Inhibitory effects of shRNA targeting Twist on epithelial-mesenchymal transition and invasion ability of human lung carcinoma cell A549 in vitro / 中国癌症杂志

ABSTRACT

Background and purpose:Epithelial-mesenchymal transitions(EMTs) occur as key steps during embryonic morphogenesis,and are now implicated in the progression of primary tumors towards metastases.We have found that the transcription factor Twist,a master regulator of EMT,was expressed markedly in human lung carcinoma cell line(A549).This study is to investigate the effects of short hairpin RNA(shRNA) targeting Twist on EMT and invasion ability of A549 cells in vitro.Methods:RNA interference plasmid that can express short hairpin RNA(shRNA) targeting Twist(positive RNAi plasmid) or express shRNA that does not match any known human coding mRNA(negative RNAi plasmid)was designed,constructed,and transfected into A549 cells line.Positive RNAi A549 cells(Pi group) expressing Twist suppressed or negative RNAi A549 cells(Ni group) expressing Twist uninfluenced was selected by neomycin resistance.In normal(control,C),Pi and Ni group A549 cells,Twist,alpha-smooth muscle actin(?-SMA) and E-cadherin expression were examined by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot,invasion ability were examined by Boyden chamber model.Results:1,In C group,both Twist and ?-SMA expressions were strongly positive,but E-cadherin was poorly expressed.The number of A549 cells permeating septum of Boyden chamber(NCS) was equal to 57.4?3.4.2,In Pi group,compared with that in C group,Twist and?-SMA expression,and NCS were all decreased,but E-cadherin expression was significantly increased(P