 48 h after transfection, the expression of cyclin D1 at the levels of mRNA and protein was detected by real time PCR and Westernblot.Assay the cell cycle by flow cytometry.ResultsThe shRNA expression vectors were constructed and confirmed after the enzyme digestion analysis and DNA sequencing.The shRNA expression vectors were successfully constructed.Pgenesil-1-CCND13 was most effective which down-regu1ated 62% mRNA and 60% protein of cyclin D1 at 48 h after transfection.Conclusion The shRNA expression vectors targeting cyclin D1 were successfully constructed.The expression of cyclin D1 genewas inhibited effectively in A549 cells transfected by Pgenesil-1-CCND13 and Pgenesil-1-CCND13 can inhibite effectively the proliferation of A549 cells, Which laid a basis for its application in treatment of lung cancer.