Establishment of pGPH1-GFP-RKIP plasmid vector and its effect on sensitivity to chemotherapy of leukemia cell line CCRF-CEM
LI Qing
DUO Li-kun
Abstract：Objective To establish plasmid vectors pGPH-green fluorescent protein ( GFP)-Raf kinase inhibitor pro-tein ( RKIP) expressing RKIP small interfering RNA ( siRNA) and to observe the effect of RKIP RNA interference on sen-sitivity to chemotherapy of pediatric leukemia cells CCRF-CEM.Methods Plasmid RKIP interference vectors pGPH-GFP-RKIP were established and transfected into CCRF-CEM cells by using electroporation technique .RKIP, phosphorylated ex-tracellular regulated protein kinase (p-ERK) and phosphorylated nuclear factor κB inhibitor (p-IκB) levels in the trans-fected cells were detected by using Western blotting .9-nitrocamptothecin (9NC) at various concentrations were used to in-duce CCRF-CEM cells apoptosis , and Western blotting was used to detect the RKIP expression , caspase-3 activities ( apop-tosis rates) were detected by using flow cytometry .Results The pGPH1-GFP-RKIP was successfully established .Trans-fection rates of pGPH1-GFP-RKIP and pGPH1-GFP plasmids were 90.04% and 91.32%, respectively.The expression levels of RKIP were effectively down-regulated and both p-ERR and p-IκB levels were up-regulated in CCRF-CEM cells transfected with pGPH-GFP-RKIP plasmids as compared with the cells transfected with pGPH 1-GFP plasmids ( all P<0.05).RKIP levels in the CCRF-CEM cells exposed to 9NC were up-regulated and the CCRF-CEM cells transfected with pGPH-GFP-RKIP had down-regulated apoptosis rates (all P<0.05).Conclusions RKIP siRNA plasmid vectors are suc-cessfully established .RKIP expression changes may be associated with the sensitivity to chemotherapy of leukemia cells .
Keywords：Raf kinase inhibitor proteinRNA interferencepediatric leukemiasensitivity to chemotherapy
Publication Date：2014-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：4( 20-23 )