Establishment of a human lung adenocarcinoma A549 cell line with RNA interference for Y-box binding protein-1 gene silencing and the influence on A549 cell proliferation

Abstract

Objective To establish a human lung adenocarcinoma A549 cell line with RNA interference for YB-1 gene silencing which can stably express green fluorescent protein (GFP) and to observe the effects of silencing Y-box binding protein (YB-1) gene on proliferation in lung adenocarcinoma A549 cells.Methods The human lung adenocarcinoma A549 cells were transfected with shRNA plasmid specific for the YB-1 gene,and were then selected through G418.G418-resistant clones were generated and screened using reverse transcription polymerase chain reaction (RT-PCR),real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting analysis.Cell proliferation was examined by methyl thiazolyl tetrazolium.Results The G418-resistant clones (A549-746 and A549-326) showed much lower level of YB-1 mRNA (0.41 ± 0.01 and 0.39 ± 0.06) than A549-shNC (P ＜ 0.05) under RT-PCR.A549-746 and A549-326 cell clones showed much lower level of YB-1 mRNA (0.43 ±0.10 and 0.20 ± 0.24) and protein expression (0.49 ± 0.06 and 0.24 ± 0.02) than A549-shNC (P ＜ 0.05) under FQ-PCR and Western blotting analysis.Meanwhile,the G418-resistan clones (A549-746 and A549-326) stably expressed GFP in vitro.The cell growth rate in YBX1-homo-746 and YBX1-homo-326 groups was decreased significantly at the first 24 h (P ＜ 0.05).Conclusion The lung cancer cell lines A549-746 and A549-326 with stable YB-1 gene silencing and GFP expression were successfully established,and silencing YB-1 gene could inhibit the proliferation of lung adenocarcinoma A549 cells. Key words: Lung adenocarcinoma; Y-box binding protein-1 ; Short hairpin RNA; Green fluorescent protein