Construction Of An Expression Vector Carrying ShRNA Targeting CK1α Gene And Its Effects On A549 Cells' Proliferation

Objective:Recent researches show that Casein kinase 1 alpha (CKla) is closely related to tumorigenesis. In this study, RNA interference was used to silence CKla and investigate the effect on A549 cells' growth and proliferation for CKla's further research.Methods:Three shRNA plasmids specifically targeting different sites of CKla (pGenesil-CKla-S1, pGenesil-CKla-S2, pGenesil-CKla-S3) and one nonsense shRNA plasmid (pGenesil-NK) were constructed and transfected into A549 cells, then sorted out by G418. The expression of mRNA and protein of CKla was detected by semi-quantitative RT-PCR and western blotting. Cell growth curve was drawn by cell counting, cell proliferation was measured by CCK-8, cell cycle distribution was assessed by flow cytometry.Results:pGenesil-shRNA expression plasmid was constructed successfully, and the expression of CKla was dramatically down-regulated in A549 cells. Expression of A549-S1,A549-S2 and A549-S3 dereased by 84.3%,57.9% and 61.8% at mRNA level (P<0.001), and by 78.4%,51.3% and 59.9% at protein level (P<0.001), respectively. Therefore, A549-S1 was chosen to do further experiment. Cell counting experiment and CCK-8 assay showed that proliferation of A549-S1 compared with A549 and A549-NK was obviously suppressed, and those IR were 37.7±2.2%(P< 0.01) and 26.2±1.5%(P<0.05), respectively. Cells at S and G2/M phases in the A549-S1 compared with those in the A549 and A549-NK were significantly reduced by 75.0±4.0%(P<0.01) and 45.2±5.4%(P<0.05).Conclusion:The growth and proliferation of A549 cells were inhibited efficiently, and arrested in G0/G1 phase after silencing CKla gene.